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Advances in Experimental Medicine and Biology 1166

Elisabetta Baldi
Monica Muratori Editors

Genetic Damage
in Human
Spermatozoa
Second Edition
Advances in Experimental Medicine
and Biology

Volume 1166

Editorial Board

IRUN R. COHEN, The Weizmann Institute of Science,

Rehovot, IL, Israel
ABEL LAJTHA, N.S.Kline Institute for Psychiatric Research,
Orangeburg, NY, USA
JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA
RODOLFO PAOLETTI, University of Milan, Milan, IT, Italy
NIMA REZAEI, Children’s Medical Center Hospital,
Tehran University of Medical Sciences, Tehran, IR, Iran
More information about this series at http://www.springer.com/series/5584
Elisabetta Baldi • Monica Muratori
Editors

Genetic Damage in
Human Spermatozoa
Second Edition
Editors
Elisabetta Baldi Monica Muratori
Department of Clinical and Department of Experimental and
Experimental Medicine Clinical Biomedical Sciences “Mario
University of Florence Serio”
Florence, Italy University of Florence
Florence, Italy

ISSN 0065-2598     ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-030-21663-4    ISBN 978-3-030-21664-1 (eBook)
https://doi.org/10.1007/978-3-030-21664-1

© Springer Nature Switzerland AG 2014, 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or
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This book is dedicated to Prof. Gianni Forti, who guided our
activities with great sapience.
Preface

The goal of the male gamete is to deliver a fully intact and functioning pater-
nal genome to the oocyte. To fulfill this aim, the process of chromatin matura-
tion during spermiogenesis must be correctly completed to guarantee DNA
protection during the long journey to reach the oocyte and to properly de-­
condense and form the male pronucleus after fertilization. Genetic abnor-
malities in spermatozoa can be generated in any phase of the sperm production
and life and may be due to endogenous and exogenous conditions, the latter
including in vitro manipulation for assisted reproduction and gonadotoxic
therapies. In addition, emerging studies point out the importance of the dam-
age to the sperm epigenome and address the mechanisms involved in generat-
ing it. All these abnormalities may have profound consequences for male
fertility status and even for the health of the progeny. This book presents an
updated overview of the various types of damage that may affect sperm chro-
matin. Besides the main mechanisms involved in the generation of de novo
mutations and DNA strand breaks and oxidation, two chapters of the book are
dedicated to sperm epigenome and epigenetic damage and their consequences
for the progeny. In addition, as one of the most important issues regards the
possible medical interventions to reduce or prevent sperm DNA fragmenta-
tion, one chapter faces the important aspect of pharmacological and surgical
treatments, lifestyle modifications, and prevention against exposure to envi-
ronmental and occupational toxicants.
We wish to thank all the authors for their invaluable contributions to the
book. They are all expert scientists in the field, and we appreciate their will-
ingness to offer their knowledge in this important branch of reproductive
medicine. We hope that this book will help the researchers in the topics of
reproduction and serve as a reference for medical and technical staff working
in assisted reproduction laboratories.

Florence, Italy Elisabetta Baldi

Monica Muratori

vii
Contents

1 Genetic Factors Affecting Sperm Chromatin Structure��������������   1

Mélina Blanco and Julie Cocquet
2 Age-Dependent De Novo Mutations During Spermatogenesis
and Their Consequences������������������������������������������������������������������ 29
Francesca Cioppi, Elena Casamonti, and Csilla Krausz
3 The Sperm Epigenome: Implications for Assisted
Reproductive Technologies�������������������������������������������������������������� 47
Douglas T. Carrell
4 Epigenetic Transgenerational Inheritance������������������������������������ 57
Joan Blanco Rodríguez and Cristina Camprubí Sánchez
5 Sperm DNA Fragmentation: Mechanisms of Origin�������������������� 75
Monica Muratori, Sara Marchiani, Lara Tamburrino,
and Elisabetta Baldi
6 Sperm DNA Fragmentation: Consequences
for Reproduction������������������������������������������������������������������������������ 87
Luke Simon, Benjamin Emery, and Douglas T. Carrell
7 Oxidative Damage to Sperm DNA: Attack and Defense�������������� 107
Joel R. Drevet and R. J. Aitken
8 Interventions to Prevent Sperm DNA Damage Effects on
Reproduction������������������������������������������������������������������������������������ 119
Sandro C. Esteves
9 Cryopreservation of Sperm: Effects on Chromatin
and Strategies to Prevent Them������������������������������������������������������ 149
Donatella Paoli, Marianna Pelloni, Andrea Lenzi,
and Francesco Lombardo
10 Effect on Sperm DNA Quality Following Sperm
Selection for ART: New Insights ���������������������������������������������������� 169
Nicoletta Tarozzi, Marco Nadalini, and Andrea Borini

ix
x Contents

11 Sperm DNA Damage in Cancer Patients �������������������������������������� 189

Hermance Beaud, Amelie R. Tremblay, Peter T. K. Chan,
and Geraldine Delbes

Index���������������������������������������������������������������������������������������������������������� 205
Contributors

R. J. Aitken Priority Research Centre for Reproductive Science, Faculty of

Science, University of Newcastle, Callaghan, NSW, Australia
Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
Elisabetta Baldi Department of Experimental and Clinical Medicine, Unit
of Sexual Medicine and Andrology, Center of Excellence DeNothe, University
of Florence, Florence, Italy
Hermance Beaud Institut national de la recherche scientifique, Centre
INRS – Institut Armand-Frappier, QC, Canada
Mélina Blanco INSERM, U1016, Institut Cochin, Paris, France
CNRS, UMR8104, Paris, France
Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris,
France
Joan Blanco Rodríguez Genetics of Male Fertility Group, Unitat de
Biologia Cel·lular (Facultat de Biociències), Universitat Autònoma de
Barcelona, Bellaterra(Cerdanyola del Vallès), Spain
Andrea Borini 9.baby Family and Fertility Center, Bologna, Italy
Cristina Camprubí Sánchez GenIntegral, Barcelona, Spain
Reference Laboratory Genetics, L’Hospitalet de Llobregat, Spain
Unitat de Biologia Cel·lular i Genètica Mèdica (Facultat de Medicina),
Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès),
Spain
Douglas T. Carrell Andrology and IVF Laboratories, Department of
Surgery, and Department of Human Genetics, University of Utah School of
Medicine, Salt Lake City, UT, USA
Elena Casamonti Department of Biomedical, Experimental and Clinical
Sciences “Mario Serio”, University of Florence, Florence, Italy
Peter T. K. Chan Division of Urology, McGill University Health Center,
QC, Canada
Francesca Cioppi Department of Biomedical, Experimental and Clinical
Sciences “Mario Serio”, University of Florence, Florence, Italy

xi
xii Contributors

Julie Cocquet INSERM, U1016, Institut Cochin, Paris, France

CNRS, UMR8104, Paris, France
Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris,
France
Geraldine Delbes Institut national de la recherche scientifique, Centre
INRS – Institut Armand-Frappier, QC, Canada
Joel R. Drevet GReD Laboratory, CNRS UMR6293 – INSERM U1103 –
Université Clermont Auvergne, Faculté de Médecine, Clermont-Ferrand,
France
Benjamin Emery Department of Surgery (Urology), University of Utah
School of Medicine, Salt Lake City, UT, USA
Sandro C. Esteves ANDROFERT, Andrology and Human Reproduction
Clinic, Referral Center for Male Reproduction, Campinas, SP, Brazil
Department of Surgery (Division of Urology), University of Campinas
(UNICAMP), Campinas, SP, Brazil
Faculty of Health, Aarhus University, Aarhus, Denmark
Csilla Krausz Department of Biomedical, Experimental and Clinical
Sciences “Mario Serio”, University of Florence, Florence, Italy
Andrea Lenzi Laboratory of Seminology – Sperm Bank “Loredana
Gandini”, Department of Experimental Medicine, University of Rome “La
Sapienza”, Rome, Italy
Francesco Lombardo Laboratory of Seminology – Sperm Bank “Loredana
Gandini”, Department of Experimental Medicine, University of Rome “La
Sapienza”, Rome, Italy
Sara Marchiani Department of Experimental and Clinical Medicine, Unit
of Sexual Medicine and Andrology, Center of Excellence DeNothe, University
of Florence, Florence, Italy
Monica Muratori Department of Experimental and Clinical Biomedical
Sciences “Mario Serio”, Unit of Sexual Medicine and Andrology, Center of
Excellence DeNothe, University of Florence, Florence, Italy
Marco Nadalini 9.baby Family and Fertility Center, Bologna, Italy
Donatella Paoli Laboratory of Seminology – Sperm Bank “Loredana
Gandini”, Department of Experimental Medicine, University of Rome “La
Sapienza”, Rome, Italy
Marianna Pelloni Laboratory of Seminology – Sperm Bank “Loredana
Gandini”, Department of Experimental Medicine, University of Rome “La
Sapienza”, Rome, Italy
Luke Simon Department of Surgery (Urology), University of Utah School
of Medicine, Salt Lake City, UT, USA
Contributors xiii

Lara Tamburrino Department of Experimental and Clinical Medicine, Unit

of Sexual Medicine and Andrology, Center of Excellence DeNothe, University
of Florence, Florence, Italy
Nicoletta Tarozzi 9.baby Family and Fertility Center, Bologna, Italy
Amelie R. Tremblay Institut national de la recherche scientifique, Centre
INRS – Institut Armand-Frappier, QC, Canada
 Genetic Factors Affecting Sperm
Chromatin Structure
Mélina Blanco and Julie Cocquet
Abstract
Spermatozoa genome has unique features that
make it a fascinating field of investigation:
first, because, with oocyte genome, it can be
transmitted generation after generation; sec-
ond, because of genetic shuffling during meio-
sis, each spermatozoon is virtually unique in
terms of genetic content, with consequences
for species evolution; and finally, because its
chromatin organization is very different from
that of somatic cells or oocytes, as it is not
based on nucleosomes but on nucleoprot-
amines which confer a higher order of packag-
ing. Histone-to-protamine transition involves
many actors, such as regulators of spermatid
gene expression, components of the nuclear
envelop, histone-modifying enzymes and
readers, chaperones, histone variants, transi-
tion proteins, protamines, and certainly many
more to be discovered.
In this book chapter, we will present what
is currently known about sperm chromatin
structure and how it is established during sper-
miogenesis, with the aim to list the genetic
factors that regulate its organization.
M. Blanco · J. Cocquet (*)
INSERM, U1016, Institut Cochin, Paris, France
CNRS, UMR8104, Paris, France
Université Paris Descartes, Sorbonne Paris Cité,
Faculté de Médecine, Paris, France
e-mail: julie.cocquet@inserm.fr
© Springer Nature Switzerland AG 2019
E. Baldi, M. Muratori (eds.), Genetic Damage in Human Spermatozoa, Advances in Experimental
Medicine and Biology 1166, https://doi.org/10.1007/978-3-030-21664-1_1
1
Keywords
Spermatozoa · Chromatin · Protamine ·
Nucleosome · Histone · Gene expression ·
Nucleus · Spermatids · Spermiogenesis
Introduction
Spermatozoa are produced through a multi-step
process called spermatogenesis, during which
spermatogonial stem cells at the base of the semi-
niferous tubules enter the differentiation pathway
to ultimately give rise to spermatozoa, released in
the lumen of the testicular seminiferous tubules.
Spermatogenesis can be divided into three
phases: mitotic phase, meiosis, and post-meiotic
phase or spermiogenesis. During mitotic phase,
spermatogonial stem cells undergo mitotic divi-
sions to maintain the spermatogonial stem cell
pool; some of them differentiate into primary
spermatocytes. Each primary spermatocyte
undergoes DNA replication and meiotic division
to produce four haploid round spermatids. Round
spermatids then differentiate into elongated sper-
matids in a process that involves dramatic mor-
phological changes including cytoplasm removal,
acrosome biogenesis, development of flagellum
for motility, accumulation of mitochondria in the
midpiece, and extensive chromatin remodeling
that results in nuclear condensation and tran-
scriptional silencing (Russell et al. 1990). The
1
2 M. Blanco and J. Cocquet
post-meiotic differentiation of round spermatids
into spermatozoa is called spermiogenesis.
During this step, spermatid chromatin is exten-
sively modified and remodeled to give rise to a
chromatin organization only found in spermato-
zoa. Indeed, in all other cells (somatic cells,
female germ cells, and male germ cells until
spermatid stage), the nucleosome is the core par-
ticle of chromatin structure (Luger et al. 1997).
Histone proteins H2A, H2B, H3, and H4 assem-
ble into an octamer around which 146 base pairs
of DNA are wrapped, and this nucleosome struc-
ture occurs every 200 base pairs in the eukaryotic
genome (Mcghee and Felsenfeld 1980; Luger
et al. 1997). In sperm chromatin, the basal unit is
not the nucleosome but the nucleoprotamine,
formed of smaller, more basic proteins (richer in
arginine) than histones: the protamines. Sperm
chromatin is organized as toroids containing
~50–100kb of DNA, leading to a chromatin
structure 5–10 times more condensed than
nucleosome-based chromatin (Ward and Coffey
1991; Balhorn 2007). This tight compaction is
essential to allow DNA to fit into a nucleus that is
seven times smaller than an interphasic somatic
cell nucleus (Ward and Coffey 1991) and to pro-
tect the paternal genome from physical and
chemical damages. It is also possible that a small
nucleus is a hydrodynamic advantage that con-
fers a higher speed to spermatozoa during their
transit (Braun 2001).
Briefly, the process of replacement of histones
by protamines requires (i) opening of the histone-­
based chromatin structure facilitated by histone
posttranslational modifications (PTM) – in par-
ticular histone hyperacetylation – and incorpora-
tion of histone variants, (ii) binding of
bromodomain proteins to acetyl residues and
recruitment of chromatin-remodeling proteins
and of transition proteins, (iii) formation and
repair of DNA breaks, and (iv) incorporation of
protamines leading to a protamine-based com-
pact chromatin structure. At the end of this pro-
cess, most histones have been replaced by
protamines. A small portion of histones (~1% in
mice, ~10% in humans) is retained in the sperma-
tozoa genome and contributes to the epigenetic
program of the embryo (Balhorn et al. 1977;
Gatewood et al. 1990; Hammoud et al. 2009;
Brykczynska et al. 2010; Erkek et al. 2013; Ihara
et al. 2014; Carone et al. 2014; Samans et al.
2014; Royo et al. 2016; Yoshida et al. 2018;
Yamaguchi et al. 2018). [For review, see
Champroux et al. (2018).]
Studying animal models (mostly knockout
mice) and patient cases, researchers and clini-
cians have found many genes involved in histone-­
to-­
protamine transition, and many more will
certainly be discovered. Each of them is a genetic
factor which could alter chromatin structure
when mutated. In this review, we will present
their known or predicted roles while describing
the key steps leading to the transition from a
histone-­
based chromatin to protamine-based
chromatin (see also Table 1.1).
 egulation of Spermatid Gene
R
Expression
The differentiation of round spermatids into sper-
matozoa involves profound morphological and
functional changes and requires a very specific
genetic program with thousands of genes only
expressed at that time and regulated at the tran-
scriptional and post-transcriptional levels (Steger
1999; White-Cooper and Davidson 2011; Kleene
2013). Studies of gene expression dynamic
throughout spermatogenesis have shown that this
program starts as early as the pachytene phase of
meiosis [see, for instance, da Cruz et al. (2016)
and Chen et al. (2018)].
Among the genes of which expression is acti-
vated/upregulated during spermiogenesis are
those required for histone-to-protamine transi-
tion such as histone variants, chaperones, histone-­
modifying enzymes, transition proteins, and, of
course, protamines themselves. Hence, transcrip-
tion regulators which control the spermatid gene
expression program can indirectly impact on
sperm chromatin structure via deregulating key
genes of this process.
This is particularly true for regulators of
Protamine 1 (Prm1) and Protamine 2 (Prm2)
gene expression: in the mouse, Prm1 and Prm2
are transcribed into mRNAs that can be detected
 1
Table 1.1 List of genes of which mutations have been shown to result in abnormal sperm chromatin structure
Molecular role in Evidence of role in sperm chromatin
Gene name Protein spermiogenesis Phenotype when mutated structure References
Genes encoding chromatin proteins
Prm1/2 Protamine 1/ 2 DNA compaction in Prm1+/− and Prm2+/− chimeric male mice Acridin orange assay on Prm1+/− and Cho et al. (2001,
male germ cells are infertile with abnormal chromatin Prm2+/− chimeric mice and Comet 2003);
compaction and sperm DNA damage assay on Prm2+/− chimeric mice Schneider et al.
Another study found that Prm2+/− males sperm (2016)
are fertile and that Prm2−/− males are Electron microscopy on sperm from
infertile with chromatin compaction Prm2+/− chimeric mice and
defect Prm2−/− mice
Tnp1/2 Transition protein 1/2 Intermediates in Tnp1−/− and Tnp2−/− mice are hypofertile Electron microscopy and western blot Yu et al. (2000);
histone-to-protamine but present chromatin compaction defect on spermatids at different stages Zhao et al.
transition and high level of unprocessed PRM2- (2001); Zhao
precursor in sperm et al. (2004)
Tnp1−/− Tnp2−/− double knockout mice
are infertile with chromatin compaction
defect and unprocessed PRM2 precursor
protein
Genetic Factors Affecting Sperm Chromatin Structure

H1fnt (H1t2) Testis-specific histone Testis specific Histone Knockout male mice are infertile with Quantification of propidium iodide in Tanaka et al.
H1 H1 sperm chromatin compaction defect, sperm DNA by FACS, western blot (2005);
nuclear abnormalities in spermatids, and on sperm and electron microscopy on Martianov et al.
low protamine level in sperm elongated spermatids (2005)
Th2a TH2A (histone cluster 1 Testis specific Histone In Th2b−/− mouse, fertility is not altered. Electron microscopy on sperm, Montellier et al.
(Hist1h2aa) H2A family member a, 2 variants The absence of TH2B is compensated by MNase digestion in condensed (2013);
and Th2b Hist1h2aa) and TH2B the overexpression of H2B in testes spermatids, and immunostaining of Shinagawa et al.
(Hist1h2ba) (histone H2B type 1-A, However, transgenic mice, in which spermatids at different stages (2015)
Hist1h2ba) TH2B is fused to a C-terminal tag, are Histone liquid chromatography and
infertile, and elongating spermatids fail mass spectrometry
to differentiate and to compact their
chromatin. TH2B is incorporated into
chromatin but is not replaced by
transition proteins or protamines in
elongating spermatids
In Th2a−/− Th2b−/− double mutant mice,
TNPs and PRMs also fail to incorporate
into chromatin, and H2B is
overexpressed
(continued)
3
 4
Table 1.1 (continued)
Molecular role in Evidence of role in sperm chromatin
Gene name Protein spermiogenesis Phenotype when mutated structure References
H3f3a, Histone H3.3 Histone H3 variant Knockout male mice are infertile with Phase-contrast microscopy on sperm, Yuen et al.
H3f3b abnormal sperm head shape, increase in ChIP-seq, and RT-qPCR on testes (2014)
H3K9me3, and decrease in H3K4me3 at
Prm1/2/3, and Tnp1 promoters are
associated with decreased expression
H2al2 H2A.L.2 (histone Spermatid specific Knockout male mice are infertile with Electron microscopy on sperm, Barral et al.
(H2al2a) H2A-Bbd type 1) histone variant chromatin compaction defect and immunoprecipitation, and (2017)
unprocessed PRM2 protein. Transition immunostaining on condensing
proteins are not bound to chromatin spermatids
Regulators of gene expression: Direct or indirect effect on the expression of genes encoding chromatin proteins
Act (Fhl5) Activator of cAMP-­ Activator of Crem, a Knockout male mice are fertile but with Electron microscopy on Act-null Kotaja et al.
responsive element major regulator of low sperm count, abnormal head shape, spermatozoa (2004)
modulator in testis spermatid gene and chromatin compaction defect in
expression sperm
Brdt Bromodomain testis-­ Driver of testis-specific Knockout male mice are infertile with Electron microscopy on elongating Pivot-Pajot et al.
specific protein gene expression altered sperm morphology, failure of spermatids and epididymal (2003); Shang
program, histone spermatids to elongate, and chromatin spermatozoa, microarray-based gene et al. (2007);
acetylation reader compaction defect. At the beginning of expression profiling, and RT-qPCR on Gaucher et al.
meiosis, BRDT regulates the expression (juvenile and adult) whole testis (2012)
of hundreds of meiotic and post-meiotic RNA, immunostaining on whole testis
genes. After meiosis, during spermatid sections, and elongating spermatids
elongation, BRDT’s first bromodomain is
involved in the recognition of histone
hyperacetylation prior to histone removal
Brwd1 Bromodomain and WD Regulator of gene Knockout male mice are infertile, with Electron microscopy on spermatids, Philipps et al.
repeat containing expression chromatin compaction defect and microarray-based gene expression (2008);
protein 1 decreased expression of Prm1, Tnp1, and profiling, and RT-qPCR on whole Pattabiraman
Tnp2 in testes testis RNA et al. (2015)
Chd5 Chromodomain- Helicase Chd5 deficient mice are subfertile or sterile Electron microscopy on sperm and Li et al. (2014);
helicase-­DNA-binding with chromatin compaction defect and sperm chromatin structure assay, Zhuang et al.
protein 5 spermiogenesis impairment in elongating western blots on spermatids, (2014)
spermatids. In spermatids, Prm1 is immunostaining on testis section,
upregulated and CHD5 appears to be ChIP-qPCR and RT-qPCR on round
enriched at its promoter. In late spermatids, spermatids
histones are retained, and the level of
non-processed PRM2 precursor is higher
M. Blanco and J. Cocquet
 1
Cnr1 Cannabinoid receptor 1 Guanine nucleotide-­ Knockout male mice are fertile but their Acridine orange assay, Comet assay, Chioccarelli
binding protein-­ spermatozoa present abnormal chromatin Aniline blue staining, western blot et al. (2010)
coupled receptor compaction and higher rate of DNA detection of TNPs and PRMs on
damage and of retained histones. Tnp2 whole testis extract. Densitometry
expression is downregulated in testis analysis of Tnp2 cDNA
Ctcf Transcriptional Architectural protein. Male germ cell-specific knockout of Ctcf Electron microscopy on elongated Hernandez-­
repressor CTCF Regulates the 3D in mice leads to infertility with low spermatids, microarray on whole Hernandez et al.
structure of chromatin sperm count, seminiferous tubule testis RNA, and western blot (2016)
atrophy, and defects in sperm head detection of PRM1 and PRM2 in
formation and chromatin compaction. spermatozoa
H1fnt expression is downregulated. KO
sperm present reduced PRM1 level and
defective histone retention
Epc1 Enhancer of polycomb Component of the Knockout male mice are infertile with Immunofluorescence on testicular Dong et al.
homolog 1 NuA4 histone spermiogenesis arrest. At the molecular section, western blot, and RNA-seq (2017)
acetyltransferase level, histone acetylation is lower, on round spermatids
(HAT) complex ubiquitination of H2A and H2B is
increased, and Tnp1, Tnp2, Prm1, and
Prm2 are upregulated
Genetic Factors Affecting Sperm Chromatin Structure

Kdm3a Lysine demethylase 3A H3K9 demethylase. Knockout male mice are infertile with Electron microscopy on spermatids, Okada et al.
(Jhdm2a) Binds Tnp1 and Prm1 low sperm count and chromatin ChIP qPCR and RT-qPCR on round (2007)
promoter compaction defects. Decreased KDM3A spermatids
at the promoter of Prm1 and Tnp1 in
round spermatids is linked with a
decreased expression of these genes
Pygo2 Pygopus 2 Unclear (belongs to a Mice with mutations in Pygo2 are sterile Immunohistochemistry on testis Nair et al.
family of a with defective elongation process and section, RT-qPCR, and western blot (2008)
co-activators of clear reduction of Prm1, Prm2, Tnp2, and on round spermatids
β-Catenin/Wnt H1fnt expression. In elongated
signaling pathway) spermatids, histone H3 acetylation is
reduced, and a higher proportion of
histones is retained indicative of defective
histone-to-­protamine transition. In testis,
Pygo2 protein co-immunoprecipitates
with a histone acetyl transferase (HAT)
activity. The phenotype induced by Pygo2
mutations appears to be independent of
the β-Catenin/Wnt signaling pathway
(continued)
5
 6
Table 1.1 (continued)
Molecular role in Evidence of role in sperm chromatin
Gene name Protein spermiogenesis Phenotype when mutated structure References
Setd2 Histone-lysine H3K36 methyl Setd2 conditional knockout in RNA-seq and RT-qPCR on round Zuo et al. (2018)
N-methyltransferase transferase murine male germ cells leads to spermatids
SETD2 infertility with a spermiogenic arrest at
step 8 round spermatids and decreased
expression of Tnp1/2 and Prm1/2/3
Sly Sycp3 like Y-linked Regulator of gene Knock-down male mice are hypofertile Immunostaining on testis section, Riel et al.
expression with reduced histone H3K79 RT-qPCR, ChIP-qPCR, and western (2013); Moretti
dimethylation and H4 acetylation in blot on spermatids, western blot on et al. (2017); El
elongating spermatids, histone retention spermatozoa, comet assay, CMA3 Kennani et al.
in spermatozoa, and abnormal chromatin staining, and electron microscopy on (2018)
compaction. Sly KD leads to the sperm
deregulation of many genes including
downregulation of the H3K79 histone
methyl transferase Dot1l and
upregulation of X- and Y-encoded H2al
genes
Sox30 Transcription factor Transcription factor Knockout male mice are infertile with a ChIP-seq and RNA-seq on Bai et al. (2019);
SOX-30 binding Tnp1 promoter spermiogenic arrest at the early round spermatogonia, spermatocytes, and Zhang et al.
spermatid stage and reduced expression round spermatids (2018)
of H1fnt, Hils1, H2afb1, H2al1n, H2al3,
Tnp2, and Prm1/2/3 in spermatids
Taf7l TATA-binding protein Activation complex of Oligozoospermia and post-meiotic Taf7l and Pol II ChIP-qPCR on adult Cheng et al.
associated factor 7l RNApol II. Binds spermatogenesis arrest in knocked-out testes (2007);
Prm1 promoter mice. Mutations of Taf7l have been found Akinloye et al.
in oligozoospermic patients (2007); Sediva
et al. (2007);
Zhou et al.
(2013)
Direct or indirect regulators of chromatin proteins at the translational/posttranslational level
Camk4 Calcium/calmodulin-­ Serine threonine Knockout male mice are infertile with Immunostaining on testis section and Wu et al. (2000)
dependent protein kinase kinase, phosphorylates spermiogenesis defect in elongating western blot on testes protein extract
type IV PRM2 spermatids, retention of TNP2, and
absence of PRM2 in elongating
spermatids
M. Blanco and J. Cocquet
 1
Cdyl Chromodomain Y-like Crotonyl-CoA Overexpression of Cdyl in transgenic Immunostaining on testis section, Liu et al. (2017)
protein hydratase (i.e., mice decreases male fertility, with lower western blot on testes, and ChIP-­
negatively regulates sperm count and motility. In elongating qPCR on spermatocytes and round
histone crotonylation) spermatids, Kcr level (in particular spermatids
H2BK12cr) is lower, and the levels of
chromatin-bound TNP1 and PRM2 are
decreased
Dcr1 Dicer Endoribonuclease Male germ cell-specific knockout of Optic microscopy and electron Korhonen et al.
acting in short Dcr1 in mice leads to infertility with low microscopy of elongating spermatids, (2011)
dsRNA-mediated sperm count and disruption of spermatid immunostaining of H3 acetylated and
post-transcriptional elongation. KO spermatids have reduced PRM1 on testis section
gene silencing H3 acetylation and PRM1 levels
Kat5 (Tip60) Histone Catalytic subunit of the Kat5 conditional mouse knockout Immunofluorescence on testicular Dong et al.
acetyltransferase KAT5 NuA4 histone (induced at postnatal day 15) produces section, western blot on germ cells (2017)
acetyltransferase degenerative tubules characterized by
complex loss of spermatocytes and spermatids.
Acetylated histone H4 and TNP2 levels
appear decreased
Nut Nuclear protein in testis Regulator of histone Knockout male mice are infertile with Immunostaining in spermatids, Shiota et al.
Genetic Factors Affecting Sperm Chromatin Structure

acetylation via spermiogenesis arrest at condensing high-performance liquid (2018)

recruitment of P300 spermatid stage, histone-to-protamine chromatography-tandem mass
and CREBBP transition failure, and decrease in H4 and spectrometry on round and elongating
H2A acetylation spermatids
Parg Poly(ADP-ribose) Degradation of Parg deficient mouse sperm presents CMA3 immunostaining on sperm, Meyer-Ficca
glycohydrolase poly(ADP-ribose) chromatin compaction defect and H1T western blot detection of histone et al. (2011a)
and TH2B retention marks, variants, and transition
proteins in spermatozoa
Piwil1 (Hiwi Piwi-like protein 1 Endoribonuclease In humans, R218A/L221A mutation in Electron microscopy on sperm, Gou et al.
in humans repressing the Hiwi is associated with azoospermia immunostaining of histone on sperm, (2017)
and Miwi in mobilization of Male mice with the same mutation are western blot of PRM1/2, and TPNP1
mice) transposable element infertile but produce spermatozoa with on spermatozoa
abnormal motility, chromatin compaction
defect, and increased nucleosome
retention
Psme4 Proteasome activator Proteasome activator Knockout male mice are hypofertile with Immunostaining on testis section and Qian et al.
(Pa200) complex subunit 4 acetylation dependent lower sperm count. Core histones are not western blot on testis extracts (2013); Khor
degraded in KO elongating spermatids, et al. (2006)
and their disappearance is delayed
(continued)
7
 8
Table 1.1 (continued)
Molecular role in Evidence of role in sperm chromatin
Gene name Protein spermiogenesis Phenotype when mutated structure References
Rfx2 DNA-binding protein Key transcription Knockout male mice are infertile with Immunohistochemistry on testis Kistler et al.
RFX2 factor of spermatid elongation failure and TNP2 section (2015)
spermatogenesis accumulation in round spermatid
nucleus. Spermiogenesis arrests at step
7 in round spermatids
Rnf8 E3 ubiquitin protein H2A and H2B Knockout male mice are sterile with Immunostaining and electron Lu et al. (2010);
ligase RNF8 ubiquitination, defective histone removal and protamine microscopy on sperm, western blot Sin et al. (2012)
recruitment of KAT8 insertion, resulting in less condensed (TNPs and PRMs) on testicular
acetyltransferase to sperm chromatin protein extracts
chromatin Another group generated a similar KO
model but did not observe chromatin
compaction defects
Sirt1 NAD-dependent protein NAD-dependent Male germ cell-specific knockout of Sirt1 Electron microscopy on elongating Bell et al. (2014)
deacetylase sirtuin-1 protein deacetylase in mice leads to male hypofertility with spermatids, western blot on testis
spermatogenesis failure at round extract for H4 acetylation, H3
spermatid stage and sperm chromatin acetylation and methylation, and H2B
condensation defect. Globally (in whole acetylation and ubiquitination.
testes) levels of histone posttranslational Western blot detection of TH2B on
modifications are altered (H4ac, spermatozoa. Immunofluorescence on
H3K4me, H2BK16ac, and H2BK120ac squash slides of stages X to XII
levels are decreased and H2BK120ub is tubules
increased). A higher proportion of
histones is retained in KO spermatozoa
By immunostaining, TNP2 and PRM1
appear to be perinuclear rather than
nuclear in Sirt1-KO elongated spermatids
Tarbp2 RISC-loading complex Required for Knockout male mice are infertile with Gene reporter and immunostaining of Lee et al.
subunit TARBP2 translational activation severe oligozoospermia, and elongated testicular section (1996); Zhong
of protamine mRNAs spermatids present a mosaic pattern for et al. (1999)
Prm1 expression
Tssk6 Testis-specific serine/ Serine threonine kinase Knockout male mice are infertile with Immunostaining on testis section and Jha et al. (2017)
threonine-protein kinase high level of PRM2 precursor and western blot detection of γH2AX and
6 retained histones in sperm. H2AX of histones in spermatids and
phosphorylation in elongating spermatids spermatozoa, respectively
is impaired
M. Blanco and J. Cocquet
 1
Regulators of the nuclear structure
Dpy19l2 Probable Nuclear envelope Mutations in humans lead to Immunostaining on testis section and Yassine et al.
C-mannosyltransferase component globozoospermia spermatozoa, acidic aniline blue, and (2015)
DPY19L2 In Dpy19l2 knockout mice, spermatid CMA3 staining on spermatozoa
elongation is defective with chromatin
compaction defect due to defective
protamination and histone retention
Gmcl1 Germ cell-less protein- Nuclear matrix Knockout male mice are infertile with Electron microscopy on testis section. Kimura et al.
(Mgcl-1) like 1 component multiple heads and flagella spermatozoa, Western blot detection of sperm (2003)
spermatid chromatin compaction defect, proteins
disruption of nuclear structure of
elongating spermatids, and accumulation
of immature PRM2 in sperm
Lis1 Platelet-activating factor Non-catalytic Disruption of the testis-specific Lis1 Immunostaining and electron Nayernia et al.
(Pafah1b1) acetylhydrolase IB component of transcript in mice leads to male infertility microscopy on testis section (2003)
subunit alpha platelet-activating caused by a spermiogenesis block.
factor acetylhydrolase Spermatids present multiple defects:
1b (PAF-AH 1B). acrosome and flagellum malformations,
Associates with defective nuclear condensation, and
Genetic Factors Affecting Sperm Chromatin Structure

microtubules abnormal TNP2 location

Others
Aurkc Aurora kinase C Serine threonine kinase Mutations in humans induce polyploid Electron microscopy on sperm Kimmins et al.
spermatozoa. Knockout male mice are (2007);
fertile but present sperm chromatin Dieterich et al.
compaction defect (2007)
NB. Genes have been grouped according to their expected molecular role, but it is sometimes difficult to discriminate between an effect on regulation of gene expression and on
regulation of chromatin structure
9
10
in round spermatids and stored as nonpolysomal,
ribonucleoproteins in the cytoplasm until transla-
tion 3–7 days later (Hecht 1990; Kleene 1989).
Among the key regulators of Protamine tran-
scription and translation are TATA box proteins
(TBPs), CREM transcription factor, Y box pro-
teins (such as MSY2), and TARBP2 (aka PRBP
in mice and TRBP in humans) [see Carrell et al.
(2007) for review and also below]. For instance,
mice lacking Tarbp2 gene fail to translate
Protamine mRNA which results in delayed
replacement of transition proteins, oligozoosper-
mia, and male infertility (Lee et al. 1996; Zhong
et al. 1999) (see Table 1.1).
Taf7l is a component of a protein complex
required for transcription of genes by RNA poly-
merase II in spermatids, such as Prm1/2 (Zhou
et al. 2013). Its knockout leads to decreased
sperm count, reduced sperm motility, and
hypofertility/sterility (Cheng et al. 2007; Zhou
et al. 2013). Its impact on chromatin sperm struc-
ture has not been described, but chromatin immu-
noprecipitation experiments (ChIP-Seq) showed
that TAF7L directly binds to the promoter of
Prm1 gene (Zhou et al. 2013) and as such could
influence its expression and thus sperm chroma-
tin structure. Mutations in Taf7l gene have been
found in patients with oligozoospermia (Akinloye
et al. 2007; Sediva et al. 2007) (see Table 1.1).
It is worth noting that, in cases of regulators of
spermatid gene expression, mutations can induce
a plethora of spermatid differentiation defects,
with sometimes a block leading to no sperm at all
(azoospermia). This is the case, for instance, of
TATA box-binding protein-like 1 Tbpl1 knockout
(aka Trf2) which leads to a spermiogenic block at
stage 7 (Zhang et al. 2001). Papolb (aka Tpap)
encodes a testis-specific enzyme responsible for
poly(A) tail extension of specific mRNAs in
round spermatids (i.e., a poly(A) polymerase)
and thus involved in post-transcriptional regula-
tion of mRNAs. Its knockout also induces a stage
7 block during spermiogenesis (Kashiwabara
et al. 2002). Crem encodes a master regulator of
spermiogenesis as it controls the expression of
many spermatid genes including Tnp1 (Transition
protein 1), Tnp2 (Transition protein 2), Prm1,
M. Blanco and J. Cocquet
and Prm2. In its absence, spermatids fail to fully
differentiate, and no spermatozoa are produced
(Blendy et al. 1996; Nantel et al. 1996). Male
mice lacking Crem activator, Act (aka Fhl5), pro-
duce spermatozoa in reduced number, with
abnormal flagellum and heads yet are fertile
(Kotaja et al. 2004). KIF17B is a regulator of
CREM-ACT activity (Macho et al. 2002) and as
such can influence expression of genes coding for
protamines and other proteins essential for sperm
structure (see Table 1.1). It is worth noting that
KIF17B has been shown to interact with MIWI,
an important regulator of sperm chromatin struc-
ture (Wang et al. 2015).
The arginine methyltransferase CARM1 has
recently been shown to also control gene expres-
sion in spermatids: Carm1-KO male germ cells
present post-meiotic gene deregulation associ-
ated with multiple spermiogenesis defects lead-
ing to male hypofertility. Investigation of the
underlying mechanism has revealed that CARM1
negatively controls the transcriptional activity of
CREM-ACT via methylating their co-activator,
the histone acetyl transferase P300 protein (Bao
et al. 2018). Yet, Carm1-KO does not seem to
affect mRNA levels of genes known to be essen-
tial for histone-to-protamine transition such as
Prm1, Prm2, Tnp1, and Tnp2 but could impact on
sperm chromatin condensation via another path-
way. The consequence of Carm1-KO on sperm
chromatin structure has not been described.
The histone H3K36 methyltransferase SETD2
is another protein that appears to control the
expression of genes essential to histone-to-­
protamine transition. SETD2 is highly expressed
in spermatocytes and spermatids in the mouse
testis and localizes to the nucleus of spermato-
cytes and round spermatids. Setd2 conditional
knockout male mice are sterile and have arrested
spermatogenesis at step 8 round spermatids. It
leads to the downregulation of Tnp1, Tnp2, Prm1,
Prm2, Prm3, H1fnt, and H2afb1 genes in round
spermatids (Zuo et al. 2018) (see Table 1.1).
Kdm3a (also known as Jhdm2a) encodes the
lysine-specific demethylase 3A, a histone
demethylase that is highly expressed in post-­
meiotic germ cells. Jhdm2a-null male mice are
1 Genetic Factors Affecting Sperm Chromatin Structure
infertile due to failure of round spermatids to dif-
ferentiate into elongated spermatids. Jhdm2a is
required for Tnp1 and Prm1 transcription, and
ChIP experiments showed that JHDM2A is
recruited to the promoter of Tnp1 and Prm1 in
round spermatids (Okada et al. 2007). Thus loss
of Jhdm2a in the testis leads to decreased expres-
sion of Tnp1 and Prm1, resulting in chromatin
condensation defects such as indistinct chromo-
center, loss of heterochromatin polarity in steps
7–9 spermatids and defective chromatin conden-
sation in step 13 spermatids (Okada et al. 2007)
(see Table 1.1).
CTCF is an architectural protein that regulates
gene expression via the 3D organization of the
genome. The specific knockout of Ctcf gene in
male germ cells leads to spermiogenesis defects
and male infertility due to abnormal histone-to-­
protamine transition, in particular defective prot-
amine incorporation (Hernandez-Hernandez
et al. 2016) (see Table 1.1).
There are many other examples of regulators
of spermatid gene expression, at the transcrip-
tional or post-transcriptional level, of which
knockout leads to an arrest during spermiogene-
sis, such as Sox30 (Bai et al. 2019; Zhang et al.
2018; Feng et al. 2017) and Rfx2 (Kistler et al.
2015). Their absence in mice precludes the for-
mation of spermatozoa; however, mutations in
these genes that do not induce a complete loss of
function (heterozygous mutations, for instance)
could lead to the production of sperm with an
abnormal chromatin structure.
Small RNAs and associated proteins also con-
tribute significantly to gene regulation during
spermiogenesis. The chromatoid body is a peri-
nuclear cloud-like/granule structure that starts to
be formed in late pachytene spermatocytes and is
predominant in round spermatids. It is involved
in post-transcriptional gene regulation and mostly
composed of RNA-binding proteins and small
RNA (i.e., mostly piRNA and miRNA) (Meikar
et al. 2014).
Dicer1 is a critical regulator of microRNA and
siRNA biogenesis (Ha and Kim 2014). Recent
studies in mouse models revealed that the ubiqui-
tously expressed Dicer1 gene has an essential
11
role in spermatogenesis. Dicer1 mRNA was
found to be highest in spermatogonia and sper-
matocytes and decreases in spermatids. Germ
cell-specific Dicer1 knockout male mice are ster-
ile, with reduced testis size, and spermatid elon-
gation is severely affected indicating abnormal
spermiogenesis. Elongating spermatids show
abnormal head shapes and disrupted chromatin
condensation and organization: elongating sper-
matids retain hyperacetylated H3, and protamine
deposition in elongating spermatids is severely
reduced; expression and localization of the his-
tone variant H1T2 (see also below) are disrupted
in knockout spermatids (Korhonen et al. 2011)
(see Table 1.1).
Mutations in regulators of the piRNA pathway
or in genes involved in the piRNA pathway could
also affect sperm chromatin structure via their
effect on gene expression. Absence of MIWI
(encoded by Piwil1/Miwi in mice) leads to an
arrest of germ cell differentiation at the begin-
ning of the round spermatid stage, and therefore
no sperm are produced (Deng and Lin 2002). In
infertile patients, R218A/L221A mutation in
Hiwi (human homolog of Piwil1) has been found
associated with azoospermia (Gou et al. 2017).
Yet, knock-in male mice generated to mimic this
mutation are infertile with a different phenotype:
spermatozoa are produced (albeit in reduced
number) and present abnormal motility and chro-
matin compaction (Gou et al. 2017). The under-
lying mechanism producing this phenotype
appears to be independent of the piRNA path-
way: the mutation in the D-Box element prevents
MIWI protein ubiquitination and degradation and
leads to sequestration of the histone ubiquitina-
tion ligase RNF8. As a consequence, in mutant
sperm, H2A and H2B posttranslational modifica-
tions are impaired which leads to nucleosome
stabilization and retention, defective protamine
deposition, and defective chromatin compaction
(Gou et al. 2017) (see Table 1.1). This study
exemplifies the complexity to predict the conse-
quence of mutations found in human patients
based on the study of animal models and, there-
fore, to provide an exhaustive list of the genes
affecting sperm chromatin structure.
12 M. Blanco and J. Cocquet

Importance of the Nuclear Envelope terized by deformed round sperm heads without
an acrosome [see Ray et al. 2017 for review].
Nuclear and chromatin remodeling during sper- Dpy19l2-null male mice are infertile and have
miogenesis also depends on the nuclear envelope abnormal sperm head and chromatin condensa-
and its components, via their effect on gene tion defects due to abnormal protamine deposi-
expression and/or on nucleus architecture. Gmcl1 tion (Yassine et al. 2015) (see Table 1.1).
(also known as Mgcl-1) encodes a protein During spermiogenesis, acrosome develop-
expected to regulate gene expression in male ment is tightly linked to nuclear shaping and
germ cells via its association with the nuclear nucleus compaction via its anchoring to a struc-
envelope (Nili et al. 2001). It is highly expressed ture formed of F-actin and keratin called the
from pachytene spermatocytes and localizes to acroplaxome (Kierszenbaum et al. 2003).
nuclear lamina; its deletion results in male infer- Moreover, acrosome development and chromatin
tility, abnormal nuclear architecture, and abnor- remodeling appear to be interconnected (De
mal chromatin condensation (Kimura et al. Vries et al. 2012), and there have been several
2003). Reduced levels of PRM1 and PRM2 pro- reports of reduced ICSI (intracytoplasmic sperm
teins were observed in Mgcl-1-null sperm, and injection) success rates (i.e., lower fertilization,
immature precursor PRM2 accumulate indicat- pregnancy, and live birth rates) in patients with
ing abnormal posttranslational processing of globozoospermia which could be due to sperm
protamines. It is yet unclear whether abnormal DNA damage resulting from poor chromatin con-
protamine processing and chromatin condensa- densation (Davila Garza and Patrizio 2013). It is
tion are direct or indirect consequences of nuclear therefore worth taking a closer look at genes
envelope abnormality (Kimura et al. 2003) (see other than Dpy19l2 which have been linked to
Table 1.1). globozoospermia phenotype.
LIS1 (encoded by Lis1 also known as Mutations in two other genes have been sug-
Pafah1b1) associates with dynein and microtu- gested to be involved in human globozoospermia,
bules and has been shown to affect nuclear struc- though with a much lower prevalence than
ture; disruption of the testis-specific Lis1 Dpy19l2 mutations: Pick1 (Liu et al. 2010) and
transcript leads to abnormal spermiogenesis, Spata16 (Dam et al. 2007). Pick1 knockout leads
with abnormal formation of acrosome and flagel- to male infertility with a phenotype similar to
lum and defective nuclear condensation in sper- human globozoospermia with defective acro-
matids. As a result, spermiogenesis is almost some formation, reduced sperm count, and
fully blocked with only a few sperm found in the deformed sperm nuclei (Xiao et al. 2009). A
epididymides (Nayernia et al. 2003) (see mouse model mimicking Spata16 human muta-
Table 1.1). LIS1 and two other subunits compose tion does not have spermatogenesis defect, but
PAF acetylhydrolase 1b. Disruption of LIS1-­ the deletion of its exon 4 produces male infertil-
associated proteins has been shown to reduce ity with severe spermiogenesis defects and only
LIS1 protein level; PAFAH α1 and α2 could few spermatozoa (Fujihara et al. 2017). The stud-
therefore have an impact on spermatid nuclear ies of Pick1 knockout and of Spata16 knockout
condensation though knockout studies have mice did not present a detailed characterization
found earlier spermatogenesis defects, at the mei- of the nuclear morphology and composition of
otic stage (Yan et al. 2003a; Koizumi et al. 2003). the produced sperm cells. So, the involvement of
DPY19L2 is also presumed to be involved in those factors in sperm chromatin structure is
nuclear envelop structure. It is highly expressed unclear (Xiao et al. 2009; Fujihara et al. 2017).
in human and mouse germ cells and co-localizes In the mouse, several other genes have been
with the region of the inner nuclear membrane shown to cause globozoospermia-like pheno-
facing the acrosome (Pierre et al. 2012). Deletion types when knocked out and could therefore also
of Dpy19l2 gene is a major cause of globozoo- be involved in chromatin compaction. This is the
spermia, a rare male infertility condition charac- case for Mfsd14a (aka Hiat1) which encodes the
1 Genetic Factors Affecting Sperm Chromatin Structure 13

hippocampus abundant transcript 1 protein of been shown to be essential for histone degrada-
which function is unknown: a mouse model with tion and eviction though it is not the sole mecha-
a LacZ gene insertion that disrupts the expression nism for histone eviction during spermatid
of the Mfsd14a gene displays spermiogenesis differentiation (Marushige et al. 1976; Oliva
defects characterized by failure of acrosome for- et al. 1987; Oliva and Mezquita 1986; Sassone-­
mation abnormal sperm head condensation (as Corsi 2002; Awe and Renkawitz-Pohl 2010).
observed by electron microscopy) and mitochon- Several histone acetyl transferases (HAT) have
drial mislocalization (Doran et al. 2016). Other been suggested to be involved in this process,
genes of which KO in mice leads to globozoo- such as CREB-binding protein (encoded by
spermia, such as Csnk2a2 (Xu et al. 1999), Atg7 Crebbp), P300 (encoded by Ep300), KAT8
(Wang et al. 2014), Gba2 (Yildiz et al. 2006), (encoded by Kat8 also known as Mof), enhancer
Golga2 (Han et al. 2017), Gopc (Yao et al. 2002), of polycomb homolog 1 (encoded by Epc1), and
Hrb (Kang-Decker et al. 2001), Hsp90b1 KAT5 (encoded by Kat5 also known as Tip60)
(Audouard and Christians 2011), Zpbp1 (Lin (Boussouar et al. 2014, Dong et al. 2017, Lu et al.
et al. 2007), Vps54 (Paiardi et al. 2011), or Smap2 2010). Yet the production of conditional KO
(Funaki et al. 2013), could also be directly or mouse models of the genes encoding these HAT
indirectly involved in sperm chromatin conden- did not allow to fully demonstrate their implica-
sation, but data on this particular phenotype are tion, either (i) because gene KO was only partial
lacking or not always conclusive. and did not show the expected phenotype
(Boussouar et al. 2014) or (ii) induced a pheno-
type or blockade of spermatogenesis before the
Remodeling of Chromatin Structure stage at which histones are hyperacetylated
During Spermatid Differentiation (Dong et al. 2017; Jiang et al. 2018). It is also
important to add that histone-modifying enzymes
As mentioned, during spermatid differentiation, and histone PTMs are involved in the regulation
the chromatin is extensively remodeled with mul- of gene expression and contribute to sperm chro-
tiple posttranslational modifications (PTM) of matin structure via their impact on spermatid
histone residues and incorporation of many his- gene expression program (as described in the pre-
tone variants. These changes in the spermatid vious paragraph).
chromatin structure aid in destabilizing nucleo- Acetylation of H3 coincides with H4 acetyla-
somes and loosening chromatin in preparation tion and could also participate in histone
for histone-to-protamine transition (Braun 2001; removal as suggested by the study of PYGOPUS
Sassone-Corsi 2002). Deregulation of this pro- 2. PYGOPUS 2 (encoded by Pygo2), a co-acti-
cess such as abnormal expression of histone-­ vator of the beta-catenin/Wnt signaling path-
modifying enzymes, histone variants, associated way, has been studied during spermatid
factors, and chaperones can lead to abnormal his- elongation: it is expressed in steps 8–12 elon-
tone eviction and is therefore expected to affect gated spermatids and co-immunoprecipitates
sperm chromatin structure. with a histone acetyl transferase activity in the
testis. Reduced levels of Pygo2 in mice lead to
male infertility associated with a decrease of H1
Histone Posttranslational histone variant H1FNT level and of histone
Modifications and Enzymes H3K9/K14 acetylation in elongating spermatids
but not of H4K8/K12 acetylation (see Table 1.1).
One hallmark of histone-to-protamine transition Though its precise role remains unclear,
is hyperacetylation of histones, predominantly of PYGOPUS 2 appears to be an essential co-regu-
histone H4, but also, to a lesser extent, of other lator of histone PTM in spermatids (Nair et al.
nucleosomal histones in stages 9–11 mouse sper- 2008) and as such could impact spermatid chro-
matids (Hazzouri et al. 2000). Acetylation has matin structure.
14
KAT6B (encoded by Kat6b also known as
Myst4) is another HAT. In bovine testes, it has
only been detected in the nuclei of elongating
spermatids and therefore has been suggested to
contribute to histone hyperacetylation in sperma-
tids (Mcgraw et al. 2007). Functional studies
remain to be performed.
Histone lysine crotonylation (Kcr) is also
observed during spermatid differentiation in steps
9–11 spermatids, coincident with histone hyper-
acetylation (Tan et al. 2011; Liu et al. 2017). Kcr
was also found enriched at transcription start sites
of genes that are post-meiotically activated (Tan
et al. 2011). It has recently been found that the
histone acetyltransferase P300 can both acetylate
and crotonylate histone lysine H3K18 depending
on the intracellular concentration of acetyl-CoA
or crotonyl-CoA (Sabari et al. 2015). Similarly,
P300 and CREBBP are able to catalyze the addi-
tion of propionyl and butyryl (Chen et al. 2007)
(which are other types of acyl groups together
with acetyl, crotonyl, 2-­ hydroxyisobutyryl,
β-hydroxybutyryl, succinyl, malonyl, glutaryl)
onto histone lysines [see Sabari et al. (2015) for
review]. Goudarzi et al. have found that P300 can
also butyrylate H4K5 and K8 (Goudarzi et al.
2016). Tight regulation of acylation of histone
lysines during spermiogenesis is therefore
expected to have a critical role in histone-to-prot-
amine transition; this is exemplified by the fact
that BRDT, which is essential to histone removal
(see below), can bind to acetylated H4K5 but not
to butyrylated H4K5 and that butyrylated histones
at H4K5/K8 persist longer than acetylated ones
(Goudarzi et al. 2016).
The dynamic of acyl-CoA availability
throughout spermatogenesis and the influence it
has on histone modifications remain to be stud-
ied, but enzymes involved in acyl-CoA synthesis
(acyl-CoA synthetases) could have an impact on
sperm chromatin structure.
Collectively, the abovementioned studies
point to a role of the histone acetyltransferase
P300 in modulating multiple histone modifica-
tions depending on acyl-CoA availability. It
remains to be demonstrated if other lysine resi-
dues can also be acylated by the same enzyme or
by others. HATs are therefore essential in the
M. Blanco and J. Cocquet
setup of spermatozoa chromatin structure.
Similarly, enzymes that regulate the removal of
acyl are important in the regulation of this pro-
cess: it has been shown that blocking histone
deacetylases (with TSA) could mimic the role of
HAT in vitro (Hazzouri et al. 2000); deregulation
of histone deacetylases could therefore also affect
histone-to-protamine transition. Chromodomain
Y-like protein (CDYL) was initially described to
have HAT activity with a predominance for H4
(Lahn et al. 2002). Shortly after, another study
did not recapitulate this finding (Caron et al.
2003), and it was recently confirmed that in fact
CDYL is not a HAT but a crotonyl-CoA hydra-
tase that converts crotonyl-CoA into
b-­hydroxybutyryl-CoA, thereby inhibiting his-
tone crotonylation (Liu et al. 2017). To better
understand the role of CDYL during spermato-
genesis, and since CDYL KO is embryonically/
perinatally lethal (Wan et al. 2013), Liu et al. pro-
duced a transgenic mouse model in which CDYL
is overexpressed. In these mice, histone lysine
crotonylation (Kcr) levels and, in particular,
H2BK12cr level were found reduced. Males were
hypofertile with decreased sperm count and
decreased sperm motility. TNP1 and PRM2 were
significantly enriched in soluble testis fraction
and significantly reduced in chromatin-bound
fraction indicating defects in histone-to-­
protamine transition (Liu et al. 2017) (see
Table 1.1).
Besides acylation, other posttranslational
modifications of histone residues such as ubiqui-
tination, phosphorylation, and methylation have
been observed in elongating spermatids, for
instance, H3K4me2/3 (Rathke et al. 2007;
Godmann et al. 2007; Song et al. 2011) and
H3K79me2/3 (Dottermusch-Heidel et al. 2014a,
b).
Those PTM are also expected to facilitate his-
tone eviction. For instance, ubiquitinated his-
tones H2A and H2B are highly expressed in
elongated spermatids just prior to histone removal
(Baarends et al. 1999). Mouse knockout of the
ubiquitin-conjugating enzyme E2 B (encoded by
Ube2b aka Hr6b) leads to male infertility, with
reduced testis weight and low number of
­spermatozoa which mostly appeared abnormally
1 Genetic Factors Affecting Sperm Chromatin Structure
shaped such as immobility and abnormal head
morphology (Roest et al. 1996). It was initially
thought that Ube2b knockout leads to abnormal
histone-­to-­
protamine transition (Roest et al.
1996) via its effect on H2A ubiquitination, but in
Ube2b-KO elongating spermatids, H2A ubiquiti-
nation is normal (Baarends et al. 1999) suggest-
ing that other enzymes are involved in this
process. Since then, UBE2B has been involved in
meiotic recombination (Baarends et al. 2003) and
the regulation of genes encoded by the sex chro-
mosomes during male meiosis and beyond
(Mulugeta Achame et al. 2010).
In 2010, Lu et al. have shown that, in male
germ cells, the E3 ubiquitin protein ligase RNF8
is required for H2A and H2B ubiquitination
which is itself required for recruitment of KAT8
acetyltransferase (also known as MOF) to chro-
matin and subsequent H4K16 acetylation that
facilitates histone eviction. The Rnf8 KO mice
they produced were infertile showing abnormal
chromatin condensation, with a failure of histone
eviction/protamine deposition (Lu et al. 2010)
(see Table 1.1). However, in 2012, Sin et al. pro-
duced Rnf8-KO mice which did not have the
same phenotype, as they display a deregulation
of post-meiotic XY gene expression but no
defects in chromatin compaction during sperma-
tid elongation (Sin et al. 2012).
The NAD-dependent protein deacetylase sir-
tuin-­1 (encoded by Sirt1) is highly expressed in
meiotic germ cells and to a lesser extent in sper-
matids (Bell et al. 2014). Sirt1-null male mice are
hypofertile with abnormally shaped sperm heads
presenting a higher incidence of chromatin con-
densation and compaction defects. SIRT1 appears
to be required for histone acetylation and subse-
quent histone eviction and protamine deposition
(Bell et al. 2014) (see Table 1.1). Interestingly,
SIRT1 has both histone and non-histone deacyl-
ase activities and has been shown to regulate the
activity of the acetyl-CoA synthetase (encoded
by Acss2), a key enzyme in the production of the
substrate required for histone acylation, via its
ability to de-acetylate this enzyme [for review,
see Sabari et al. (2017)].
Calcium/calmodulin-dependent protein kinase
type IV (CaMK IV encoded by Camk4) is a ser-
15
ine/threonine kinase involved in transcriptional
regulation. In testes, it is highly expressed in
elongating spermatids and associated to the chro-
matin and nuclear matrix (Wu and Means 2000).
Camk4-KO mice are infertile with severe sper-
matogenesis defects (dramatic reduction of testis
weight and sperm count) suggesting problems
occurring before spermatid differentiation.
CaMK IV has been suggested to be important for
histone-to-protamine transition because
Camk4-KO spermatids retain TNP2 longer and
have lower level of PRM2 (see Table 1.1).
Interestingly CaMK IV has been shown to phos-
phorylate PRM2 in vitro (Wu et al. 2000).
In mice, downregulation of the H3K79 methyl
transferase DOT1L and of H3K79me2 level in
spermatids is associated with hypofertility, chro-
matin condensation defects, and a higher propor-
tion of retained histones than in wild-type mouse
spermatozoa (Moretti et al. 2017). This has been
observed in mice knocked down for Sly, a multi-
copy gene located on the mouse Y chromosome
long arm (MSYq), which controls the expression
of many spermatid-expressed genes (Moretti
et al. 2017) (see Table 1.1). The sole impact of
Dot1l knockout remains to be determined, as Sly
knock down leads to the deregulation of many
genes in addition to Dot1l, including X- and
Y-encoded H2al genes (El Kennani et al. 2018)
which could contribute to the chromatin remod-
eling defects of this mouse model (see below). It
is worth adding that Sly is not conserved in
humans, but its partners and target genes are and
could therefore influence expression of genes
involved in sperm chromatin organization in
humans.
Histone Variants and Transition
Proteins
In addition to multiple changes of histone PTM,
the process of histone-to-protamine transition
involves expression and incorporation of many
histone variants, many of which are testis-­
specific or testis-enriched. Their incorporation
into the chromatin is expected to result in weaker
interaction with DNA, destabilization of the
16
nucleosomal structure, and finally histone dis-
placement (Govin et al. 2006). Each nucleosome
contains one molecule of the linker histone H1
that binds DNA in the nucleosome and linker
DNA between nucleosomes (Wolffe 1997); sev-
eral testis-specific H1 variants exist. The
spermatid-­specific linker histone H1-like protein
(HILS) is specifically expressed in elongating
spermatids and has been hypothesized to be
involved in chromatin condensation at this stage
(Yan et al. 2003b), yet a recent study demon-
strated it is a poor condenser of chromatin
(Mishra et al. 2018). ChIP-Seq experiments on
elongating/condensing spermatids revealed
HILS1 is preferentially located in the regions
encoding LINE elements and specific histone
PTM such as H3K9me3, H4K20me3, and
H4K5ac (Mishra et al. 2018). Its molecular effect
during spermiogenesis remains to be identified.
H1FNT (H1 histone family member N, testis-­
specific, encoded by H1fnt aka H1t2) is another
histone variant highly expressed in spermatids.
Immunohistochemical examination of histone
H1FNT localization showed that it is expressed
in steps 5–13 mouse spermatids (Tanaka et al.
2005) and appears as cap-like structure at the
inner periphery of the nuclear membrane
(Martianov et al. 2005). H1fnt-null male mice are
infertile, and H1fnt-null sperm have abnormal
sperm heads and abnormally condensed chroma-
tin due to defective deposition of protamines
(Martianov et al. 2005; Tanaka et al. 2005) (see
Table 1.1).
Several spermatid-specific histone variants
appear to be dispensable for this process as their
knockout does not lead to any defects in sperma-
tid differentiation nor sperm abnormalities. For
instance, H1T (encoded by Hist1h1t) is a testis-­
specific variant of histone linker H1 only found
in spermatocytes and round spermatids, but the
knockout of its gene does not have any phenotype
(Lin et al. 2000).
TH2B is a histone variant that replaces most
of canonical H2B in male germ cells from the
spermatocyte stage (Montellier et al. 2013).
Surprisingly, sperm production and male fertility
are normal in its absence (in Th2b-null mice), but
spermatid chromatin structure is changed with a
M. Blanco and J. Cocquet
higher expression of H2B and changes in the
level of multiple histone PTMs (including of
H2B and of other nucleosomal histones such as
H4) compared to WT (Montellier et al. 2013).
Transgenic male mice expressing TH2B fused to
a His, Flag, and HA C-terminal tag are however
infertile due to a block at elongating/condensing
spermatid stage. In this model, tagged TH2B
appears to be assembled into nucleosomes of
spermatocytes and round spermatids, normally.
But later, in elongating spermatids, histone evic-
tion and replacement by transition proteins are
abnormal leading to defective chromatin com-
paction (Montellier et al. 2013). In another study
that investigated the function of both TH2A and
TH2B, double knockout male mice were found to
be sterile, and their spermatids displayed abnor-
mal nuclear morphology and chromatin structure
despite overexpression of canonical H2B
(Shinagawa et al. 2015) (see Table 1.1).
Collectively, these data show that TH2A and
TH2B are required for proper histone-to-­
protamine transition and that compensation
mechanisms are at work and can induce a chro-
matin re-organization without impacting on prot-
amine deposition and male fertility. One cannot
exclude that those compensations could differ
among species and that the deletion of one gene
could have an impact in one species but no effect
in another.
H3.3 histone variant appears to play multiple
roles during spermatogenesis. H3.3 protein is
encoded by two genes, H3f3a and H3f3b, (Yuen
et al. 2014) and is incorporated into the chroma-
tin of mouse sex chromosomes during meiosis
when sex chromosomes are transcriptionally
inactive (Van Der Heijden et al. 2007). It has
been shown that a small proportion of H3.3 is
retained in sperm chromatin and correlates with
genes important for development of the early
embryo (Erkek et al. 2013). In mice H3f3a KO
leads to male infertility with reduced number of
germ cells and abnormal sperm heads indicating
defects in chromatin condensation (Yuen et al.
2014) (see Table 1.1). Another study reported
that H3f3b KO mice died shortly after birth but
that heterozygous mice were viable with male
infertility characterized by spermatogenesis
1 Genetic Factors Affecting Sperm Chromatin Structure
arrest at the spermatid stage; in the same study,
H3f3a KO was found to produce abnormally
shaped spermatozoa and reduced male fertility
(Tang et al. 2013). The exact molecular role of
H3.3 during spermiogenesis remains unclear.
Transition proteins (TNPs) are the protein
intermediates which are transiently incorporated
in the spermatid chromatin (in steps 12–13 mouse
spermatids) after histones have been removed.
The biochemical properties of TNPs and prot-
amines make them suitable for condensing DNA
in the sperm nucleus. TNPs are rich in arginine,
lysine, and serine (Dadoune 2003; Brewer et al.
2002). TNP1 and TNP2 are capable of condens-
ing DNA at a rate similar to those of protamines,
while dissociation rates from DNA for TNP1 and
TNP2 are faster than those of protamines
(Dadoune 2003, Brewer et al. 2002). Thus, TNPs
can destabilize nucleosomes and facilitate his-
tone eviction. They are then rapidly replaced by
protamines. TNP1 and TNP2 are essential for
male fertility, and the deletion of one TNP is par-
tially compensated by overexpression of the
other remaining TNPs. Tnp1- or Tnp2-null male
mice are fertile though with reduced litter sizes,
and spermatozoa present with abnormal chroma-
tin condensation (Yu et al. 2000; Zhao et al.
2001). Double knockout leads to male infertility
with abnormal sperm head morphology, abnor-
mal nuclear shape and uncondensed nucleus, and
abnormal chromatin condensation (Zhao et al.
2004) (see Table 1.1), yet, in the absence of
TNPs, histones are removed and protamines
deposited. Protamine 2 remains however as an
uncleaved precursor, and overall protamine-­
mediated genome compaction is not normal since
spermatozoa are unable to fertilize oocytes by
ICSI (Zhao et al. 2004).
Like histones, TNP1 and TNP2 have been
shown to carry posttranslational modifications,
such as lysine methylation, arginine methylation,
lysine acetylation, and serine phosphorylation.
The histone-arginine methyltransferase CARM1
(encoded by Carm1 also known as Prmt4) and
the histone lysine N-methyltransferase SETD7
appear to be responsible for methylation of tran-
sition proteins on arginine or lysine residues,
respectively (Gupta et al. 2015). Phosphorylation
17
has been shown to be mediated by PKA and PKC
kinases and to affect TNP properties (Levesque
et al. 1998; Meetei et al. 2002; Ullas and Rao
2003). Acetylation of TNP2 by the P300 has been
shown to reduce its DNA condensation ability
(Pradeepa et al. 2009). Abnormal posttransla-
tional modification(s) of TNPs could impair
histone-­to-protamine transition and thus sperm
chromatin structure.
H2A.L.2 is a H2A variant specifically
expressed in spermatids that is critical for sperm
chromatin structure, as it is required for TNP
incorporation into the chromatin during histone-­
to-­
protamine transition. And indeed,
H2A.L.2-­null male mice have a phenotype very
similar to that of TNPs double knockout males:
they are sterile and H2A.L.2-null sperm are
unable to fertilize oocytes in vitro. Besides,
H2A.L.2-null sperm contain unprocessed PRM2
protein, and, despite incorporation of protamines,
genome compaction is defective in these mice
(Barral et al. 2017) (see Table 1.1). H2A.L.2 vari-
ant was shown to be incorporated, together with
TH2B, onto nucleosomes by Nap1L4 (nucleo-
some assembly protein 1-like 4) in elongating
spermatids, which leads to a more open chroma-
tin. H2A.L.2 is critical for TNP incorporation
and then efficient protamine-mediated genome
compaction (Barral et al. 2017).
Collectively, these data show that TNP’s
molecular role is not to displace histones, as pre-
viously thought, but rather to recruit and process
PRM, which in turn displaces histones and com-
pacts the paternal genome.
Chaperones and Readers
A recent study has identified the testis-specific
NUT protein as an essential regulator of histone
acetylation as it recruits P300 and/or CREB-­
binding protein to enhance histone acetylation. In
its absence, histone H4 (in particular at K5 and
K8) and H2A acetylation levels are dramatically
decreased, and spermatid elongation is arrested
(Shiota et al. 2018) (see Table 1.1).
Acetylated/acylated histones are recognized
by histone readers before being displaced; in par-
Another random document with
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 legislation, but from the circumstances that the existing laws
were hastily framed or were the outcome of party rancour. If
we had formerly had a Senate composed of men of experience and
good patriots, they would never have consented to the conclusion
of so many onerous loans, to the application of so many
iniquitous measures, nor to the convocation of the special
tribunal, 'le tribunal extraordinaire,' of 1899.

"At first the Radical party was not favourable to the

institution of an Upper Chamber, but it now recognizes the
great advantages it will offer, and has rallied to my project.
The Progressist party has always been favourable to it. The
majority of the Liberal party has also adhered to it. I
therefore believe that this new institution will be of the
greatest service to the country. All that is required, and
with a little good will it can be easily done, is that the
members of the two Chambers should endeavour honestly,
sincerely, and loyally to work for the good of the State and
of the nation. If I have not thought right to raise the
qualification for the suffrage, as desired by some people, it
is because I did not wish to disfranchise any of those who
have enjoyed the right of voting during the last 35 years. I
do not wish to restrict any of the rights of the nation.

{451}

"The application of the new Constitution will be the great

task of my Government, in which I have every confidence. The
Prime Minister, Dr. Vuitch, has the sympathy and support not
only of his own party but of all who would like to see the
country governed in a liberal spirit. His presence at the head
of the Ministry is a pledge for the active and sincere
co-operation of all elements of order and progress. … As soon
as the new Constitution has been promulgated, the Government
will invite the co-operation of all those which admit its
necessity and fitness. A large Conservative party will thus be
formed which will have the requisite power and authority for
 all purposes of government, for the application of the
Constitution, and for the elaboration of financial and
economic laws necessary for the progress of the country.

"As regards the question of the succession to the Throne, I

wanted to settle it finally, as the members of the reigning
dynasty are not numerous unless the remote collateral lines be
included, which is not possible. Moreover, everybody wished me
to take in this matter such decisions as I might think proper
in view of securing the continuation of the Servian Monarchy.
The first thing to be done was to safeguard the rights of the
direct line without seeking to bind ourselves by the Salic
Law, which there is really no reason to apply in our country.
I should add here that there are no anti-dynastic elements in
Servia, with the exception, perhaps, of a few hare-brained
individuals who really do not enter into account. My people
are profoundly attached to the reigning dynasty, and never
lose an opportunity of showing me their loyalty. It is the
same with all the political parties.

"Before promulgating the Constitution I decided to consult the

most influential members of the parties in office. They agreed
with me, and promised me to assist harmoniously in the work. I
have also consulted the leading members of the Liberal party,
and with two or three exceptions they have given me the same
assurances. Such being the case, I may say that the
Constitution of 1901 is not a production of my will or of my
good pleasure, but that it is the result of an understanding
between the Sovereign and the leaders of the three political
parties. I consequently reckon upon their sincere and active
co-operation, and I trust they will not fail me. I am firmly
convinced that the new Constitution will act as a fresh and
vigorous stimulus to my country, and that it will bring it
that calm and stability which it sorely needs. I sincerely
regard it as a source of prosperity and welfare for Servia."

SEVERALTY ACT, The Indian.

 See (in this volume)
INDIANS, AMERICAN: A. D. 1899-1900.

SEYMOUR, Vice-Admiral Sir Edward:

Expedition to relieve Peking.

See (in this volume)

CHINA: A. D. 1900 (JUNE 10-26).

SEYYIDIEH, The province of.

See (in this volume)

BRITISH EAST AFRICA PROTECTORATE: A. D. 1895-1897.

SHAFTER, General:
Commanding the expedition against Santiago de Cuba.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1898 (JUNE-JULY).

SHAFTER, General:
Surrender of Spanish forces at Santiago and all eastern Cuba.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1898 (JULY 4-17).

SHAFTER, General:
Report of sickness in army.
Removal of troops to Montauk Point.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1898 (JULY-AUGUST:
CUBA).

SHANGHAI.
"Shanghai is the New York of China. It occupies a position on
the coast quite similar to that of New York on our own eastern
coast, and its percentage of importations into China is about
the same as that which New York enjoys in the United States.
The large share of the foreign trade of China which Shanghai
controls is due largely to its position at the mouth of the
great artery through which trade flows to and from China—the
Yangtze-Kiang. Transportation in bulk in China up to the
present time having been almost exclusively by water, and the
Yangtze being navigable by steamers and junks for more than
2,000 miles, thus reaching the most populous, productive, and
wealthy sections of the country, naturally a very large share
of the foreign commerce entering or leaving that country
passes through Shanghai, where foreign merchants, bankers,
trade representatives, trade facilities, and excellent docking
and steamship conveniences exist. The lines of no less than
eight great steamship companies center at Shanghai, where they
land freight and passengers from their fleets of vessels which
are counted by hundreds, while the smaller vessels, for river
and coastwise service, and the native junks are counted
literally by thousands. The Yangtze from Shanghai westward to
Hankow, a distance of 582 miles, is navigable for very large
steamships that are capable of coasting as well as river
service. Hankow, which with its suburbs has nearly a million
people, is the most important of the interior cities, being a
great distributing center for trade to all parts of central
and western China and thus the river trade between Shanghai
and Hankow is of itself enormous, while the coastwise trade
from Shanghai, both to the north and south, and that by the
Grand Canal to Tientsin, the most important city of northern
China, is also very large."

United States, Bureau of Statistics,

Monthly Summary, March, 1899, page 2191.

"When the English chose this position, in 1842, for their

mercantile settlement, it seemed difficult to believe that
 they would ever succeed in making the place a rival of Canton
or of Amoy. It is true that Shanghai possessed important
commercial relations already, and the great geographical
advantage of commanding the entrance to the navigable river
which traverses the whole empire from west to east; but the
builders of the city there had to struggle with enormous
difficulties of soil and climate. They had to solidify and
drain the land, dig canals, dry up marshes, cleanse the air of
its miasms, besides incessantly dredging and clearing the
channel, to keep it open for their ships. The first European
merchants established at Shanghai were favored in fortune by
the national disasters of China. The Taiping war drove
fugitives in multitudes to the territory conceded to
foreigners, and when the town of Soutcheou was destroyed, in
1860, Shanghai succeeded it as the great city of the country."

É. Reclus,
Nouvelle géographie universelle,
volume 7, page 455.

SHANGHAI: A. D. 1898.
Rioting consequent on French desecration of a cemetery.
Extension of foreign settlements.

See (in this volume)

CHINA: A. D. 1898-1899.

SHANTUNG, The "Boxer" outbreak in.

See (in this volume)

CHINA: A. D. 1900 (JANUARY-MARCH).

SHIMONOSEKI, Text of the Treaty of.

See (in this volume)

CHINA: A. D. 1894-1895.
{452}

SHIPPING OF THE WORLD: In 1900.

Statement of number and net and gross tonnage of steam and

sailing vessels of over 100 tons of the several countries of
the world, as recorded in Lloyd's Register for 1900-1901
[dated July 1, 1900].

United States, Commissioner of Navigation,

Annual Report, 1900, page 125.

FLAG STEAM.
SAIL. TOTAL.
------------------------- -
----------------- -------------
Number. Net tons. Gross tons.
Number. Net tons. Number. Tonnage.

British:
United Kingdom. 7,020 7,072,401 11,513,759
1,894 1,727,687 8,914 13,241,446
Colonies. 910 378,925 635,331
1,014 384,477 1,924 1,019,808
Total. 7,930 7,451,326 12,149,090
2,908 2,112,164 10,838 14,261,254

American
(United States):
Sea. 690 594,237 878,564
2,130 1,156,498 2,820 2,035,062
Lake. 242 436,979 576,402
73 138,807 315 715,209
Total. 932 1,031,216 1,454,966
2,203 1,295,305 3,135 2,750,271

Argentine. 95 36,938 57,239

106 30,407 201 87,646
Austro-Hungarian 214 240,808 387,471
56 28,613 270 416,084
Belgian. 115 111,624 162,493
2 420 117 162,913
Brazilian. 215 85,799 133,507
117 29,580 332 163,087
Chilean. 52 38,960 62,872
75 48,106 127 110,978
Chinese. 48 41,847 65,721
1 573 49 66,294
Colombian. 1 555 877
5 1,110 6 1,987
Danish. 369 240,599 412,273
433 106,738 802 519,011
Dutch. 289 307,574 467,209
117 63,068 406 530,277
French. 662 542,305 1,052,193
552 298,309 1,214 1,350,562
German. 1,209 1,344,605 2,159,919
501 490,114 1,710 2,650,033
Greek. 139 111,797 178,137
230 65,957 369 245,094
Haitian. 5 912 1,750
2 414 7 2,164
Italian. 312 343,020 540,349
864 443,306 1,176 983,655
Japanese. 484 303,303 488,187
582 86,370 1,006 574,557
Mexican. 25 6,562 11,460
13 3,081 38 14,541
Montenegrin. 1 1,064 1,857
14 3,513 15 5,370
Norwegian. 806 467,123 764,683
1,574 876,129 2,380 1,640,812
Peruvian. 3 3,204 4,869
33 9,607 36 14,476
Portuguese. 48 37,153 57,664
156 53,391 204 111,055
Roumanian. 17 9,686 17,361
3 659 20 18,020
Russian. 496 292,277 469,496
750 251,405 1,246 720,901
Sarawakian. 2 244 418
2 418
Siamese. 4 821 1,435
1 294 5 1,729
Spanish. 422 416,882 642,231
175 52,549 597 694,780
Swedish. 678 260,023 418,550
755 218,722 1,433 637,272
Turkish. 135 58,974 94,781
170 48,709 305 143,490
Uruguayan. 17 6,438 10,468
19 4,032 36 14,500
Venezuelan. 12 2,450 4,246
8 1,185 20 5,431
Zanzibarian. 3 1,871 2,808
3 2,808
Other countries:
Hawaii. 23 11,185 16,922
24 29,707 47 46,629
Cuba. 35 17,651 27,040
11 2,410 46 29,450
Philippine Islands 69 19,587 31,099
42 8,236 111 39,335
Various:
Arabia,
Salvador,
Ecuador,
Liberia,
Samos,
Nicaragua,
Bulgaria,
Costa Rica,
Egypt,
Persia,
Porto Rico,
etc. 31 10,130 17,717
22 9,127 53 26,844

Total. 15,898 13,800,513 22,309,358

12,524 6,674,370 28,422 29,043,728

SHIRE HIGHLANDS, The.

See (in this volume)

BRITISH CENTRAL AFRICA PROTECTORATE.

SHOA.

See (in this volume)

EGYPT: A. D. 1885-1896.

SHUN-CH'ING, Anti-missionary insurrection at.

See (in this volume)

CHINA: A. D. 1898-1899 (JUNE-JANUARY).
SIAH CHAI, or Vegetarians, The.

See (in this volume)

CHINA: A. D. 1895 (AUGUST).

SIAM: A. D. 1896-1899.
Declaration between Great Britain and France
with regard to Siam.

A declaration of agreement, in part as follows, between Great

Britain and France, was signed at London, January 15, 1896:

"I.
The Governments of Great Britain and France engage to one
another that neither of them will, without the consent of the
other, in any case, or under any pretext, advance their armed
forces into the region which is comprised in the basins of the
Petcha Bouri, Meiklong, Menam, and Bang Pa Kong (Petriou)
Rivers and their respective tributaries, together with the
extent of coast from Muong Bang Tapan to Muong Pase, the
basins of the rivers on which those two places are situated,
and the basins of the other rivers, the estuaries of which are
included in that coast; and including also the territory lying
to the north of the basin of the Menam, and situated between
the Anglo-Siamese frontier, the Mekong River, and the eastern
watershed of the Me Ing. They further engage not to acquire
within this region any special privilege or advantage which
shall not be enjoyed in common by, or equally open to, Great
Britain and France, and their nationals and dependents. These
stipulations, however, shall not be interpreted as derogating
from the special clauses which, in virtue of the Treaty
concluded on the 3rd October, 1893, between France and Siam,
apply to a zone of 25 kilometers on the right bank of the
Mekong and to the navigation of that river.

{453}
II.
Nothing in the foregoing clause shall hinder any action on
which the two Powers may agree, and which they shall think
necessary in order to uphold the independence of the Kingdom
of Siam. But they engage not to enter into any separate
Agreement permitting a third Power to take any action from
which they are bound by the present Declaration themselves to
abstain.

III.
From the mouth of the Nam Huok northwards as far as the
Chinese frontier the thalweg of the Mekong shall form the
limit of the possessions or spheres of influence of Great
Britain and France. It is agreed that the nationals and
dependents of each of the two countries shall not exercise any
jurisdiction or authority within the possessions or sphere of
influence of the other."

In a despatch to the British Ambassador at Paris, written on

the same day, Lord Salisbury explained the intent and purpose
of the agreement as follows: "It might be thought that because
we have engaged ourselves, and have received the engagement of
France, not under any circumstances to invade this territory,
that therefore we are throwing doubt upon the complete title
and rights of the Siamese to the remainder of their kingdom,
or, at all events, treating those rights with disregard. Any
such interpretation would entirely misrepresent the intention
with which this arrangement has been signed. We have selected
a particular area for the application of the stipulations of
this Treaty, not because the title of the King of Siam to
other portions of his dominions is less valid, but because it
is the area which affects our interests as a commercial
nation. The valley of the Menam is eminently fitted to receive
a high industrial development. Possibly in course of time it
may be the site of lines of communication which will be of
considerable importance to neighbouring portions of the
British Empire. There seems every prospect that capital will
flow into this region if reasonable security is offered for
its investment, and great advantage would result to the
commerce and industry of the world, and especially of Great
Britain, if capitalists could be induced to make such an
application of the force which they command. But the history
of the region in which Siam is situated has not in recent
years been favourable to the extension of industrial
enterprise, or to the growth of that confidence which is the
first condition of material improvement. A large territory to
the north has passed from the hands of the Burmese Government
to those of Great Britain. A large territory to the east has
passed from the hands of its former possessors to those of
France. The events of this recent history certainly have a
tendency to encourage doubts of the stability of the Siamese
dominion; and without in any degree sharing in those doubts,
or admitting the possibility, within any future with which we
have to deal, of the Siamese independence being compromised,
Her Majesty's Government could not but feel that there would
be an advantage in giving some security to the commercial
world that, in regard to the region where the most active
development is likely to take place, no further disturbances
of territorial ownership are to be apprehended."

Great Britain, Parliamentary Publications

(Papers by Command: France, Number 2, 1896, pages 1-3).

Perhaps the above explanation can be better understood after

reading the following:

"In the early eighties France commenced the subjugation of

Tonquin. … It was not until 1893 that France openly attacked
Siam. The demand was subtly formulated—on behalf, not of the
Government of the French Republic, but of 'the Empire of
Annam.' But even so the French had been in Annam for perhaps a
quarter of a century, whereas Siam could show an undisturbed,
undisputed tenure of the Mekong River's 'rive gauche' for at
least ninety years. … The cession to France of territory
amounting to rather more than one-third of the entire kingdom
was insisted upon; and in March 1893 that Power sent the
ship-of-war Lutin to Bangkok, where she remained for months a
standing menace. A rigorous blockade of the Siamese seaboard
followed, resulting in a few short days in complete surrender
of the disputed territory to France and the payment of a heavy
war indemnity. … By the Anglo-French Convention of last year
[as given above] the King of Siam's position became, to say
the least, slightly anomalous. That agreement practically
amounted to the fair division, between France and England, of
the whole of Siam save that portion situate in the fertile
valley of the Meinam, whose autonomy they still guarantee to
preserve. … France holds, in addition to the long-coveted port
of Chantabûn, that part of the province of Luang Phrabang
which is situate upon the right bank of the Mekong. … The
Siamese king is 'nulli secundus' among Oriental monarchs as a
progressive ruler. And fate has been unkind to him indeed! He
has encouraged English customs and the English language by all
the means in his power—has taken the kindliest possible
interest in the introduction of electric light, electric
tramways, &c., into his capital—has endeavoured to model his
army and navy, his prison and other systems, upon the English
method—and has in person opened the first railway (that
connecting Bangkok with Pâknam) in Siam. It is, indeed, one of
the strangest and most interesting sights, as you stroll
through the streets of the capital, to witness the 'riksha and
gharry of comparative barbarism travelling in juxtaposition to
the electric tramcar and the bicycle! And for his broad and
enlightened views the King of Siam has been requited by the
wholesale and utterly unjustifiable plunder of his most
fertile lands."
Percy Cross Standing,
The Significance of the Siamese Visit
(Nineteenth Century, June, 1897).

Frequent collisions between French and Siamese in the

so-called "neutral zone" on the right bank of the Mekong
continued, until a new convention was agreed upon in May,
1890. This gave to France the province of Luang-Phrabang, in
return for which she agreed to withdraw entirely from the
neutral territory and from the port of Chantabûn.

SIAM: A. D. 1898.
Gift of relics of Buddha.

See (in this volume)

BUDDHA.

SIAM: A. D. 1899 (May-July).

Representation in the Peace Conference at The Hague.

See (in this volume)

PEACE CONFERENCE.

SIAN FU,
SI-NGAN-FU,
The Chinese Imperial Court at.

See (in this volume)

CHINA: A. D. 1900 (AUGUST-SEPTEMBER).

SIBERIA.

See (in this volume)

RUSSIA IN ASIA.

SIBERIAN ARCTIC EXPLORATION.

 See (in this volume)
POLAR EXPLORATION, 1805, 1896, 1897, 1898, 1899, 1900.

{454}

SIERRA LEONE PROTECTORATE.

Extension of British authority over the Hinterland of the
Colony of Sierra Leone.
The hut tax.
Insurrection of natives.

"Immediately adjoining the Colony of Sierra Leone, lying to

the northward and eastward, is the Hinterland, the boundaries
of which were defined by the Agreement between Great Britain
and France which was concluded 21st January 1895. The extreme
depth from south to north is about 210 miles, lying between 7°
and 10° north latitude, and 180 miles from east to west, lying
between 10° 40' and 13° 20' of west longitude. The estimated area
is rather more than 30,000 square miles—about the size of
Ireland. … Unlike many regions on the west coast of Africa,
the country is, for the most part, well watered by rivers and
running streams. The population of the Hinterland has not been
ascertained. It has been variously estimated, before the
present troubles, at from about 750,000 up to about 2,000,000.
The trade and revenue of the Colony depend almost entirely on
the Hinterland. A very large proportion of the goods imported
into the Colony are carried into and consumed in the
Hinterland. These goods are paid for by means of the products
of the Hinterland, which are exported, and the profits derived
from the exchange enable the merchants to pay the Customs
duties, which constitute the bulk of the Colonial revenue. The
territories forming the Hinterland are, according to the
native organisation, ruled over by a large number of Chiefs
(or Kings, as they used to be, and still in native parlance
are, called). The portions of country under each Chief are
well ascertained, and recognised by the various Chiefs and
their subjects. …

"The relations between the English Government and the Chiefs

at the time of the conclusion of the Agreement between France
and England in 1895 was … that some of the Chiefs whose
territories lay most adjacent to the Colony of Sierra Leone
had contracted with the English Crown certain treaties of
cession, and treaties directed to definite objects of amity
and good offices. In addition there had sprung up by usage a
limited consensual and advisory jurisdiction, under which
Chiefs as well as persons not Chiefs would bring their
differences (mainly as to territorial boundaries) before the
Governor of Sierra Leone as a sort of arbitrator, and
implicitly follow his awards. This jurisdiction was exercised
over an area of no defined limits, so far as any rules were
concerned. As a fact, it was limited by conditions of distance
and facility of travel, so that whilst the usage was most
established in the countries nearest to Freetown, there was
none in the more distant regions, or if there was any it was
at most so rudimentary as to be jurally of no account. … I
have not been able to trace any instance in which, either
under treaty or any other form of consent, or without consent,
the English Government has imposed, or endeavoured to impose
any direct taxation upon the Chiefs or people of the
Hinterland prior to 1896.

"The agreement between France and Great Britain delimited the

respective spheres of interest of the two countries south and
west of the Middle or Upper Niger, and thus defined for
England in the Hinterland of Sierra Leone a territory within
which, so far as concerned any question between France and
England, England was at liberty to exercise whatever species
or extent of jurisdiction she might consider proper. It made,
of course, no alteration on the existing native organisation,
nor upon the existing relations between England and the native
Chiefs, who were not parties to the agreement in any sense. …
On 31st August 1896 a Proclamation was published setting forth
that Her Majesty had assumed a Protectorate over the
territories adjacent to the Colony of Sierra Leone in which
Her Majesty had acquired power and jurisdiction. For purposes
of administration the Hinterland was divided into five
districts, intended to be of about equal size, avoiding
severance as far as possible by the district boundary of the
territories of Paramount Chiefs. These districts have been
named as the Karene, Ronietta, Bandajuma, Pangmua, and
Koinadugu districts. In anticipation of the arrangements that
might become necessary for the government of the Protectorate,
an Order of the Queen in Council had been made on 24th August
1895, … whereby, … Her Majesty was pleased, by and with the
advice of her Privy Council, to order that it shall be lawful
for the Legislative Council, for the time being, of the Colony
of Sierra Leone, by Ordinance or Ordinances, to exercise and
provide for giving effect to all such jurisdiction as Her
Majesty may at any time, before or after the passing of the
Order in Council, have acquired in the said territories
adjacent to the Colony of Sierra Leone. … Following upon the
Order of the Queen in Council, an Ordinance, entitled 'An
Ordinance to Determine the mode of exercising Her Majesty's
Jurisdiction in the Territories adjacent to the Colony of
Sierra Leone,' was passed by the Legislative Council and
Governor of Sierra Leone for the Government of the
Protectorate, on 16th September 1896."

Great Britain,
Report and Correspondence on Insurrection in
the Sierra Leone Protectorate
(Parliamentary Publications:
Papers by Command, 1899, C. 9388, pages 10-17).

The Ordinance above mentioned, which was reenacted, with some

changes, in September, 1897, provided, among other things, for
the imposition of a house tax, or hut tax, upon the natives,
and this proved to be the main cause of a serious native
revolt in the Protectorate. "By way of asserting the Crown's
 ownership of all lands, whether in use and occupation or
not—and also of attempting to make the people defray the cost
of governing them by methods they resent—the Protectorate
Ordinance imposes a 'house tax' of five shillings a year, and,
in the case of 'houses with four rooms or more,' of ten
shillings a year, on every 'householder'; the same to be paid
in 'sterling coin' on or after the 1st January in each year,
or, in default of payment on demand, to be distrained for with
so much addition as will defray the cost of removing the
property and disposing of it for 'the price current at the
nearest market.' The absurdity of thus importing the mechanism
of civilisation into 'house tax' levying among these ignorant
savages matches the injustice of the tax itself. The mud
hovels to be taxed are rarely worth more than the equivalent
of two or three shillings apiece, and shillings or other
'sterling coin' are rarely seen or handled by the natives,
such wages as they earn being generally paid in kind, and such
trade as they carry on being nearly always in the way of barter.
{455}
Few who are not chiefs or headmen own property worth as much
as five shillings, and property for which five shillings could
be obtained 'at the nearest market' might be worth the
equivalent of five pounds to them. There was no attempt to
raise the proposed house or hut tax before last January
[1898], and perhaps none of the natives have even yet any
understanding of the clauses of the Protectorate Ordinance
providing them with new-fangled 'courts of Justice,' and
taking from them all proprietary rights in their land. But as
soon as a proclamation was issued on 21st August, 1896,
notifying the contemplated changes, all who heard of them were
reasonably alarmed, and wherever the news spread seeds of fresh
discontent were sown. …

"There were burning of huts, buffeting of chiefs, and so

forth, in the south and east, as well as in the north, where,
owing to the alleged recalcitrancy of Bai Bureh and the zeal
of Captain Sharpe, the District Commissioner, the havoc was
 greatest. Early in February several chiefs and headmen were
brought to Freetown from Port Lokko in manacles, to be tried,
or punished without trial, on a charge of 'refusing to comply
with the provisions of the Protectorate Ordinance, and
inciting their subjects to resist the law.' 'The most
affecting part of the matter,' says the newspaper report, 'is
that the natives all loudly affirm their unswerving loyalty to
the Government, and say that they do not refuse to pay the hut
tax because they do not wish to, but because they really
cannot pay.' Their apologies were not listened to. Instead, a
detachment of the West India Regiment was sent up to assist
Captain Sharpe in the little war on which he had already
embarked. A futile attempt to arrest Bai Bureh on 18th
February led to a general uprising, and the first battle was
fought on 3rd March, when the town of Karina was recovered
from the 'insurgents' who had occupied it, and over sixty of
them were killed. Another fight occurred at Port Lokko, on 5th
March, when the 'insurgents' lost about forty more. These
victories being insufficient, fresh troops were sent up in
batches, until the entire force of conquerors numbered 800 or
upwards. They found it easier to cow than to conquer the
people, and the unequal struggle went on for three months. At
the end of May operations had to be suspended during the rainy
season, and before they can be renewed it may be hoped that
peace will be patched up. Already, indeed, the 'rebellion'
appears to be practically crushed, and with it all the
civilisation and all the commerce that had been planted in the
Karina district. Hundreds of natives have been shot down, many
more hundreds have died of starvation. Nearly all the huts
that it was proposed to tax have been destroyed, either by the
owners themselves, or by the policemen and soldiers."

H. R. Fox Bourne,
Sierra Leone Troubles
(Fortnightly Review, August, 1898).

SILVER QUESTION, The: A. D. 1895 (January-February).

 Attitude of Free Silver majority in the U. S. Senate
towards the Treasury gold reserve.

See (in this volume)

UNITED STATES OF AMERICA:
A. D. 1895 (JANUARY-FEBRUARY);
and 1895-1896 (DECEMBER-FEBRUARY).

SILVER QUESTION, The: A. D. 1896.

In the American Presidential election.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1896 (JUNE-NOVEMBER).

SILVER QUESTION, The: A. D. 1896-1898.

The Indianapolis Monetary Commission report and
Secretary Gage's plan in Congress.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1896-1898.

SILVER QUESTION, The: A. D. 1897.

Negotiations by envoys from the United States for an
international bi-metallic agreement.

See (in this volume)

MONETARY QUESTIONS: A. D. 1897 (APRIL-OCTOBER).

SILVER QUESTION, The: A. D. 1900.

Practical settlement of the issue in the United States.
Attempted revival in the Presidential canvass.

See (in this volume)

UNITED STATES OF AMERICA:
A. D. 1900 (MARCH-DECEMBER), and (MAY-NOVEMBER).

SILVER REPUBLICANS.