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Testis Physiology—Overview and Histology

Chapter · January 2018

DOI: 10.1016/B978-0-12-801238-3.64567-1

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 Testis Physiology—Overview and Histology
Nathália LM Lara, Guilherme MJ Costa, Gleide F Avelar, and Samyra MSN Lacerda, Federal University of Minas Gerais,
Belo Horizonte, Brazil
Rex A Hess, University of Illinois at Urbana-Champaign, Urbana, IL, United States
Luiz R de França, Federal University of Minas Gerais, Belo Horizonte, Brazil; National Institute for Amazonian Research, Manaus, Brazil
© 2018 Elsevier Inc. All rights reserved.

Functional Organization of the Testis 1

Spermatogenesis 2
Germ Cell Syncytium and Apoptosis 3
Spermatogonial Phase 5
Undifferentiated spermatogonia 5
Differentiated spermatogonia 5
Meiotic Phase 5
Spermiogenic Phase 6
Sertoli Cell 8
Stages and Cycle of the Seminiferous Epithelium and Spermatogenic Wave 9
Leydig Cells and Steroidogenesis 11
References 12

Functional Organization of the Testis

Testis is the male reproductive gonad. It yields two main functions: the production of testosterone, the male sexual hormone, and
sperm. These functions are crucial for the maintenance of male characteristics, but also for the preservation of species, which will be
further described in this article. The mammalian testis is covered by a protective fibrous capsule, the tunica albuginea, composed
mainly of collagen with some fibroblastic and smooth muscle elements. The thickness, composition and amount of connective
tissue shows tremendous variation among species and its percentage in the testis ranges from
4% in mice to
20% in humans. The
capsule extends from the tunica inward, forming the testicular parenchyma, but also delineates the rete testis and the mediastinum.
Besides its protective function, this capsule is also involved in regulating blood flow, temperature, pressure and sperm movement
(Russell and França, 1995).
Functionally, the testis parenchyma is organized into two major compartments: tubular and intertubular, also called the
interstitium (Fig. 1). The tubular compartment is composed by seminiferous tubules that can be subdivided from its exterior
wall to its center into tunica propria, seminiferous epithelium and tubular lumen. The tunica propria contains the peritubular
smooth muscle-like myoid cells, that form one (ex: mice and rat) to several layers (ex: humans and marsupials) according to the
species, containing also collagen and laminin fibers. The seminiferous epithelium is a highly organized tubular lining that is
integrated with germ cells and the somatic, supporting Sertoli cells, which also secrete the fluid responsible for tubular lumen
formation and sperm transport. No blood vessels, nerves or other cell types are present in the seminiferous epithelium. The tubular
diameter shows high variation among mammals (Table 1), ranging from
160 to
450 mm in mammals. In the intertubular
compartment, the steroidogenic Leydig cells are usually the most frequent cell type present. Besides these cells, blood and lymphatic
vessels, nerves, mast cells, macrophage and dendritic cells, as well as fibroblasts and connective tissue fibers are found in the
interstitium, and their amount varies significantly according to the species. Although spermatogenic and steroidogenic activities
occur in two distinct functional compartments of the testis, their full accomplishments require very complex cellular and
physiological interactions between both, an important aspect that is advanced in other chapters.
As shown in Table 1, although the testis compartments are similarly organized among the mammalian species already
investigated, high variation is noted for several representative parameters, such as the gonadosomatic index (testis mass/body
mass), the percentage occupied by seminiferous tubules and Leydig cells in testicular parenchyma, tubular diameter, Leydig and
Sertoli cell numbers, Sertoli cell efficiency and, as a consequence, spermatogenic efficiency (daily sperm production per testis gram).
For instance, human spermatogenic efficiency is very low, particularly when compared to rodents. Large variation is also observed
for the percentage of Leydig cells, ranging from
1% in Akodon montensis to
30% in capybara, the biggest rodent worldwide, with a
predominant investment in male dominance through androgens, instead of sperm production.
Fig. 2 illustrates testis histology in some mammalian species depicted in Table 1. In this figure, it is easy to recognize the large
area occupied by connective tissue within the intertubular compartment in human testis, whereas the percentage of interstitial space
taken by Leydig cells in capybaras can reach up to 80% in some males. In the collared peccary, these steroidogenic cells present a
unique and peculiar arrangement in the testis parenchyma, forming dark stripes around the seminiferous tubules lobes, due to the
high lipid content in the cytoplasm of these cells. It is considered that these functional differences among species are related, for
instance, to their reproductive strategies. Besides that, the knowledge of these peculiar characteristics makes a given species an

Encyclopedia of Reproduction, 2nd Edition https://doi.org/10.1016/B978-0-12-801238-3.64567-1 1

2 Testis Physiology—Overview and Histology

Fig. 1 Testis parenchyma composition in adult mouse, depicting the two main compartments of the testis: tubular and intertubular. The tubular compartment is
constituted by the tunica propria, constituted by peritubular myoid cells and basal lamina, and seminiferous epithelium, composed by Sertoli cells and germ cells in
different stages of development, with a central tubular lumen. In the intertubular compartment, Leydig cells are found adjacent to blood vessels, lymphatic vessels
(not shown) and fibers and cells of connective tissue (not shown).

Table 1 Comparative parameters related to testis stereology and spermatogenic events in mammalian species

Tubular
diameter Seminiferous LC volume LC per testis SC per testis SC DSP per testis
GSI a (%) (mm) tubule (%) LC (%) (mm3) gram (106) gram (106) efficiency b gram (106)

Mouse 0.55–0.76 216 91–93 3.7–5.3 1021–1450 29–49 39–41 10.5–11.5 45–48
Rat 0.76 354 89 2.8 1207 12.6 27 8–10.3 17–24
Hamster 2.13 275 92.5 2.7 1092 55 44.5 8.2 24
Capybara 0.12 205 50 32.1 2029 186 19 5.6 10
Akodon montensis 1.1 233 96.6 1.1 807 14.6 54.7 10.3 62
Collared peccary 0.21 255 77.4 12.8 1170 120 28 11.1 23.4
Boar 0.4 224 83–85 7–19 1100–2200 60–120 16–39 12.4 20–27
Cat 0.078 223 88 6 2044 30 32 5.1 16
Dog 0.07–0.33 209–237 85–90 4–9 756–1064 46–111 33–41 6–8 14–22
Horse 0.09 230 61–73 18.4 4300 21–24 28 8.7–10.5 16–21
Marmoset 0.36 256 92.4 2.0 1416 13.9 35 8 18
Human 0.08 280 62 6.4 3000–4300 9 49 3 4–4.5

LC, Leydig cell; SC, Sertoli cell; DSP, daily sperm production.
a
Gonadosomatic index ¼ testis mass divided by body mass.
b
Round spermatids per Sertoli cell (SC).

interesting model for the investigation of testis function. For example, studies were developed in our laboratory using the collared
peccary to evaluate Leydig cell influence on the spermatogonial niche and spermatogonial differentiation (Campos-Junior et al.,
2012), an aspect that will be presented in more detail in the last section of this book chapter.

Spermatogenesis

Spermatogenesis is a highly organized process that occurs in the seminiferous epithelium and is orchestrated by Sertoli cells. Based
mainly on morphological and functional criteria, this process has been classically divided into three well-characterized phases,
named spermatogonial (proliferative), spermatocytary (meiotic) and spermiogenic (differentiation). However, due to several recent
findings related to spermatogonial stem cell biology and regulation, the spermatogonial phase could be subdivided into two
functionally different categories or subsets, comprising the undifferentiated and differentiated spermatogonial pools (Fig. 3). Germ
 Testis Physiology—Overview and Histology 3

Fig. 2 Comparative testis histology of four representative mammalian species: Akodon montensis, capybara, collared peccary and human. Observe the differences
in volume density of the two testis compartments—the seminiferous tubules (ST) and intertubular compartments (IC). Leydig cell volume density (%) is much higher
in capybara, whereas very few Leydig cells are observed in the wild rodent Akodon montensis. In the peccary, these steroidogenic cells present a unique and
peculiar arrangement in the testis parenchyma, forming dark stripes around the seminiferous tubules lobes, due to the high lipid content in their cytoplasm (insert).
In man, connective tissue of the intertubular compartment is quite extensive.

cells are not randomly distributed in the seminiferous epithelium, but organized in distinct and regular cellular associations known
as stages of the seminiferous epithelial cycle (Hess and França, 2007). In this article, the germ cell types as well as spermatogenic
phases and the basic Sertoli cell functions will be briefly described.

Germ Cell Syncytium and Apoptosis

Incomplete cytokinesis during mitosis is a typical feature of germ cells and spermatogenesis. This morphofunctional phenomenon
forms an intercellular communication by creating stable intercellular bridges, also called cytoplasmic bridges, which are maintained
throughout spermatogenesis. The intercellular bridge is large enough to allow the passage and sharing of cell organelles, such as
mitochondria, and several molecular signals. The type A single (As) spermatogonium is considered the only dividing germ cell
without intercellular bridges. It was shown that Tex14 knockout mice present defects in the intercellular bridges formation and,
consequently, the germ cells were unable to complete meiosis, making these rodents infertile. As reviewed by Greenbaum et al.
(2011), several roles for mammalian intercellular bridges in spermatogenesis have been proposed, including the following:

• sharing products of both sexual chromosomes, so haploid cells can remain “phenotypically diploid” even after meiosis;
• allowing the transference of cytoplasmic signals, important to maintain the synchrony in cell divisions; and
• producing a uniform population of sperm, detecting possible errors during germ cell development and facilitating the
elimination of the entire syncytium by apoptosis.

Germ cell loss is normally observed in the adult testis, contributing therefore to the maintenance of healthy spermatogenesis and
determining the total sperm output. Thus, an intricate homeostatic control of germ cell renewal, proliferation and apoptosis is
necessary to maintain normal sperm production. In this regard, significant germ cell loss occurs during the spermatogonial phase,
mainly regulating the density of these cells, which possibly represents an early mechanism to limit the number of germ cells that can
4 Testis Physiology—Overview and Histology

Fig. 3 Schematic representation of the mouse spermatogenic process: spermatogonial, meiotic and spermiogenic phases. Spermatogonial phase can be
subdivided into two distinct functional categories, that is, undifferentiated and differentiated spermatogonia. The type A single spermatogonium (As) is considered
the true stem cell, while type A paired (Apr) and A aligned (Aal4–16) are considered amplifying stem cells. There is a fine balance between differentiation and self-
renewal in the undifferentiated spermatogonial pool before the final differentiation (dotted green line) to type A1 spermatogonium (A1). After that, these cells are
considered definitively committed to sperm production, going through meiotic and spermiogenic phases. Spermatogonial cells: type A undifferentiated [single (As),
paired (Apr) and aligned (Aal4–16)] and differentiated (A1–4); intermediate (In); and type B (B) spermatogonia. Primary spermatocytes: preleptotene (Pl), leptotene (L),
zygotene (Z), pachytene (P) and diplotene (D) spermatocytes. Meiotic divisions (MI and MII). Secondary spermatocyte (II). Round (R) and elongated spermatids (E).

Fig. 4 Schematic illustration of germ cell loss (red) during spermatogenesis and the potential reasons for apoptosis of these cells in each phase of the
spermatogenic process.

be supported by each Sertoli cell. Apoptosis is also frequently observed during meiosis, probably serving as a defense mechanism for
the preservation of the species and as quality control for monitoring chromosomal damage in the spermatocytes. The extent and
purpose of spermatid apoptosis is less clear, but may also be associated with quality control, especially as related to DNA integrity
after chromatin condensation (Fig. 4) (Murphy and Richburg, 2014), or represent a mechanism related to the finite number of germ
cells that each individual Sertoli cell is capable of supporting. The rate of apoptosis, the most affected cell types affected, as well as
the pathways involved in this process vary significantly among species.
 Testis Physiology—Overview and Histology 5

Spermatogonial Phase
Undifferentiated spermatogonia
This category or subset, as mentioned above, is responsible for maintaining the balance between self-renewal and differentiation,
which is fundamental for ensuring continuous sperm production in mammals. This category is comprised of single (As), paired
(Apr) and aligned (Aal) type A spermatogonia. In the past, single spermatogonia were considered the true stem cell among germ
cells. However, because Apr and Aal spermatogonia have the ability to return to a more undifferentiated state through syncytial/
clonal fragmentation in a steady state, several recent studies have demonstrated that these cells are considered as both progenitors
and amplifying cells of the spermatogenic process. This characteristic is very important in regulating the number of cells that can
progress and be sustained throughout the next steps of spermatogenesis. These undifferentiated cells are normally present in all
stages of the seminiferous epithelial cycle and the size of the syncytium/clones (Apr, Aal 4, 8, 16) in mice determines the type and
prevalence of the membrane receptors involved in self-renewal and/or differentiation (Yoshida, 2012).
Several studies, mainly in mice, have shown that As and Apr spermatogonia present higher expressions of GFRA1 (GDNF family
receptor alpha-1) and Nanos2 (Nanos homolog 2), which are factors involved in self-renewal. As soon as these cells start to
differentiate and become Aal spermatogonia (Al 4, 8, 16), a gradual reduction of these factors is observed, while the expression of
Neurogenin-3 and RAR (retinoic acid receptor) increases, concomitantly with the spermatogonial syncytium/clones enlargement.
Lower expression of RAR on As and Apr spermatogonia ensures that these cells do not differentiate at the stages nearing spermiation,
when the levels of retinoic acid (which stimulates germ cell differentiation) are elevated. This control prevents the depletion of
spermatogonial stocks for the next cycle, ensuring continuous sperm production. Aal spermatogonia normally express the RAR and
receive retinoic acid stimuli, which starts the expression of a classic stem cell factor receptor, known as c-kit, and is considered crucial
for spermatogonial proliferation and differentiation (Fig. 5).

Differentiated spermatogonia
This category of spermatogonia is observed after the differentiation of As, Apr or Aal spermatogonial types, under retinoic acid
influence. Because the newly formed cells are committed to sperm production, losing their reversibility characteristic is a functional
process crucial for spermatogenesis. The differentiation process occurs concomitantly with spermiation and type B spermatogonia
division to preleptotene spermatocytes, and this germ cell composition in the seminiferous epithelium determines the quantity of
retinoic acid available to promote spermatogonial differentiation. After differentiation, spermatogonia pass through successive
mitotic divisions until the formation of preleptotene cells. The number of mitotic divisions is related to the number of differentiated
spermatogonial generations in a species, which seems to be phylogenetically determined and highly correlated with the magnitude
of sperm production. For example, due to the fact that most rodents present five more generations of differentiated spermatogonia
than humans, the expected number of sperm produced could be up to 32-fold higher (Fig. 6). In contrast to the previous subset
(undifferentiated spermatogonia), the presence of differentiated spermatogonia occurs in specific germ cell associations, called
stages of the seminiferous epithelial cycle, which presents germ cells at different states of differentiation (please see below) (Hess,
1990; Hess and França, 2007). It is worth mentioning that the characterization of differentiated spermatogonial cell types is based
on the presence of heterochromatin, whereas their generation number is based on the presence of mitotic figures and the
quantification of spermatogonial cells per seminiferous tubule cross-section.

Meiotic Phase
Similar to the transition of undifferentiated to differentiated spermatogonia, due to the capacity of type B spermatogonia to express
retinoic acid receptors and yield preleptotene spermatocytes, the transition from mitosis to meiosis also depends of retinoic acid.

Fig. 5 Molecular expressions that are characteristic of undifferentiated and differentiated spermatogonial cells. Single (As) and paired (Apr) spermatogonia express
high levels of self-renewal factors (GFRA1 and Nanos2), while aligned (Aal4–16) spermatogonia show higher expression of proteins involved in differentiation (RAR-Y
and Neurog3). After differentiation, the type A1, A2, A3, A4, Intermediate (In) and B spermatogonia predominantly express a protein (c-kit) involved in germ cell
proliferation and differentiation.
6 Testis Physiology—Overview and Histology

Fig. 6 Number of differentiated spermatogonia generations and the magnitude of sperm production. Contrasting with the number of cellular divisions (two) that are
constant during meiosis, the number of differentiated spermatogonial generations, which seems to be considered phylogenetically determined, significantly varies
among the mammalian species; directly influencing therefore the magnitude of sperm production. Spermatogonial phase: type A (A1–4); intermediate (In); and type
B (B) spermatogonia. Meiotic phase: preleptotene (Pl), leptotene (L), zygotene (Z), pachytene (P) and diplotene (D) spermatocytes; meiotic divisions (M1 and M2);
secondary spermatocyte (S2). Spermiogenic phase: Round (R) and elongated spermatids (E). The number present in the tip of the bar represents the possible
number of cells yield, whereas 2 illustrates the cell divisions after the differentiated spermatogonial phase.

Thus, Stra8 (stimulated by retinoic acid gene 8) is activated after the direct action of retinoic acid in the spermatogonium and
triggers the expression of important transcription factors, assuring DNA replication and the subsequent events of meiotic prophase.
During meiosis, pairing, synapsis, crossing-over and segregation of homologous chromosomes are observed. This complex and
crucial process starts with the division of the type B spermatogonia, generating preleptotene spermatocytes (Pl) (Fig. 7). Pl are the
first meiotic cells to be formed and are actively involved in DNA synthesis, being usually the last germ cells to synthesize this
molecule. Pl are followed by leptotene spermatocytes, when the chromosomes become more apparent as long strands and form a
peculiar arrangement called bouquet. Chromosomal pairing and synaptonemic complex formation start in zygotene and are
completed in pachytene spermatocytes. In this last step, thickening and shortening of the chromosomes and exchange of
chromosomal material occur, in a process called crossing-over, which is responsible for genetic variability among individuals
within the same species. The end of synaptonemic complex occurs in the diplotene spermatocyte, when the chromosomes are
partially separated. Diakinesis involves the separation of chromosomes into two sister chromatids. This first meiotic prophase is
followed by metaphase, when the paired chromatids concentrate at the equator of the germ cell. Anaphase involves the movement
of paired chromatids towards the cell poles, and telophase gives rise to daughter cells called secondary spermatocytes. Different
from primary spermatocytes, these cells have a very short life span, with haploid number of chromosomes, although they have
duplicated DNA content. The secondary spermatocytes pass through the second meiotic division very quickly, forming the round
spermatids, which present a haploid content of chromosomal DNA (Fig. 7).

Spermiogenic Phase
The spermiogenic phase is a long process in which haploid, round spermatids pass through striking morphological changes until
spermatozoa formation. The structural and functional changes that occur in the developing spermatids are usually subdivided into
steps. During this phase, the following cellular events can be observed: (a) formation of new organelles, including the chromatoid
body composed by RNA; (b) nuclear chromatin compaction; (c) acrosome formation by the Golgi apparatus; (d) flagellum
formation; (e) development of ectoplasmic specializations and tubulobulbar complexes at the interface between Sertoli cells and
elongating/elongated spermatids; (f ) elimination of cytoplasm by spermatids (residual body); and (g) release of spermatids from
the seminiferous epithelium (spermiation). Therefore, during spermiogenesis, many important genes/proteins have specific and
crucial roles in the development of spermatozoa.
This spermatogenic phase is highly dependent on androgens (steroids) and the presence of androgen binding proteins (ABP)
located in the Sertoli cell processes promotes high androgen concentrations near the elongated spermatids. Through the mainte-
nance of spermatid adhesions to Sertoli cells, androgens induce elongation of the final steps of spermatids. Furthermore, several
studies have demonstrated that testosterone is able to sustain complete spermatid differentiation by itself. In addition, follicle-
stimulating hormone (FSH) has also an important role in sperm cytostructural modifications, acting on round spermatid flagellum
extrusion and in the replacement of histones by protamines during spermiogenesis, assuring therefore the correct condensation of
spermatid nuclei (Smith and Walker, 2014). Recent studies have also shown estrogen pathways to be involved in the preparation of
elongating spermatids for release during spermiation (Kumar et al., 2017).
 Testis Physiology—Overview and Histology
Fig. 7 Morphology and characterization of mouse spermatocytes at different stages of the meiotic phase of spermatogenesis. During meiosis, pairing, synapsis, crossing-over and segregation of homologues chromosomes are
observed. The main characteristics/events presented in each cellular phases or steps (preleptotene, leptotene, zygotene, pachytene, diplotene, meiosis I, secondary spermatocytes, meiosis II and spermatids) are listed in the
boxes placed below each considered spermatocyte type.

7
8 Testis Physiology—Overview and Histology

Sertoli Cell
Sertoli cells orchestrate germ cells development and differentiation, being therefore crucial for the completion of spermatogenesis.
Among the innumerable Sertoli cells functions the following should be mentioned: (a) delivery of nutrients and growth factors for
spermatogenic cells; (b) physical support of germ cells; (c) release of the spermatids towards the tubular lumen during spermiation;
(d) secretion of tubular fluid; (e) phagocytosis of cytoplasm (the residual body) derived from late spermatids; and (f ) phagocytosis
of apoptotic germ cells. In addition, due to the presence of FSH, androgens, estrogens and thyroid hormones receptors, Sertoli cells
also intermediate most of the hormonal regulation of spermatogenesis (Fig. 8A). Sertoli cells are able to accomplish these
numerous, and sometimes simultaneous, functions because each cell is tall and extends through the entire height of the epithelium,
from basement membrane to the lumen (Hess and Vogl, 2015). Cytoplasmic arms of each Sertoli cell is capable of extending
around four different generations of developing germ cells, as it forms intricate membrane organelles. Its surface area can reach
16,000 mm2. Sertoli cells change dramatically in morphology and physiology over the course of the seminiferous epithelial cycle,
with the translocation of numerous organelles, expression of numerous different proteins for specific functions, and changes in the
location of these proteins to meet different needs of the changing germ cell associations.

Fig. 8 Main Sertoli cell functions and the balance between Sertoli cell proliferation and differentiation. (A) Schematic illustration of the key factors related to the
Sertoli cell functions. As it can be observed, the Sertoli cell is involved in crucial spermatogenic events, such as germ cell nutrition and support, signaling, hormonal
intermediation, luminal fluid secretion, endocytosis and spermiation. (B) Length of the Sertoli cell proliferation window (pink boxes) is crucial to the determination of
their final number. The major factors controlling proliferation and differentiation phases of Sertoli cells are FSH and thyroid hormones, respectively. In laboratory
rodents, experimental manipulation of these factors can shorten (B1) or prolong (B3) Sertoli cell proliferation, resulting respectively in smaller and larger testis/sperm
production. (B2) Animals under normal conditions, exhibit an equilibrium between proliferative and differentiation phases.
 Testis Physiology—Overview and Histology 9

In mammals, Sertoli cells are known to proliferate actively during the fetal period, presenting, at least in well-investigated
laboratory rodents (mice and rats), a peak in proliferation just before birth. Postnatally, its proliferation extends for around
2–3 weeks after birth in rats and 10–15 days in mice, and the end of its mitotic phase is correlated with the initiation of primary
spermatocyte differentiation, Sertoli cell-barrier development and fluid secretion/lumen formation. In humans, Sertoli cells
proliferate during the perinatal and neonatal periods, and it is believed that this somatic cell becomes quiescent for several years,
followed by a second proliferation peak in response to increased gonadotrophins at puberty. A similar pattern is observed in pigs,
but the second proliferation peak occurs in a matter of months and not years, because puberty in pigs occurs around 4 months of
age. Except for the transitional area between the seminiferous tubules and the rete testis, in adulthood the number of Sertoli cells in
the testis is rather stable. However, recent in vitro and in vivo studies, in laboratory animals, seasonal animals (hamsters) and
humans, suggest that there is a fine modulation of Sertoli cell differentiation. Therefore, these cells seem to be able to return to a
more immature state or, additionally, a specific population of Sertoli cells can keep their immature/stemness state, which could be
the situation for Sertoli cells located in the transitional area between seminiferous tubules and the rete testis (Figueiredo et al., 2016).
Although the mechanisms that regulate Sertoli cell proliferation are not yet completely elucidated, it is strongly suggested that
FSH is the main factor involved in this process. However, other factors such as for instance androgens, estrogens, activin, TGF-b,
BMP2, BMP7, IL1 and TNFalpha, probably play important roles on Sertoli cell proliferation. In contrast to FSH, thyroid hormones
are responsible for the differentiation/maturation of Sertoli cells, changing them functionally from a mitotic to a non-mitotic state.
As each Sertoli cell is capable of supporting a relatively fixed number of germ cells, in a species-specific manner, experimental
manipulation of the Sertoli cell population will affect the spermatogonial niche functionality and, ultimately, the magnitude of
sperm production (Fig. 8B–D).
As already mentioned, the stabilization of Sertoli cell proliferation and their functional maturation coincide with the develop-
ment of the blood–testis or Sertoli cell-barrier (SCB). The anatomical basis of SCB consists of the establishment of tight junctions
between Sertoli cells, in addition to the presence of basal ectoplasmic specializations, gap and adherens junctional proteins, which
also contribute to this structural and functional junctional complex. Therefore, at the molecular level, the SCB provides a physical
barrier to segregate the cellular events of spermatogenesis, particularly controlling and restricting the paracellular transport of several
substances (paracrine factors, hormones and biological molecules) across the seminiferous epithelium. Additionally, the SCB also
helps to establish an immunoprivileged microenvironment for the development of the spermatogenic process, which is especially
important as meiotic germ cells and developing spermatids can produce several immunogenic autoantigens. Furthermore, studies
have shown that the SCB is also involved in different cellular events during spermatogenesis; for example, spermatogonial
differentiation, initiation of meiosis, or reinitiation of spermatogenesis after a toxic injury (França et al., 2016).

Stages and Cycle of the Seminiferous Epithelium and Spermatogenic Wave

Stage classification methods are very useful and enable studies involving toxicological and comparative analyses, making it possible
to follow specific cellular alterations caused by the administration of drugs that promote changes in spermatogenesis. In a tubular
cross-section, it is observed that the seminiferous epithelium is composed of Sertoli and germ cells that are closely associated. It can
also be noted that, differing from Sertoli cells, germ cells are organized in a well-defined manner in concentric layers, where the most
immature germ cells (spermatogonia and early spermatocytes) are located at the base of the seminiferous epithelium, adjacent to
the basement membrane, while more advanced germ cells (leptotene/zygotene to spermatids) occupy successive layers towards the
tubular lumen.
In 1952, Leblond and Clermont described a classical method in which they accurately defined the germ cell associations present
in seminiferous tubules of rats and found that these associations, although different, were constant and with a specific time
duration. Analyzing the development of the spermatid acrosomal system, these scientists classified rat spermatogenesis into
14 cellular associations, called stages. In rodents and most non-primate species, one stage occupies a cross section of a seminiferous
tubule. Based on the same methodology, 12 different stages were described in mice (Fig. 9) and 6 in man. At the same time, another
method for stage classification, named the tubular morphology system, was already described. In the tubular method, only eight
stages of the seminiferous epithelium cycle are described for any species evaluated, but these stages include all cells and steps seen
with the acrosomal system of classification. This latter classification, used for several mammalian species, takes into account the
spermatid nuclear morphology and localization, their association with other germ cell types (i.e., differentiated spermatogonia and
spermatocytes) and the presence of meiotic figures. More recently, another classification that could integrate both acrosomal and
tubular morphology systems was developed. In this simpler and useful classification, the spermatogenic process was divided into six
representative phases: (I) before meiosis; (II) after meiosis; (III) presence of acrosomal granule; (IV) presence of acrosome; (V) pre-
spermiation; and (VI) post-spermiation. A novel immunohistochemical approach was also recently introduced, in which the stages
are visualized using immunolocalization of proteins that identify specific germ cells and Sertoli cells and label the acrosomal
formation (Nakata et al., 2015). As more and more antibodies become available, this latter method of classification may become
more popular, as it provides a powerful method for tracing gene/protein expressions through the cycle of spermatogenesis.
Independent of the classification criteria used, in humans and some other primate species more than one stage is observed per
cross section of the seminiferous tubule. The functional meaning for this finding is still unknown but might be related to the germ
cell clonal size, an aspect that surely deserves an accurate investigation.
Development of the stages is not related to any movement in the longitudinal axis of the seminiferous tubule. However, there is
a distinct order of cellular associations (stages) along the length of the tubule, forming linear segments. The continuity of these
10 Testis Physiology—Overview and Histology

Fig. 9 Stages I–XII of the seminiferous epithelial cycle in the mouse, according to the development of the acrosomic system. The individual germ cell nuclei shown
in the right column represent the germ cells found in association at each particular stage. A, type A differentiated spermatogonia; In, intermediate spermatogonia; B,
type B spermatogonia; Pl, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene spermatocytes; meiosis, meiotic figure; S2, secondary spermatocyte; R,
round spermatids; E, elongating/elongated spermatids; SC, Sertoli cells.

segments is usually observed in a descending sequence from the proximal connections to the rete testis to the approximate center of
the tubule, where the stage sequence is reversed (Nakata et al., 2017); thus, stage VII would be adjacent to stage VI, and so on. This
special arrangement will eventually lead to a complete series of stages, defined as a spermatogenic wave. However, unfortunately,
very few species were investigated in this particular aspect, making the spermatogenic wave still a not very well understood
functional aspect of spermatogenesis. Nevertheless, it is considered that the importance of wave is a continuous commitment of
germ cells to the cycle, as well as the release of sperm along the seminiferous tubules in a steady way in non-seasonal breeders,
ensuring therefore constant sperm production and maturation (Griswold, 2016).
In association with the concept of spermatogenic phases, the cycle of the seminiferous epithelium was defined as a series of
transformations that occur, within the same stage, leading to the differentiation of germ cells in a vertical way. It means that one
cycle can be represented by the differentiation of spermatogonia into spermatocytes and spermatocytes into spermatids at the same
stage. Thus, the cycle of the seminiferous epithelium was characterized as a temporal and sequential event (time and space) of
changes in cellular associations in a given area of the seminiferous tubule. Several studies have suggested that Sertoli cells cyclically
change their functions, coordinating the differentiation of germ cells. One of the most important candidates to play a role in this
regulation is the retinoic acid, which acts as a stimulator of the periodic germ cells differentiation. For instance, it was shown that an
increase in retinoic acid signaling is able to induce type A spermatogonia differentiation and influence Sertoli cell gene expression,
modulating their action on the other germ cells (Hess and França, 2007; Griswold, 2016).
Another very important functional aspect of spermatogenesis is duration of the seminiferous epithelium cycle. Although
controlled by the germ cell genotype (França et al., 1998), it is considered species-specific and does not have a conserved feature
among species or orders. Nevertheless, several studies, mainly from our research group, have demonstrated that phylogeny is
usually associated with stage frequencies, particularly when considering the combination of stages in pre-meiotic and post-meiotic
phases among closely related species. Therefore, when the frequencies of these two combined phases are considered, three patterns
 Testis Physiology—Overview and Histology 11

Table 2 Frequencies of pre-meiotic, meiotic and post-meiotic stages of the seminiferous epithelium cycle and the duration of each spermatogenic cycle
and the total spermatogenesis in several mammalian species

Pre-meiotic (%) a Meiotic (%) b Post-meiotic (%) c Spermatogenic cycle length (days) Total duration of spermatogenesis (days)

Mouse 18–23 9–11 66–71 8.6–8.9 38.7–40.1

Rat 24 5 71 12.9 58
Hamster 26–29 5.5–7.5 65–66 9–17 39–76
Akodon montensis 28 8.2 63.8 8.9 40
Capybara 44.8 14.6 40.6 11.9 53.5
Collared peccary 38 11 51 12.6 55.1
Boar 28–31 12 56–60 9 40.5
Opossum 48.9 4.8 46.3 17.3 77.9
Cat 45.5 17.6 36.9 10.4 46.8
Dog 37–41 7–11 50–57 12.6–13.8 56–62
Horse 35 15.8 49.2 12.2 54.9
Bovine 64.7 8.8 26.5 14 63
Buffalo 64.8 9.2 26.0 8.6 38.7
Goat 58.7 9.4 31.9 10.6 47.7
Marmoset 61 8 31 15.4 69.1
Human 45–75 5–7 20–50 16 72
a
After spermiation and prior to metaphase.
b
Meiosis I through meiosis II.
c
After completion of meiosis until spermiation.

can be recognized among species: (1) a relative equilibrium between pre-meiotic and post-meiotic phase frequencies (e.g.,
marsupials); (2) pre-meiotic phase corresponding to two thirds of the spermatogenic cycle (e.g., ruminants); and (3) pre-meiotic
frequency lasting about one-quarter of the entire spermatogenic cycle (e.g., laboratory rodents) (Table 2). As also observed in
Table 2, in most mammalian species investigated up-to-date the total duration of spermatogenesis is situated in the range of
40 to

60 days. It means that, from undifferentiated spermatogonia, usually less than 2 months are required for the formation and
release (spermiation) of sperm from the seminiferous epithelium.

Leydig Cells and Steroidogenesis

As previously noted in Table 1, the steroidogenic Leydig cell is the most frequent cell type observed in the intertubular compartment.
The Leydig cell exhibits huge variations in individual volumes among mammalian species. For instance, in humans and horses the
Leydig cell individual volume reaches more than 4000 mm3, whereas in Akodon montesis this value is around 800 mm3, being only
400 mm3 in rams. Interestingly, in a comparative study not yet published from our laboratory, involving 10 different breeds of dogs,
we observed that the more aggressive breeds (pitbull and pincher) have a much greater occupancy of Leydig cells, compared to other
breeds such as the poodle, whose testis has a much larger tubular compartment and spermatogenic efficiency. These findings
illustrate the importance of evaluating the testis compartments, in order to have a better understanding of reproductive strategies
and animal behavior.
The pattern of distribution and organization (cytoarchitecture) of Leydig cells in the intertubular compartment is highly variable
among the mammalians species already investigated (see Fig. 2) and probably represents important functional aspects that are yet
unknown or have not been clearly elucidated. The last comprehensive review of this subject was published more than four decades
ago (Fawcett et al., 1973). A recent publication from our laboratory showed that in the collared peccary Leydig cells present a unique
cytoarchitecture, forming a cord-like structure surrounding the seminiferous tubules lobes, and only very few Leydig cells are present
among the seminiferous tubules. This pattern markedly differs from those already described by Fawcett and colleagues in 1973.
Taking advantage of this knowledge, we developed studies regarding spermatogonial stem cell biology and niche in this species, in
which we found that, in contrast to differentiated type A spermatogonia, the undifferentiated spermatogonial cells were located
closer to the intertubular compartment areas without Leydig cells (Campos-Junior et al., 2012). In another investigation, we
observed that, during postnatal testis development in pigs, and somewhat similar to horses, seminiferous tubule maturation and
lumen formation follow an asymmetric pattern, being more advanced in the intermediate areas distant from the tunica albuginea
and closer to the central area, where the rete testis and the testis mediastinum are located (Avelar et al., 2010). Interestingly,
individual Leydig cell volume in the intermediate areas was always much larger at all investigated ages, maintaining this functional
pattern even in sexually mature pigs. As similar results are now being observed for the seminiferous tubules in rodents, having the
rete testis as a reference point, these striking finding open new venues for testis function investigation and, once more, reinforces the
functional interactions between the two different testis compartments.
 12 Testis Physiology—Overview and Histology

Regarding androgen production in the testis, it has been suggested that testosterone levels are more related to mitochondria and
smooth endoplasmic reticulum present in the cytoplasm than in the Leydig cells number and size. In addition, in comparison to the
plasma levels, intratesticular testosterone concentration is strikingly higher, meaning that normal spermatogenesis demands a
rather high amount of androgens. As already mentioned, testis steroidogenesis is crucial for maintenance of androgen levels, being
essential for the completion of full spermatogenesis and for the development and preservation of sexual male characteristics.
Overall, androgen controls spermatogenesis through Sertoli cell intermediation, as germ cells per se do not express receptors for
these hormones. Androgens and probably other steroids, including estrogen, are especially necessary for spermiogenesis and sperm
release.
The production of androgens and steroids in general begins as early as gonadal differentiation takes place and it is maintained up
to senescence, when its levels decrease. In this context, it has been demonstrated that Leydig cell capacity for producing testosterone
varies along with the different phases of testis development and is associated with the enzymatic composition of these cells. Hence,
studies in the literature reported that during fetal life a particular Leydig cell population is present in the testis, producing androgen
as an intermediate product. At this phase, it was recently shown that Sertoli cells are remarkably necessary for completion of
testosterone synthesis, once these supporting somatic cells express 17b-hydroxysteroid dehydrogenase 3 (Hsd17b3), the last
enzymatic step necessary for testosterone production. Hsd17b3 is not detected in Fetal Leydig cells. Thus, the androgen produced
by Fetal Leydig cell is delivered to the Sertoli cell that will provide the final conversion to testosterone (Shima and Morohashi,
2017). Postnatally, the Leydig cell population changes dramatically and the Adult type Leydig cell develops the enzymatic
machinery necessary to accomplish testosterone production. Concomitantly, Hsd17b3 gene is silenced in Sertoli cells and, in this
respect, the only source of testosterone in post pubertal testis is the Leydig cell (Ye et al., 2017). In this context, the rather substantial
interactions between Leydig cells and macrophages and the possibility of the coexistence of distinct Leydig cells populations in the
testis of some species represent an interesting aspect of testis function that might be taken into consideration in future studies.

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