20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
| |
Received: 12 January 2020    Revised: 7 March 2020    Accepted: 7 April 2020

DOI: 10.1111/andr.12796

ORIGINAL ARTICLE

Phospholipase C zeta profiles are indicative of optimal sperm

parameters and fertilisation success in patients undergoing
fertility treatment

Junaid Kashir1,2,3  | Bhavesh V. Mistry3 | Lujain BuSaleh1,3 | Reema Abu-Dawas1,3 |

Michail Nomikos4 | Ahmed Ajlan5 | Raed Abu-Dawud3 | Nadya AlYacoub3 |
Saad AlHassan5 | F. Anthony Lai4,6 | Abdullah M. Assiri1,3,6,7 | Serdar Coskun5

1
College of Medicine, Alfaisal University,
Riyadh, Kingdom of Saudi Arabia Abstract
2
School of Biosciences, Cardiff University, Background: Oocyte activation is driven by intracellular calcium (Ca2+) oscillations
Cardiff, UK
induced by sperm-specific PLCζ, abrogation of which causes oocyte activation defi-
3
Department of Comparative Medicine,
King Faisal Specialist Hospital and Research
ciency in humans. Clinical PLCζ investigations have been limited to severe male infer-
Center, Riyadh, Kingdom of Saudi Arabia tility conditions, while PLCζ levels and localisation patterns have yet to be associated
4
College of Medicine, QU Health, Qatar with general sperm viability.
University, Doha, Qatar
5 Materials and Methods: PLCζ profiles were examined within a general population of
Department of Pathology and Laboratory
Medicine, King Faisal Specialist Hospital and males attending a fertility clinic (65 patients; aged 29-53), examining PLCζ throughout
Research Centre, Riyadh, Kingdom of Saudi
various fractions of sperm viability. Male recruitment criteria required a minimum sperm
Arabia
6
Biomedical Research Centre, Qatar count of 5 × 106 spermatozoa/mL, while all female patients included in this study yielded at
University, Doha, Qatar least five oocytes for treatment. Sperm count, motility and semen volume were recorded
7
Institute for Research and Medical
according to standard WHO reference guidelines and correlated with PLCζ profiles ex-
Consultations, Imam Abdulrahman Bin Faisal
University, Dammam, Saudi Arabia amined via immunoblotting and immunofluorescence. Appropriate fertility treatments
were performed following routine clinical standard operating protocols, and fertilisation
Correspondence
Junaid Kashir, College of Medicine, Alfaisal success determined by successful observation of second polar body extrusion.
University, Riyadh, Kingdom of Saudi Arabia.
Results and Discussion: Four distinct PLCζ patterns were observed at the equatorial,
Email: junaidkashir@hotmail.com
acrosomal + equatorial regions of the sperm head, alongside a dispersed pattern, and
Funding information
a population of spermatozoa without any PLCζ. Acrosomal + equatorial PLCζ corre-
JK was supported by a Healthcare Research
Fellowship Award (HF-14-16) made by lated most to sperm health, while dispersed PLCζ correlated to decreased sperm vi-
Health and Care Research Wales (HCRW).
ability. Total levels of PLCζ exhibited significant correlations with sperm parameters.
This study was also supported by a National
Science, Technology and Innovation plan PLCζ variance corresponded to reduced sperm health, potentially underlying cases
(NSTIP) project grant (15-MED4186-20)
of male sub-fertility and increasing male age. Finally, significantly higher levels of
awarded by the King Abdulaziz City for
Science and Technology (KACST) to JK, PLCζ were exhibited by cases of fertilisation success, alongside higher proportions of
AMA and FAL.
Ac + Eq, and lower levels of dispersed PLCζ.
Conclusions: PLCζ potentially represents a biomarker of sperm health, and fertilisa-
tion capacity in general cases of patients seeking fertility treatment, and not just
cases of repeated fertilisation. Further focused investigations are required with
larger cohorts to examine the full clinical potential of PLCζ.

© 2020 American Society of Andrology and European Academy of Andrology

Andrology. 2020;8:1143–1159.  |
wileyonlinelibrary.com/journal/andr     1143
 |
1144       KASHIR et al.
KEYWORDS
fertilisation, male infertility, oocyte activation, phospholipase C zeta (PLCzeta), spermatozoa
1 |  I NTRO D U C TI O N
Infertility affects ~ 15% of couples, while male infertility affects ~ 7%
of men, worldwide.1 Although genetic causes of male infertility can
be attributed to ~ 30% of cases, ~50% of male factor infertility cases
remain unexplained, 2–4 which is the major causative factor underly-
ing infertility.1 Although some concerns have been partially allevi-
ated through the use of assisted reproductive technology (ART), it
seems increasingly clear that fertility outcome may be determined
by the efficacy of early events during fertilisation.5
At mammalian fertilisation, oocytes undergo a series of events
collectively termed oocyte activation, driven by distinct pat-
terns of intracellular calcium (Ca2+) oscillations, initiated by a tes-
tis-specific phospholipase C termed phospholipase C zeta (PLCζ).6
Immunodepletion of PLCζ from sperm extracts diminished Ca2+-
release within oocytes,6 while the presence of PLCζ was confirmed
in sperm extract fractions capable of inducing Ca2+ oscillations.7,8
Recombinant PLCζ cRNA and protein injection into mouse oocytes
initiated Ca2+ oscillations and embryogenesis to the blastocyst
stage.6,9 RNA interference (RNAi) experiments yielded mice with
disrupted testicular PLCζ expression, with spermatozoa inducing
abnormal Ca2+ oscillations, and a reduced litter size.10 Finally, sper-
matozoa from knock-out mouse models of PLCζ were unable to elicit
Ca2+ oscillations following intracytoplasmic sperm injection (ICSI;
whereby a single spermatozoon is microinjected directly into the oo-
cyte), while in vitro and in vivo fertilisation experiments exhibited
abnormally high levels of polyspermy, and a severely reduced profile
of Ca2+ release and litter size.11,12
Infertile human spermatozoa unable to activate human and
mouse oocytes, even following ICSI (oocyte activation deficiency;
OAD), are unable to elicit Ca2+ oscillations, resulting in a reduced
capacity of oscillations, if at all present.13 Mutations identified in
the PLCζ gene of such patients resulted in predicted modifications
of the enzyme fold, abrogating PLCζ activity and/or levels within
14–21
the spermatozoa. Indeed, reduced/absent levels of PLCζ are a
hallmark of OAD spermatozoa,1,13,14,18,22 with PLCζ deficiencies as-
sociated with multiple male-specific conditions including abnormal
sperm morphology,1,14,18,23–26 sperm DNA fragmentation and oxida-
tion 27,28 and abnormal pregnancies. 29 Thus, immunological analysis
of PLCζ in human spermatozoa could be a powerful diagnostic ap-
13,14,19,23,30,31 1
proach [for a detailed review, see Kashir et al ].
However, the scope of studies thus far has been limited to ex-
amining only severe cases of complete fertilisation failure, and not
whether analysis could be expanded to include more subtle alter-
ations of PLCζ in the general male population seeking fertility treat-
ment. Thus, while PLCζ exhibits great promise as a clinical prognostic
factor and has been suggested to be a causative factor in cases of
complete fertilisation failure as has been previously investigated at
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
great length 13,14,19,23,30,31
[for a detailed review, see Kashir et al1],
a methodical analysis of such potential in relation to general sperm
parameters has yet to be performed. Herein, we perform the first
systematic analysis of PLCζ profiles in human spermatozoa from a
general population of males undergoing fertility treatment to exam-
ine whether PLCζ could be used as a diagnostic measure of male
fertility, systematically characterising PLCζ levels and localisation
patterns in spermatozoa in relation to routine parameters of sperm
health such as sperm count and progressive motility, and semen
volume.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Patient recruitment
Male patients undergoing consecutive treatment cycles of assisted
reproductive technology (ART) therapy at the fertility clinic in the
King Faisal Hospital and Research Centre (KFSHRC). The study was
approved by the local research ethics committee and the office of
research affairs at the KFSHRC (RAC# 2 170 015), and written in-
formed consent was obtained from all patients before starting treat-
ment and collecting samples for experimentation. Male recruitment
criteria required a minimum sperm count of 5  ×  106 spermatozoa/
ml, while all female patients included in this study yielded at least
five oocytes for treatment. As we aimed to examine spermatozoa
from a range of patients to investigate presence of any correlations
between PLCζ and general sperm parameters, our only inclusion cri-
teria was a total sperm count ≥ 5 × 106 spermatozoa/ml.
A total of 65 patients, aged 29-53  years, were recruited for this
study. Semen samples were produced by masturbation on the same
day of oocyte aspiration. Semen was allowed to liquefy at 37°C for
15  min prior to analysis. Raw semen was used to record the sperm
counts (concentration;  × 106/mL), progressive motility (%), and semen
volume (mL) according to standard WHO reference guidelines,32 which
were then used for correlative analysis with PLCζ. Morphology (% nor-
mal morphology) was also assessed as per standard protocols. Optimal
parameters for sperm motility, sperm count and semen volume were
recorded as per WHO reference ranges. Patient recruitment, sample
processing, ovarian stimulation and fertility treatment all were per-
formed in the clinic at the KFSHRC following routine standard operat-
ing protocols. The fertility clinic laboratory at the KFSHRC is accredited
by the College of American Pathologists (CAP) and participates in the
CAP proficiency testing programme for external quality assurance
(EQA). The sperm analysis system employed is internally quality con-
trolled on a daily bases using Accubeads and a video-recorded semen
clip. Excess spermatozoon following treatment was then processed
in research laboratories. Fertilisation success was determined by

KASHIR et al.
successful observation of second polar body extrusion and fertilisation
success rates determined by the proportion of oocytes successfully
fertilised out of the total number of oocytes used for each case.
2.2 | Semen and sperm processing
Semen was subject to density gradient washing (DGW) by overlay-
ing raw semen (raw) on top of 90% density gradient medium (Isolate
Sperm Separation Medium, Irvine Scientific) at a 1:1 ratio and centri-
fuged at 300 g for 20 min at room temperature (RT). The supernatant
was collected, and the sperm pellet was then subject to ×3 washes
using 1× phosphate-buffered saline (PBS)+protease inhibitors (PI)
(Roche, Germany), by centrifugation at 500 g for 10 min. Following
washing, the sperm pellet was resuspended in an appropriate amount
of PBS  +  PI. Sperm concentration was determined microscopically
using a Neubauer improved counting chamber (Hausser Scientific,
5
USA), and single-use aliquots of 5 × 10 spermatozoa/aliquot were
made for immunoblotting using 5× sample loading (Laemmli) buffer
(10% (w/v) SDS; 10mM beta-mercaptoethanol; 20% (v/v) glycerol;
0.2M Tris-HCL, PH 6.8; 0.05% (w/v) bromophenol blue). Each ali-
quot was briefly vortexed, snap frozen in liquid nitrogen and stored
at − 80°C until required.31
To prepare spermatozoa for immunofluorescence microscopy,
the sperm suspension was fixed with 4% paraformaldehyde at RT for
15 min. Following fixation, spermatozoa were centrifuged at 500 g
for 10  min, and the pellet was washed thrice using PBS  +  PI and
resuspended in an appropriate volume of PBS  +  PI. Samples were
stored at 4°C until required (not more than a week). To examine PLCζ
in various sperm fractions, spermatozoa from the various stages of
the DGW was processed for immunoblotting and immunofluores-
cence as described above. The fractions used were obtained from
the raw ejaculate, the layer above the 90% gradient following DGW,
and the pellet after the DGW to give the raw, DGW-rejected and
DGW-selected fractions, respectively. Before fixation, each fraction
of spermatozoa was washed ×3 using PBS + PI. For each fraction, sin-
gle-use aliquots of 5 × 105 spermatozoa/aliquot were also prepared.
Unless explicitly stated, the DGW-selected fraction of sperma-
tozoa was utilised from all patients to perform our analyses using im-
munoblotting and immunofluorescence, for correlations with sperm
parameters and fertilisation outcome.
2.3 | SDS-PAGE and immunoblotting
31,33
Immunoblotting was performed as previously described . Briefly,
single-use sperm aliquots were thawed and heated at 101°C for 5 min,
followed by immediate vortexing, cooling on ice and brief centrifu-
gation. Recombinant NusA-tagged PLCζ bacterial cell lysates were
prepared, and recombinant NusA-tagged PLCζ purified, as exten-
sively previously described.33–36 This method of protein production
has been extensively characterised, and we have consistently previ-
ously demonstrated that our antibodies do not exhibit non-specific
|
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
      1145
cross-reactivity in bacterial lysates not overexpressing PLCζ. Protein
samples were separated through SDS-PAGE using 10% gels and trans-
ferred onto polyvinylidene difluoride (PVDF) membranes (Amersham
Hybond, GE Healthcare Life Sciences USA), using the wet-transfer
method at 100V for 1 hour. Transfer and protein separation efficacy
were determined by staining membranes with 0.1% (w/v) Ponceau
stain (in 5% (v/v) acetic acid). Membranes were incubated overnight
at 4°C with primary antibodies diluted appropriately, followed by
incubation with secondary antibodies conjugated with horseradish
peroxidase (HRP) for 1 hour at RT diluted at 1:1000 (anti-rabbit) or
1:5000 (anti-mouse). HRP detection was achieved using the ECL se-
lect kit, following the manufacturers recommended protocol (GE Life
Sciences). Chemiluminescence was detected using the ImageQuant
LAS 4000 (GE Healthcare Life Sciences) image system.
2.4 | Immunofluorescence microscopy
Immunofluorescence was performed as previously described.18,31
Briefly, fixed spermatozoa were added to hydrophobic moulds
drawn previously using a PAP pen (Vector Laboratories) onto slides
pre-coated with 0.01% (w/v) poly-l-lysine solution (Sigma-Aldrich).
Spermatozoa were permeabilised with PBS-1% Triton X-100 (v/v) for
1  hour at RT, and blocking was performed using PBS-10% bovine
serum albumin (BSA; Sigma-Aldrich). Cells were incubated with pri-
mary antibodies at appropriate dilutions with PBS-5% BSA overnight
at 4°C, after which cells were incubated with AlexaFluor 488-conju-
gated goat anti-rabbit secondary antibody (1:100; Life Technologies),
diluted in PBS-5% BSA 1hour at RT. Finally, cells were mounted with
VECTASHIELD Mounting Medium containing 4′-6-diamidino-2-phe-
nylindole (DAPI) (Vector Laboratories) and slides stored at 4°C in the
dark until imaging (not more than 3 days).
Immunofluorescence experiments were performed with anti-
gen unmasking/retrieval (AUM) using Acidic Tyrode's solution (AT)
(pH = 2.5-3.0; Sigma, UK) as previously described 31 and incorporated
in the immunofluorescence protocol between the permeabilisation
and blocking steps. AT solution was added to hydrophobic moulds
and incubated for 5 seconds at RT, followed by ×3 washes with PBS.
Images were captured at ×40 and 100× magnifications (with oil
immersion, type FF, Electron Microscopy Sciences, Cat. No. 16916-
04), using an OLYMPUS BX53 fluorescence microscope (Olympus).
An OLYMPUS DP73 camera (Olympus) was used to capture images
using OLYMPUS cellSens Entry software (Olympus). Brightfield im-
ages were captured alongside the corresponding fluorescence im-
ages obtained using a fluorescein isothiocyanate (FITC) filer. Care
was taken to ensure images were captured at the same exposure
time for all patients and samples.
2.5 | Antibodies and validation
Two distinct antibodies were used against PLCζ. The first was a poly-
clonal antibody (EF pAb) raised in rabbits against a 16-mer peptide
 |
1146       KASHIR et al.
sequence (8SKIQDDFRGGKINLEK 23) of human PLCζ protein. This
antibody has previously been extensively characterised and pub-
lished in the literature against mouse and human sperm and recom-
binant PLCζ.31,33–36 A further monoclonal antibody against PLCζ (I33
mAb) was generated for this study raised by injecting a recombinant
PLCζ protein fragment corresponding to position 155-465 of the
human PLCζ amino acid sequence (UniProt ID: Q86YW0) into rab-
bits, followed by cell fusion into rabbit hybridoma cells and affinity
purification (GenScript). A monoclonal antibody against alpha (α)-
tubulin raised in mouse was used as a loading control (Thermo Fisher
Scientific, Cat:62 204) for immunoblotting.
Antibody specificity was determined through blocking experi-
ments by incubating antibodies for 1 hour at RT with a molar excess
of affinity-purified recombinant NusA-PLCζ protein before addition
to membranes for immunoblotting. Recombinant NusA-PLCζ pro-
tein generation and purification were performed as extensively pre-
viously detailed.33–36 Blocking experiments were performed using
immunoblotting on human sperm extracts alongside recombinant
NusA-PLCζ bacterial lysates. For immunoblotting experiments, an-
tibodies were appropriately diluted by factors of 1:1000 (EF pAb),
1:500 (I33 mAb) or 1:10 000 (α-tubulin). Appropriate controls were
also performed by incubation of secondary antibody only in the ab-
sence of primary antibodies for both EF and I33 antibodies in both
immunofluorescence and immunoblotting protocols.
2.6 | Sperm PLCζ analysis
Sperm PLCζ fluorescence quantification was performed on images
obtained at 40× magnification as previously described.18,31 Only
spermatozoa in the same plane of view were analysed for each
image, while spermatozoa obscured by overlying debris or by tails/
heads of other spermatozoa were excluded. Analysis was limited to
the sperm head only, consisting of quantification of total PLCζ im-
munofluorescence and the proportion of PLCζ localisation patterns
observed on each patient. Such analyses were performed with the
ImageJ software package (National Institutes of Health) using the
18,31
regions of interest tool. Hundred cells per patient were analysed,
and total fluorescence for each cell was subtracted with background
fluorescence (relative fluorescence). PLCζ immunoblotting quanti-
fication was achieved through relative density quantification using
18,31
the ImageJ software package. Pixel density of PLCζ bands iden-
tified by both EF and I33 antibodies were normalised with the pixel
density of bands identified by the α-tubulin antibody to give the rela-
tive density for PLCζ for each patient.
2.7 | Statistical analyses
Mean values and the standard error of the mean (SEM) were subject
to statistical tests as appropriate to determine statistical significance
using the Prism 7.0 software package (GraphPad). The extent of vari-
ability was measured by comparisons between the average standard
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
deviations (SD) in the variables examined for each patient. To evalu-
ate differences between two variables, the t test was employed
using Welch's correction for unequal standard deviations. Statistical
comparisons between multiple variables were performed (including
multiple comparisons tests) using either the one-way or two-way
analysis of variance (ANOVA) as appropriate. One-way ANOVA anal-
ysis was also followed by Tukey post hoc analysis, while two-way
ANOVA analysis was followed by the two-stage step-up method of
Benjamini, Krieger and Yekutieli37 to control the false discovery rate
following multiple comparisons.
Data are presented as mean ± standard error of the mean (SEM),
while data represented as proportions (%) were ARCSIN transformed
prior to statistical analysis to avoid truncation of data. Pearson cor-
relation coefficients were calculated for all variables where correla-
tions were examined. All such analyses were performed on relative
fluorescence values obtained with and without antigen unmasking. A
P-value ≤ .05 was considered to be statistically significant. The level
of significance was adjusted for the analysis underlying localisation
pattern correlations, where the post hoc corrected significance value
was adjusted according to the number of distinct localisation pat-
terns observed (0.05/n, where n is the number of distinct patterns
observed). Thus for localisation pattern analyses to be considered
significant, a P-value ≤ 0.0125 was considered statistically significant.
3 | R E S U LT S
3.1 | PLCζ in human spermatozoa
Antibody validation experiments revealed that both the EF and I33
antibodies recognised specific bands corresponding to recombinant
NusA-tagged human PLCζ in bacterial lysates which have been ex-
tensively characterised in previous studies (Figure  1A), alongside
PLCζ from human spermatozoa (Figure 1B) at the expected molecular
weights (~133kDa and ~ 70kDa, respectively). Lower bands were ob-
served in bacterial NusA lysates (~100kDa and ~ 70kDa), representing
degraded protein fragments of NusA-human PLCζ as previously de-
scribed.34 These bands were depleted following blocking of antibodies
using purified recombinant PLCζ protein before exposure of antibod-
ies to membranes. Collectively, these results indicate our antibodies
specifically recognise a single band corresponding to the expected
molecular weight of PLCζ within human spermatozoa (~70 kDa).
Immunofluorescence using both antibodies identified previously
characterised succinct populations at the equatorial only (Eq) and
acrosomal  +  equatorial (Ac  +  Eq) segments of the sperm head, re-
ported by multiple previous studies. However, we also observed a
novel ‘dispersed’ pattern of localisation, not contained to a specific
location, but rather as punctate loci throughout the sperm head
(Figure  2). Our observed patterns were not identical throughout
spermatozoa as has previously been suggested by studies,18,22 with
the exact distribution of particular localisation patterns in the sperm
head exhibiting large degrees of variance, but largely able to fit
within four descriptive categories.
 |

20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
KASHIR et al.       1147

F I G U R E 1   Immunoblotting
analysis and corresponding Ponceau-
stained images indicating specificity
of EF polyclonal and I33 monoclonal
in recognising A) bacterial lysates
overexpressing recombinant NusA-human
PLCζ and B) PLCζ in human spermatozoa.
Both EF polyclonal (pAb) and I33
monoclonal (mAb) antibodies recognised
bands at the expected molecular weight
for NusA-PLCζ (~133 kDa) and human
sperm PLCζ (~70kDa). In recombinant
lysates, further lower bands were also
identified (~100 kDa and ~ 70 kDa), which
likely represent degradation product as
all bands identified in recombinant lysates
and human spermatozoa were depleted
following blocking with recombinant
NusA-PLCζ protein

We also observed fluorescence throughout the sperm tail, in-
cluding the midpiece, in all cells (100%) examined using both antibod-
ies. Such fluorescence, alongside localisation patterns in the sperm
head, was diminished following blocking of both antibodies. No sig-
nals were detected using the negative controls utilising secondary
antibody only (Figure 3). Finally, we also observed a small population
of spermatozoa which did not exhibit any PLCζ immunofluorescence
in the sperm head, classified as ‘none’ in subsequent sections.
3.2 | Proportional distribution of PLCζ in human
sperm viability fractions
PLCζ quantification throughout sperm fractions indicated dy-
namic alterations in the proportions of PLCζ localisation observed
(Figure 4). In raw (ejaculated) sperm fractions, the dispersed pattern
of localisation was predominant (81.7%), followed by the Ac  +  Eq
(14.6%) and Eq (1.3%) distributions, while 2.4% of spermatozoa ex-
hibited no fluorescence (none).
The density gradient washing rejected (DGW-rejected) frac-
tion (representing spermatozoa not utilised clinically and viewed
as non-viable) exhibited the same pattern of distribution, with
the predominant pattern being the dispersed localisation (69.6%)
followed by the Ac  +  Eq (22.1%) and the Eq (8.3%) patterns of
localisation. Spermatozoa without PLCζ fluorescence were not
observed in this fraction. The DGW-selected fraction, contain-
ing spermatozoa exhibiting highest sperm parameters which are
utilised clinically, exhibited both dispersed and Ac  +  Eq patterns
as predominant (38.5% and 38.2%, respectively), which were not
statistically significant from each other (P = .43). This was followed
by the Eq pattern (16.9%) and the spermatozoa exhibiting no PLCζ
fluorescence (6.4%). Collectively, the dispersed pattern of locali-
sation was found to decrease as spermatozoon was processed into
its different fractions, exhibiting highest distribution in the raw
(ejaculate) fraction.
3.3 | Relationship between PLCζ localisation,
relative fluorescence levels and sperm parameters
While sperm morphology was assessed for all patients examined
as per standard protocols, we could not perform a satisfactory
 |
1148       KASHIR et al.
correlative analysis with PLCζ, as the range of morphology scores in
our patient population was low, with most patients exhibiting a high
morphology score (88%-99%) as low morphology scores were not
a recruitment criteria for this study. Correlative analysis between
PLCζ localisation patterns and sperm parameters using the Pearson's
correlation coefficient (r) test indicated significant correlations
(P  ≤  .0125) between sperm count and the acrosomal  +  equatorial
(positive correlation) and dispersed (negative correlation) patterns
of localisation (r = .33 and r = −.32, respectively). A further signifi-
cant (P  ≤  .0125) negative correlation was also observed between
the dispersed population of PLCζ and sperm motility (r = −.31). All
other localisation patterns exhibited no significant correlation with
sperm parameters, with the exception of the relationship between
the equatorial pattern of PLCζ localisation and sperm motility, which
appeared to be approaching statistical significance (P = .024).
No significant relationship was observed between PLCζ local-
isation and male age (Table  1). Significant correlations (P  ≤  .0125)
were observed between PLCζ fluorescence and the Eq (negative
correlation; r  =  −.37), Ac  +  Eq (positive correlation; r  =  .51), dis-
persed (negative correlation; r  =  −.51) and no PLCζ fluorescence
(negative correlation; r  =  −.5) distributions of PLCζ in the sperm
head (Table 1).
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
F I G U R E 2   Representative
immunofluorescence images of observed
PLCζ localisation patterns in human
spermatozoa. Brightfield (leftmost
panels) and PLCζ (green fluorescence;
middle panel) images were obtained.
Predominant representative localisation
patterns were captured, and other
distributions clustered in general
localisation were also obtained (rightmost
panel). Top panel shows equatorial
localisation (white arrows), and the middle
panel indicates acrosomal + equatorial
localisation (red arrow indicates acrosomal
localisation). The bottom panel shows
the dispersed patterns of localisation.
Further examples of each pattern of
localisation are also detailed to illustrate
the variability exhibited in localisation
patterns even within the same pattern.
The bottom-most separate panel shows
an example of the extent of fluorescence
observed in the tail, with the midpiece
and entirety of the tail being identified.
Images obtained were captured at 100×,
while white scale bars represent 10 μm.
Images are representative of at least 100
cells examined per patient recruited (65
patients)
The dispersed proportion of localisation decreased significantly
(P ≤ .05) in ideal sperm motility (34.6% vs 53.9%, respectively) and
count values (36.7% vs 56.1%, respectively), while the population
of Ac  +  Eq significantly increased in the optimal values for motil-
ity and count compared to the sub-optimal range (35.5% vs 25.6%,
and 35.9% vs 22.8%, respectively). The proportion of spermatozoa
exhibiting Eq localisation increased in optimal values of sperm mo-
tility, but otherwise exhibited no significant change (P > .05) in other
parameters. Optimal ranges of semen volume did not exhibit any sig-
nificant change in any proportion of localisation patterns (Figure 5;
Figure S1).
3.4 | Relationship between acrosomal + equatorial/
dispersed localisation pattern ratios and
sperm parameters
The ratio between Ac + Eq and dispersed localisation proportions
for patients significantly (P ≤ .05) positively correlated with sperm
motility and count (r  =  .32 and r  =  .31, respectively), but did not
significantly correlate (P  >  .05) with semen volume or male age
(Table  2). The optimal parameters for sperm motility and count
 |

20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
KASHIR et al.       1149

F I G U R E 3   Representative immunofluorescence images of

negative control (secondary antibody incubation only without
primary; top panel), and antibody blocking (antibodies blocked with
a molar excess of purified recombinant NusA-PLCζ; bottom panel).
Brightfield (leftmost panels) and PLCζ (green fluorescence; middle
panel) images were obtained. Both panels indicated diminished/
absent levels of PLCζ fluorescence, which was representative
for both EF and I33 antibodies. Images obtained were captured
at 100×, while white scale bars represent 10μm. Images are
representative of at least 100 cells examined for each treatment
exhibited a significantly higher (P ≤ .05) ratio between the Ac + Eq
and dispersed patterns of localisation compared to sub-optimal
parameters (ratios of 1.5 vs 0.9, and 1.4 vs 0.5, respectively).
Conversely, however, ideal parameters of semen volume did not
exhibit any significant differences in the localisation ratio exam-
ined (Figure 6; Figure S2).
3.5 | Relationship between PLCζ quantification and
optimal sperm parameters
PLCζ immunoblotting using both antibodies identified sperm PLCζ
bands at the expected molecular weight for human sperm PLCζ
at ~ 70 kDa. Immunoblotting for alpha tubulin (α-tubulin) also iden-
tified single bands at the expected molecular weight (~50  kDa)
(Figure  7). Relative densities of both EF and I33 PLCζ bands
FIGURE 4 Histogram indicating the dynamic distribution of PLCζ
patterns of localisation throughout various human sperm fractions
following antigen unmasking. The predominant pattern in the raw
and DGW-rejected fractions (expected to contain predominantly
low-quality spermatozoa) was observed to be the dispersed pattern
of localisation, followed by the acrosomal + equatorial (Ac + Eq)
and equatorial patterns, with a smaller proportion of spermatozoa
not exhibiting any immunofluorescence. Conversely, the dispersed
localisation pattern significantly decreased in the DGW-selected
fraction (expected to contain the highest quality spermatozoa), while
the Ac + Eq pattern significantly increased. These were followed by
the equatorial only pattern, and the proportion of spermatozoa not
exhibiting any fluorescence. Data are represented as proportions of
spermatozoa exhibiting localisation patterns ± standard error of the
mean (SEM). Asterisk (*) indicates a statistically significant (P ≤ .05)
difference, while a statistically insignificant (P > .05) difference is
indicated by n.s. Data are indicative of at least 100 cells examined
from 3 technical repeats from spermatozoa pooled from 10 males
(normalised with the relative densities of corresponding α-tubulin
bands) from both raw and DGW-selected samples were then corre-
lated to semen parameters.

TA B L E 1   Analysis of correlations between the proportions of distinct patterns of PLCζ localisation observed and semen parameters from
human patients undergoing fertility treatment, observed relative fluorescence after AUM, and male age (years)

Relative fluorescence Male

Localisation pattern Motility (%) Count (106/mL) Volume (mL) (a.u) age

Equatorial P = .024 P = .36 P = .27 r = −.37 (P < .0125) P = .3

Acrosomal + Equatorial P = .13 r = .33 (P < .0125) P = .3 r = .51 (P < .0125) P = .5
Dispersed r = −.31 (P < .0125) r = −.32 (P < .0125) P = .36 r = −.51 (P < .0125) P = .42
None P = .48 P = .31 P = .1 r = −.5 (P < .0125) P = .3

Note: Statistically significant (P ≤ .0125) differences are detailed in bold, along with the corresponding Pearson's correlation coefficient (r; negative
values represent a negative correlation, while positive values indicate a positive correlation).
Abbreviation: a.u, arbitrary units.
 |
1150       KASHIR et al.
F I G U R E 5   Histograms indicating the changes in localisation
patterns observed in non-optimal and optimal A) sperm
motility (optimal motility ≥ 40%) and B) sperm count (optimal
count ≥ 15 × 106spermatozoa/mL). Data are represented as
proportions (%) with standard error of the mean (SEM). Asterisks (*)
indicate statistically significant differences (P ≤ .05). Eq: equatorial;
Ac + Eq: acrosomal + equatorial
No statistically significant (P >  .05) correlations were observed
between sperm parameters or male age and PLCζ relative band den-
sity from raw spermatozoa using either antibody. However, statisti-
cally significant positive correlations (P ≤ .05) were observed using
both antibodies between PLCζ relative band density of DGW sperm
6 the ratio of the acrosomal + equatorial
  Motility (%) Count (10 /mL)
Localisation ratio (Ac + Eq/ r = .32 (P < .05) r = .31 (P < .05)
Dispersed)
Note: Statistically significant (P ≤ .05) differences are detailed in bold, along with the corresponding
Pearson's correlation coefficient (r; positive values indicate a positive correlation).
Abbreviation: a.u, arbitrary units.
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
and motility (r = .38 and r = .4, respectively), as well as semen volume
(r = .38 and r = .4, respectively). Quantification of PLCζ using relative
fluorescence did not indicate any significant differences (P > .05) be-
tween any parameters examined (Table 3).
Examination of total fluorescence levels of PLCζ did not indicate
any significant differences (P  >  .05) between non-optimal and op-
timal ranges of semen volume, sperm motility and sperm concen-
tration (Figure S3). Similarly, PLCζ levels quantified through relative
density using both antibodies did not reveal any significant differ-
ences (P > .05) in optimal compared to non-optimal levels for motility
(Figure S4A) and count (Figure S4B). However, levels of PLCζ were
observed to be significantly higher (P ≤ .05) in semen volumes higher
than 5.5ml compared to those levels of PLCζ observed at semen vol-
umes ≤ 5.5ml, using both EF and I33 antibodies (Figure 8).
3.6 | The relationship between PLCζ variability and
immunoblotting and sperm parameters
PLCζ levels quantified using both immunofluorescence and immu-
noblotting appeared significantly variable. To determine the extent
of this variation, we performed correlative analyses between the
observed standard deviation in levels of PLCζ (quantified using im-
munofluorescence and immunoblotting) and sperm parameters.
Variance in PLCζ fluorescence exhibited significant (P ≤ .05) neg-
ative correlations with sperm motility and count (r = −.26 and r = −.4,
respectively), and a significant (P ≤ .05) positive correlation with rel-
ative fluorescence levels (r  =  .6). No other significant correlations
were observed. Interestingly, variance observed in PLCζ levels quan-
tified by relative band density exhibited significant (P ≤ .05) positive
correlations between male age and total quantified levels using both
EF (r = .34 and r = .32, respectively) and I33 antibodies (r = .41 and
r  =  .31, respectively). No other statistically significant differences
were observed (P > .05) (Table 4).
No significant differences were observed in variance levels
between optimal and non-optimal male parameters in both fluo-
rescence and relative density quantification in all sperm fractions
(data not shown). Finally, the variance observed in PLCζ fluores-
cence exhibited significant (P  ≤  .0125) positive correlation with
dispersed localisation (r = .3), while no significant correlation was
observed with the other patterns of PLCζ localisation, although
the relationship between variability and the equatorial pattern
appeared to be approaching statistical significance (P  =  .014)
(Table 5).
TA B L E 2   Correlative analysis between
Male age
Volume (mL) (years)
(Ac + Eq) and dispersed localisation
P = .38 P = .47 patterns (Ac + Eq/dispersed), and
semen parameters from human patients
undergoing fertility treatment, and male
age (years)

KASHIR et al.
F I G U R E 6   Histograms illustrating
differences in the localisation ratio
between the acrosomal + equatorial
(Ac + Eq) and dispersed patterns of
localisation observed in non-optimal
and optimal A) sperm motility (optimal
motility ≥ 40%) and B) sperm count
(optimal count ≥ 15 × 106spermatozoa/
mL). Data are represented as the mean
ratio with standard error of the mean
(SEM). Asterisks (*) indicate statistically
significant differences (P ≤ .05)
3.7 | Successful fertilisation is associated
with higher total levels of PLCζ and a higher
Ac + Eq:dispersed localisation ratio
PLCζ levels in spermatozoa quantified using both immunofluores-
cence and immunoblotting were significantly higher in cases where
successful fertilisation was observed compared to cases where fer-
tilisation failure was recorded (6.5a.u vs 4a.u and 1.3a.u vs 0.8a.u,
respectively). Similarly, the Ac + Eq:dispersed localisation ratio was
significantly higher in cases of successful fertilisation (1.4) compared
to cases of fertilisation failure (0.9), suggesting higher proportions
of spermatozoa exhibiting Ac  +  Eq in cases of successful fertilisa-
tion, and lower proportions in cases of fertilisation failure (with the
converse true for the dispersed pattern of localisation; Figure  9).
Analyses indicated no significant differences between quantifica-
tion of PLCζ levels and localisation ratios (Ac  +  Eq/dispersed) and
treatment method deployed (IVF or ICSI), nor was a significant dif-
ference observed between fertilisation success rates of both treat-
ments (Figure S5). We also observed that neither semen parameters
nor female/male age correlated significantly to fertilisation rates
(Table S1).
4 | D I S CU S S I O N
2+
Sperm PLCζ-driven Ca oscillations ensure oocyte activation and
fertilisation, while also determining the efficacy of embryogenesis.
PLCζ is thus regarded therapeutically (in cases of reduced/absent
levels within spermatozoa) and potentially as an informative diagnos-
tic to determine which cases would benefit from such therapeutic
interventions, as opposed to prognostic repeated fertility treatment
failure. However, previous attempts to examine PLCζ profiles in
human spermatozoa in relation to male conditions 24–26,28,38–41 have
|
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
      1151
been limited to only severe cases of fertilisation failure as opposed
to examination of a more general population of spermatozoa from
males attending a fertility clinic.5,36
Herein, we examine levels and localisation patterns of PLCζ in
human spermatozoa using two distinct antibodies in a general pop-
ulation of human patients seeking fertility treatment. We found that
sperm quality correlated to proportions of spermatozoa exhibiting
distinct patterns of PLCζ localisation. These insights have the tre-
mendous potential to dramatically and directly improve assisted
reproductive therapies. While immunological analyses of PLCζ in
human spermatozoa represent a powerful diagnostic approach, sig-
nificant issues remain where PLCζ antibodies used recognise multi-
ple protein bands in addition to PLCζ. The same antibodies have also
been used to identify different immunoblotting profiles by separate
studies, as well as identifying multiple patterns of predominant lo-
calisation patterns.13,14,19,23,30 This is particularly problematic since
most studies attempting to investigate potential links between PLCζ
and particular conditions of male infertility have relied solely upon
immunofluorescent quantification of PLCζ using a single antibody,
avoiding confirmation of specificity or quantification using immuno-
blotting, making it difficult to distinguish between PLCζ and other
proteins. This is particularly concerning for PLCζ localisation pat-
terns, where a dominant motif has yet to be isolated in relation to
sperm health. To this degree, previous attempts to examine PLCζ
24–
profiles in human spermatozoa in relation to male conditions
26,28,39–42
have been viewed with caution due to antibody specificity
and protocol sensitivity.30,43,44
Predictably, such shortcomings have led to conflicting results
between the association of specific PLCζ localisation patterns
and quantification levels.18,20,22,31 This is of particular concern
as some studies have either purported that PLCζ is not the oo-
cyte activation factor using such antibodies or protocols, 45 while
other studies have also suggested there is no link between PLCζ
 |
1152       KASHIR et al.
and sperm parameters, or indeed even ICSI failure. 20 Thus, while
PLCζ exhibits great promise as a clinical prognostic factor, such
applications have previously been severely limited by a lack of
specific antibodies, preventing a methodical analysis of such po-
tential. Perhaps a more recent thorough study was performed by
Rahimizadeh et al,46 who examined potential relationships be-
tween PLCζ levels and localisation patterns with male age, sperm
characteristics, DNA integrity and cellular maturity in spermato-
zoa from men exhibiting either asthenoteratozoospermia, or un-
explained infertility.
However, while a significant relationship was observed between
PLCζ and hyaluronic acid-binding ability, no other relationships
could be observed apart from fertile men exhibiting significantly
higher levels of PLCζ compared to infertile/sub-fertile men. While
a significant start, this study relied on a single commercially avail-
able antibody that has previously exhibited questionable variabil-
ity in specificity against PLCζ (a point specifically mentioned by
the authors of the study themselves who advised caution due to
this fact).46 Furthermore, this and other previous studies did not
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
F I G U R E 7   Immunoblot analysis of
human spermatozoa from different
patients obtained from raw and density
gradient washed (DGW)-selected
sperm fractions, identifying single
immunoreactive bands corresponding
to the expected molecular weight of
human sperm PLCζ (~70kDa) using EF
polyclonal antibody (pAb) (top panel)
and I33 monoclonal antibody (mAb)
(middle panel). Alpha (α)-tubulin was used
as a normalising loading control, and
specifically identified a single band at the
expected molecular weight (~50 kDa).
Rightmost panel indicates Ponceau-
stained membranes to show sufficient
protein separation and membrane transfer
utilise antigen unmasking methodology, which not only enhances
the visualisation efficacy of PLCζ, but also improves the diagnos-
tic capacity of PLCζ in spermatozoa. Herein, due to such concerns
surrounding antibody specificity and consistency, we utilised two
distinct (polyclonal and monoclonal) antibodies against PLCζ,
thoroughly confirming immunoblotting and immunofluorescent
specificity, to systematically quantify levels of PLCζ in spermato-
zoa from a general population of males seeking fertility treatment
using the two distinct methods of immunofluorescence (using anti-
gen retrieval protocols) and immunoblotting. Collectively, we show
that multiple relationships exist not only between levels of PLCζ
and sperm quality, but also between specific patterns of localisa-
tion, which is the first instance that this has been demonstrated.
Importantly, we show such relationships exist in spermatozoa from
a general population of males, and not specifically within a sub-fer-
tile or infertile population as previous studies have targeted, being
the first demonstration that PLCζ potentially represents a bio-
marker of general sperm health, and not just cases of oocyte ac-
tivation failure.
 |

20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
KASHIR et al.       1153

TA B L E 3   Analysis of correlations
Count Male age
between relative density and relative
Quantification Motility (%) (106/mL) Volume (mL) (years)
fluorescence of PLCζ following
immunoblotting and immunofluorescence EF P = .06 P = .42 P = .18 P = .25
of raw semen and DGW-selected Raw spermatozoa
spermatozoa with EF and I33 antibodies, Relative density (a.u)
and semen parameters and male age I33 P = .06 P = .33 P = .26 P = .29
from human patients undergoing fertility Raw spermatozoa
treatment Relative density (a.u)
EF r = .38 P = .2 r = .38 P = .07
DGW-selected spermatozoa (P < .05) (P < .05)
Relative density (a.u)
I33 r = .4 P = .1 r = .4 P = .08
DGW-selected (P < .05) (P < .05)
spermatozoa
Relative density (a.u)
EF P = .06 P = .244 P = .19 P = .4
DGW-selected
spermatozoa
Relative fluorescence
(a.u)

Note: Statistically significant (P ≤ .05) differences are detailed in bold, along with the corresponding
Pearson's correlation coefficient (r; negative values represent a negative correlation, while positive
values indicate a positive correlation).
Abbreviation: a.u, arbitrary units; DGW, density gradient washed.

4.1 | Specific patterns of PLCζ in the head, tail and
midpiece of human spermatozoa
We identified three distinct patterns of PLCζ localisation: equatorial
alone (Eq), acrosomal + equatorial (Ac + Eq) and a uniformly dispersed
pattern of punctate loci throughout the sperm head (dispersed). We also
observed a minority of spermatozoa not exhibiting any pattern of local-
isation (none). These results agree with previous studies reporting sim-
ilar localisation patterns, with additional tail localisation.5,13–15,18,42–44
Contrary to these reports, however, we did not detect the acrosomal
5,13–15,18,42–44
(alone) and post-acrosomal populations. We are confident
to exclude antibody specificity as an explanation for these observed
discrepancies, particularly in light of the localisation patterns that were
diminished following antibody blocking. This was also the case for the
single protein band corresponding to the estimated molecular weight
of human PLCζ (~70kDa) following immunoblotting with both our an-
tibodies. Perhaps the acrosomal only and the post-acrosomal popu-
lations previously reported are features of spermatozoa from severe
cases of OAD, while our population is more representative of a larger
male population which may exhibit a differential profile of sperm PLCζ
compared to severe OAD patients. Regardless, it is imperative that fu-
ture studies systematically investigate this potential disparity within
the particular focus.
PLCζ localisation patterns were not restricted to the sperm
head, but have also been reported in the sperm tail and mid-
piece.5,13–15,18,22,30,42–44 This appears conserved across spe-
cies,47,48 substantiating the potential importance of this localisation.
Indeed, microinjection of equine tails into mouse oocytes resulted
in high-frequency Ca2+ oscillations, suggesting that the tail may
contain functional PLCζ.47 There could be significant functionality
to such populations, as the entirety of the spermatozoa is eventually
incorporated into the oocyte,49 and such tail/midpiece populations
may serve as additional sources of sperm PLCζ to ensure sufficient
requisite levels are delivered to the oocyte for effective oocyte ac-
tivation. However, while our observations provide further support
to assertions regarding the veracity and physiological relevance of
sperm PLCζ populations, the key experiment to confirm this would
be to inject human sperm tails into mouse and/or human oocytes
and examine for patterns of Ca2+, which was beyond the scope of
this study. It is urgent now, considering our current investigations
and observations, that such experiments are performed.
Previous studies have also identified a similar punctate pattern
to ours using the same EF antibody we employed, but without AUM
protocols.34 Since AUM increases the level of observable PLCζ flu-
orescence in spermatozoa,31 perhaps the low visualisation fidelity
observed by previous studies which did not apply AUM protocols
can be linked to such dispersed PLCζ localisation. Previous stud-
18,22,30
ies report significant proportions of spermatozoa exhibiting
a complete absence of PLCζ in the sperm head, suggesting some
44
diagnostic value of a higher proportion of spermatozoa lacking
PLCζ linked to ICSI failure. We propose that this previously reported
proportion of spermatozoa lacking PLCζ in fact represents dispersed
PLCζ, previously undetectable due to a lack of AUM. Indeed, obser-
vation of higher proportions of dispersed PLCζ exhibiting spermato-
zoa corresponded to lower proportions of spermatozoa exhibiting
absent PLCζ. Furthermore, as is subsequently discussed herein, the
dispersed pattern of PLCζ also correlated to low levels of PLCζ in
spermatozoa, alongside poorer sperm quality.
 |

20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1154       KASHIR et al.

of both patterns.50 The most physiologically relevant population,

at least in humans, has always been regarded to be the equatorial
pattern, which would ensure immediate/rapid PLCζ diffusion into
the oocyte at fertilisation.5,6 However, the majority of spermatozoa
we examined exhibited acrosomal  +  equatorial patterns of PLCζ,
with a smaller proportion of spermatozoa exhibiting only the equa-
torial pattern. The occurrence of the equatorial patterns of PLCζ
corresponded to spermatozoa with intact acrosomes,13,14,42 while
Escoffier et al23 confirmed the presence of PLCζ beneath the ac-
rosomal cap in human spermatozoa following electron microscopy.
Furthermore, the DGW-selected fraction of spermatozoa we ex-
amined exhibited a higher proportion of spermatozoa with just the
equatorial pattern compared to the DGW-rejected pattern, suggest-
ing that the acrosome was intact in such spermatozoa since DGW
protocols are regarded to select a higher proportion of spermatozoa
with intact acrosomes compared to DGW-rejected fractions.

4.2 | Acrosomal + equatorial and dispersed patterns

potentially representing diagnostic indicators of
sperm fertility

Density gradient washing is a method to isolate the most motile and

morphologically relevant spermatozoa from a raw ejaculate, whereby a
raw ejaculate is overlaid on top of a discontinuous gradient. The most
motile and healthy spermatozoa are collected in a sperm pellet follow-
ing centrifugation (DGW-selected), representing the most fertile sper-
matozoa. Cellular debris and non-viable sperm cells remain at the top
of the gradient (DGW-rejected), representing the most non-viable cells.
We predominantly observed the Ac + Eq and dispersed patterns
of PLCζ. However, a more detailed analysis indicated that progres-

F I G U R E 8   Histograms depicting relative density levels
observed in non-optimal and optimal values of semen volume
(optimal volume 1.5-5.5 mL). Data are represented as proportions
(%) with standard error of the mean (SEM). Asterisks (*) indicate
statistically significant differences (P ≤ .05). Relative density levels
were recorded following immunoblotting with the i) EF and ii) I33
antibodies. a.u: arbitrary units
We found equatorial PLCζ to be most widespread throughout
spermatozoa, either by itself, or in combination with the acroso-
mal population, providing support to the physiological importance
sion through the various sperm fractions (raw, DGW-rejected, and
DGW-selected) decreased the proportions of spermatozoa exhibit-
ing the dispersed pattern of localisation, while conversely increas-
ing the proportions of spermatozoa exhibiting the Ac + Eq patterns
of PLCζ localisation. As sperm quality improved, the proportion of
spermatozoa exhibiting Ac  +  Eq PLCζ increased, while dispersed
PLCζ decreased, suggesting that Ac + Eq PLCζ is most physiologically
relevant, while the dispersed localisation patterns corresponded to
non-viable spermatozoa, or at least spermatozoa with a decreased
capacity for oocyte activation and fertilisation.

TA B L E 4   Analysis of correlations between the levels of variance in PLCζ following immunofluorescence (relative fluorescence) and
immunoblotting (relative density), and semen parameters, male age, and corresponding relative fluorescence levels of PLCζ

Volume Quantification
Variance Motility (%) Count (106/mL) (mL) Male age (years) (a.u)

Relative fluorescence (a.u) r = −.26 (P < .05) r = −.4 (P < .05) P = .47 P = .3 r = .6 (P < .05)
Relative density (EF); a.u P = .22 P = .22 P = .3 r = .34 (P < .05) r = .32 (P < .05)
Relative density (I33); a.u P = .16 P = .16 P = .49 r = .41 (P < .05) r = .31 (P < .05)

Note: Statistically significant (P ≤ .05) differences are detailed in bold, along with the corresponding Pearson's correlation coefficient (r; negative
values represent a negative correlation, while positive values indicate a positive correlation).
Abbreviation: a.u, arbitrary units.
 |

20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
KASHIR et al.       1155

TA B L E 5   Analysis of correlations
  Equatorial Acrosomal + Equatorial Dispersed None
between the levels of variance in PLCζ
following immunofluorescence (relative Variance in relative P = .014 P = .37 r = .3 (P < .0125) P = .07
fluorescence) using the EF antibody, and fluorescence (EF)
localisation patterns of PLCζ
Note: Statistically significant (P ≤ .0125) differences are detailed in bold, along with the
corresponding Pearson's correlation coefficient (r; positive values indicate a positive correlation).
Abbreviation: a.u, arbitrary units.

F I G U R E 9   Histograms depicting differences in (A) relative fluorescence, (B) relative density and (C) Ac + Eq/dispersed localisation ratio of
sperm PLCζ between cases of fertilisation failure and fertilisation success following fertility treatment. Fertilisation failure represented cases
where 0 oocytes were able to successfully exhibit fertilisation, while fertilisation success represents cases where successful fertilisation
was observed. Fertilisation success was determined by successful extrusion of the second polar body. Data are represented as means with
standard error of the mean (SEM). Asterisks (*) indicate statistically significant differences (P ≤ .05)

Previous studies have associated dispersed/punctate PLCζ to
reduced fertility outcomes.13,14,18,22 Indeed, globozoospermic sper-
matozoa (morphologically abnormal round-headed spermatozoa)
exhibit low success in oocyte activation without clinical interven-
1,51
tion. The majority of such spermatozoa do not contain PLCζ,
but those do predominantly exhibit a similar dispersed pattern that
we currently identify,52 and rarely result in successful fertilisation
without clinical intervention through assisted oocyte activation.
However, when spermatozoon from the same population exhib-
iting a small acrosomal bud was injected into oocytes, pregnancy
without artificial oocyte activation protocols could be achieved.52
Investigation of PLCζ localisation in such spermatozoa indicated an
acrosomal pattern of localisation, while PLCζ exhibited a dispersed
pattern of localisation in round-headed spermatozoa without an
acrosomal bud that resulted in oocyte activation failure.53 Thus,
dispersed PLCζ in the sperm head can be linked to morphological
aspects of spermatozoa, suggesting further physiological relevance
for the acrosomal population of PLCζ. Unfortunately, while we per-
formed morphology analysis on spermatozoa from all patients in this
study, we could not obtain a satisfactorily large range of morphology
scores to be able to perform a reliable analysis, with all patients ex-
hibiting a high morphology score. Perhaps it would be worth in fu-
ture to perform a similar study to this one with morphology being
the sole criteria for patient recruitment.
Dispersed PLCζ exhibited significant negative correlation with
both motility and sperm count, indicating that as sperm quality
improved, the proportions of spermatozoa with dispersed PLCζ
decreased. The converse was true for Ac  +  Eq PLCζ, which exhib-
ited positive correlation with sperm count, indicating that improv-
ing sperm quality led to higher proportions of with Ac  +  Eq PLCζ.
Finally, PLCζ localisation could also be associated with total levels
of PLCζ, with the equatorial and dispersed patterns correlating to
lower levels, and the Ac + Eq pattern correlating to higher levels of
PLCζ. Considering that absent/reduced levels of PLCζ are generally
associated with a reduced fidelity of successful oocyte activation,
the Ac  +  Eq pattern of localisation seems to correlate to healthier
spermatozoa with a greater capacity for inducing oocyte activation.
Optimal sperm motility and count levels exhibited higher pro-
portions of the Ac + Eq PLCζ, while the same parameters exhibited
significantly lower levels of dispersed PLCζ compared to sub-optimal
 |
1156       KASHIR et al.
parameters. Equatorial PLCζ did not correlate with parameter exam-
ined apart from optimal sperm motility. Thus, perhaps sperm health
could be measured by examination of the Ac  +  Eq and dispersed
patterns of localisation, where higher levels of Ac  +  Eq PLCζ and
lower dispersed PLCζ levels correlate to better sperm quality. The
ratio between Ac + Eq:dispersed PLCζ was significantly higher in op-
timal parameter levels (more Ac + Eq compared to dispersed) than
sub-optimal levels which exhibited lower ratios (more dispersed than
Ac + Eq).
4.3 | PLCζ levels correlate with human sperm
parameters only in DGW-selected and not raw
spermatozoa
Our analyses revealed significant positive correlations between PLCζ
levels and sperm motility and semen volume. However, this was only
observed using immunoblotting (with both EF and I33 antibodies)
and not immunofluorescence quantification and was only observ-
able in DGW-selected and not raw spermatozoa. This suggests that
DGW-selected, and not raw, spermatozoa require examination if
quantification of PLCζ is to be performed in a diagnostic capacity.
However, the relationship between PLCζ levels in raw spermatozoa
and spermatozoa motility, as well as between PLCζ fluorescence
levels and spermatozoa motility, seemed to be approaching signifi-
cance, with perhaps larger study populations exhibiting the required
levels of significance.
Examination of PLCζ levels by fluorescence and relative band
density did not exhibit any significant differences between optimal
and sub-optimal levels of sperm motility and count. The same was
true when examining semen volume using relative fluorescence lev-
els, which did not exhibit any significant differences between op-
timal and sub-optimal semen volumes. However, immunoblotting
indicated that PLCζ was significantly higher in spermatozoa from
those patients with semen volumes greater than 5.5ml compared to
those patients with lower semen volumes. However, the WHO has
recently removed the upper limit for semen volume as an optimal pa-
rameter. While the reasons for higher levels of PLCζ in such volumes
are unclear at present, such relationships merit further investigation.
Perhaps a potential explanation for this is that a certain ‘window’ of
PLCζ may be required for effective oocyte activation. Microinjection
of increasing levels of PLCζ in human oocytes resulted in increasing
frequencies and amplitudes of Ca2+ oscillations,54 which are striking
considering that frequency/amplitude of Ca2+ oscillations affects the
efficacy of early mouse embryogenesis.55,56 Precise temporal profiles
2+
of PLCζ-driven Ca oscillations may not only be necessary for oo-
cyte activation, but also equally important for subsequent embryo-
genesis. Thus, levels of sperm PLCζ outside of this specific ‘window’
may underlie not only infertility through fertilisation failure, but also
perhaps cases of male sub-fertility, whereby enough PLCζ may be de-
livered to oocytes to cause activation, but insufficient for embryonic
5
competence. Indeed, either too little, or too much, PLCζ may lead
to impaired embryogenesis despite completion of oocyte activation,
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
suggesting that the high levels of PLCζ in abnormally high semen vol-
umes may indeed represent sub-optimal parameters for fertility.
Low levels of sperm PLCζ were recently correlated to poor sperm
parameters such as motility and overall sperm count,18,22 parameters
which are widely accepted to underlie low ART success. However,
these studies examined populations of infertile, ICSI-failed males,
not examining a range of sperm parameters as we have done in this
study. Thus, we propose that PLCζ levels may only be indicative of
severe cases of OAD such as a complete absence or severe reduction
of PLCζ in the spermatozoa, but not more subtle cases resulting in
sub-fertility. However, to fully ascertain this, a dedicated study is
required examining PLCζ parameters in relation to the efficacy of
embryogenesis and fertility treatment outcomes.
4.4 | PLCζ variance may be prognostic of sperm
health and male age
Kashir et al18 suggested that examination of sperm PLCζ levels may
not be informative due to inherent variability within human sper-
matozoa, an observation repeated by subsequent investigations. 22,30
Although we observed positive correlations between total levels of
PLCζ and sperm parameters, the exact nature of this relationship
remains unclear perhaps due to this variance. Our current results
suggest that as sperm quality improved, PLCζ variability decreases.
However, as levels of PLCζ increased so did variability.
These results agree with assertions that a specific window of
PLCζ is required for optimal oocyte activation and embryogenesis,
with poor sperm parameters or levels of PLCζ outside of this window
exhibiting increased variance in levels of PLCζ, reducing the progno-
sis of successful fertilisation by decreasing the chances of a suitable
amount/profile of PLCζ being delivered to the oocyte. This is further
supported by our findings that the dispersed pattern of localisation
exhibited a significant positive correlation with PLCζ variability, indi-
cating that as levels of this localisation pattern (which we purport to
correspond to decreased capability of oocyte activation) increase, so
do levels of variability, further supporting our assertions that levels
of PLCζ variability may be prognostic of fertilisation outcome.
Increasing male age also exhibited positive correlation with
PLCζ variability as determined by relative density. Recently, Yeste
et al 30 indicated that increasing age in human males did not exert
any effect upon sperm PLCζ, a finding which we also observed,
but this time in a more general male population as opposed to
fertile vs infertile individuals. Such observations are in line with
our assertions that increasing variability in levels of PLCζ may be
indicative of a poor prognosis for oocyte activation and fertilisa-
tion, considering that our results are suggestive of variability in-
creasing in cases exhibiting poorer sperm parameters. Our results
also agree with studies indicating that older males exhibit poorer
sperm parameters and reproductive outcomes, particularly post-
40 years of age. 57–59 Thus, perhaps variability in levels of PLCζ can
be attributed to increasing amounts of dead or non-viable sper-
matozoa within such samples, or perhaps may even be indicative

KASHIR et al.
of sub-optimal spermatogenesis. However, further focused inves-
tigations are required to substantiate such assertions, while also
examining males from a larger age range.
4.5 | Higher PLCζ levels and specific localisation
patterns correspond to successful fertilisation
in a general population of patients seeking
fertility treatment
Successful fertilisation was associated with higher levels of PLCζ,
as well as higher levels of Ac + Eq PLCζ, and lower proportions of
spermatozoa exhibiting dispersed PLCζ. These observations are
strikingly similar to what we observed in relation to PLCζ levels and
localisation patterns in relation to sperm parameters and DGW frac-
tions, suggesting that perhaps levels and specific localisation pat-
terns of PLCζ could be used to predict fertilisation success, or at
least the chances of such success.
While such trends have been observed in the past (lower levels
and abnormal patterns of PLCζ localisation) in cases of repeated ICSI
failure 1,13–18,20,21 to the best of our knowledge this is the first report
describing a similar trend for a wider range of patients attending the
fertility clinic, regardless of fertility treatment utilised. Such obser-
vations suggest that a specific level of PLCζ is required to initiate
successful oocyte activation leading to completion of fertilisation,
while also support our previous assertions that the Ac + Eq pattern
of localisation corresponds to better quality spermatozoa and higher
levels of PLCζ, while the dispersed pattern correlates to lower qual-
ity spermatozoa, alongside lower levels of PLCζ. We would assert to
this degree that such PLCζ parameters could be utilised as a diag-
nostic measure to indicate potential fertilisation outcome in general
cases of fertility treatment.
However, while our cohort of 65 patients is one of the largest thus
far for studies relating to PLCζ, we were only able to examine fertilisa-
tion success in a relatively binary way, that is success or failure. As pre-
viously discussed, the amount of PLCζ delivered to the oocyte could
determine the rate of cell cycle progression, by altering the frequency/
2+ 55,56
amplitude of Ca oscillations. Thus, perhaps varying levels of
PLCζ could also in turn lead to varying degrees of fertilisation success
in terms of proportions of oocytes successfully fertilising within each
case. It is thus necessary that future studies attempt to examine such
questions within a larger and more diverse patient cohort before more
confident conclusions can be made alongside clinical application.
Collectively, the current study represents the first systematic ef-
fort to associate profiles of the oocyte activation factor PLCζ, within
a general population of sperm parameters in a fertility clinic. We
show that utilisation of two distinct antibodies identified specific
populations of PLCζ within the sperm head at the Eq and Ac  +  Eq
regions. We also identified that the dispersed pattern of PLCζ, along
with Ac  +  Eq populations, was most prevalent. Our analyses indi-
cated that the Ac  +  Eq PLCζ was most physiologically relevant to
healthy sperm parameters and fertilisation success, while dispersed
PLCζ seemed correlated to spermatozoa with a reduced capacity for
|
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
      1157
fertilisation. Finally, we also show for the first time that significantly
higher levels of PLCζ correlated to fertilisation success in general
cases of patients seeking fertility treatment, and not just cases of re-
peated fertilisation failure, indicating that perhaps PLCζ represents
a biomarker of sperm health and fertilisation capacity.
Indeed, proportions of spermatozoa exhibiting the Ac  +  Eq
pattern of PLCζ increased in DGW-selected fractions, while pro-
portions of spermatozoa exhibiting the dispersed pattern of PLCζ
decreased. We further indicate that variance in levels of sperm PLCζ
corresponded to reduced sperm health and may perhaps underlie
cases of increased fertilisation failure associated with cases of male
sub-fertility, and perhaps increasing male age. We posit that our re-
sults greatly stand to benefit the clinical utilisation of PLCζ as a ther-
apeutic and diagnostic in general cases of fertility treatment, and
not just cases of ICSI failure. However, the current study examined
a relatively small population (65 males), which may prevent robust
conclusions from being formulated, despite significant relationships
already being observed. It is now essential that similar studies are
employed to enable a multi-centre analysis of a significantly larger
population of patients to ensure that further potential relationships
between PLCζ and general sperm parameters and fertility outcomes
are fully evaluated robustly.
AC K N OW L E D G E M E N T S
Some of the work described herein was performed by LB and R
AbuDawas as part of their graduate research projects. The authors
thank Dr Evangelia Livaniou and Dr Vyronia Vassilakopoulou of the
National Center for Scientific Research ‘Demokritos' (Athens, Greece)
for their assistance in generating the EF polyclonal antibody against
PLCζ, Dr Peter MB Cahusac of Alfaisal University (Riyadh, Kingdom
of Saudi Arabia) for invaluable discussion and assistance regarding
statistical analyses, and Mr Faisal Alotaibi and the Research Centre
Administration at the KFSHRC (Riyadh Kingdom of Saudi Arabia) for
their invaluable efforts in assisting with experimental logistics.
C O N FL I C T O F I N T E R E S T
All authors have no conflict(s) of interest to declare.
AU T H O R C O N T R I B U T I O N S
JK, FAL, AMA and SC were responsible for study and experimen-
tal design, and overall scope of the study. JK, BVM, MN, LB and R
AbuDawas performed the majority of experimental procedures,
with further contributions by R Abu-Dawud and NA. JK wrote the
manuscript with input from all authors, and all authors approved the
final manuscript.
ORCID
Junaid Kashir  https://orcid.org/0000-0002-5056-8222
REFERENCES
1. Kashir J, Heindryckx B, Jones C, De Sutter P, Parrington J, Coward
K. Oocyte activation, phospholipase C zeta and human infertility.
Hum Reprod Update. 2010;16:690-703.
 |
1158       KASHIR et al.
2. Harton G, Tempest H. Chromosomal disorders and male infertility.
Asian J Androl. 2012;14:32-39.
3. Jungwirth A, Giwercman A, Tournaye H, et al. European Association
of urology guidelines on male infertility: the 2012 update. Eur Urol.
2012;62:324-332.
4. Hotaling J. Genetics of male infertility. Urol Clin North Am.
2014;41:1-17.
5. Kashir J, Nomikos M, Lai F. Phospholipase C zeta and calcium oscil-
lations at fertilisation: the evidence, applications, and further ques-
tions. Adv Biol Regul. 2018;67:148-162.
6. Cox L, Larman M, Saunders C, Hashimoto K, Swann K, Lai F. Sperm
phospholipase Czeta from humans and cynomolgus monkeys trig-
gers Ca2+ oscillations, activation and development of mouse oo-
cytes. Reproduction. 2002;124: 611–623.
7. Fujimoto S, Yoshida N, Fukui T, et al. Mammalian phospholipase Cζ
induces oocyte activation from the sperm perinuclear matrix. Dev
Biol. 2004;274:370-383.
8. Kurokawa M, Sato K-I, Wu H, et al. Functional, biochemical, and
chromatographic characterization of the complete [Ca2+]i oscilla-
tion-inducing activity of porcine sperm. Dev Biol. 2005;285:376-392.
9. Kouchi Z, Fukami K, Shikano T, et al. Recombinant phospholipase
Cζ has high Ca2+Sensitivity and induces Ca2+Oscillations in mouse
eggs. J Biol Chem. 2004;279:10408-10412.
10. Knott J, Kurokawa M, Fissore R, Schultz R, Williams C. Transgenic
RNA interference reveals role for mouse sperm phospholipase Cζ
in triggering Ca2+ oscillations during Fertilization1. Biol Reprod.
2005;72:992-996.
11. Hachem A, Godwin J, Ruas M, et al. PLCζ is the physiological
trigger of the Ca2+oscillations that induce embryogenesis in
mammals but conception can occur in its absence. Development.
2017;144:2914-2924.
12. Nozawa K, Satouh Y, Fujimoto T, Oji A, Ikawa M. Sperm-borne
phospholipase C zeta-1 ensures monospermic fertilization in mice.
Sci Rep. 2018;8:1–10.
13. Yoon S-Y, Jellerette T, Salicioni AM, et al. Human sperm de-
void of PLC, zeta 1 fail to induce Ca2+ release and are unable
to initiate the first step of embryo development. J Clin Invest.
2008;118:3671-3681.
14. Heytens E, Parrington J, Coward K, et al. Reduced amounts and ab-
normal forms of phospholipase C zeta (PLC ) in spermatozoa from
infertile men. Hum Reprod. 2009;24:2417-2428.
15. Kashir J, Jones C, Lee HC, et al. Loss of activity mutations in
phospholipase C zeta (PLC ) abolishes calcium oscillatory ability
of human recombinant protein in mouse oocytes. Hum Reprod.
2011;26:3372-3387.
16. Kashir J, Konstantinidis M, Jones C, et al. A maternally inherited au-
tosomal point mutation in human phospholipase C zeta (PLC ) leads
to male infertility. Hum Reprod. 2012;27:222-231.
17. Kashir J, Konstantinidis M, Jones C, et al. Characterization of two
heterozygous mutations of the oocyte activation factor phospho-
lipase C zeta (PLCζ) from an infertile man by use of minisequenc-
ing of individual sperm and expression in somatic cells. Fertil Steril.
2012;98:423-431.
18. Turner K, Child T, McVeigh E, et al. Variance in total levels of phos-
pholipase C zeta (PLC-ζ) in human sperm may limit the applicability
of quantitative immunofluorescent analysis as a diagnostic indica-
tor of oocyte activation capability. Fertil Steril. 2013;99:107-117.e3.
19. Escoffier J, Lee HC, Yassine S, et al. Homozygous mutation of
PLCZ1 leads to defective human oocyte activation and infertility
that is not rescued by the WW-binding protein PAWP. Hum Mol
Genet. 2016;25:878-891.
20. Ferrer-Vaquer A, Barragan M, Freour T, Vernaeve V, Vassena R.
PLCζ sequence, protein levels, and distribution in human sperm do
not correlate with semen characteristics and fertilization rates after
ICSI. J Assist Reprod Genet. 2016;33:747-756.
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
21. Torra-Massana M, Cornet-Bartolomé D, Barragán M, et al. Novel
phospholipase C zeta 1 mutations associated with fertilization fail-
ures after ICSI. Hum Reprod. 2019;34:1494-1504.
22. Yelumalai S, Yeste M, Jones C, et al. Total levels, localization pat-
terns, and proportions of sperm exhibiting phospholipase C zeta are
significantly correlated with fertilization rates after intracytoplas-
mic sperm injection. Fertil Steril. 2015;104:561-568.e4.
23. Escoffier J, Yassine S, Lee HC, et al. Subcellular localization of phos-
pholipase Cζ in human sperm and its absence in DPY19L2-deficient
sperm are consistent with its role in oocyte activation. Mol Hum
Reprod. 2015;21:157-168.
24. Azad N, Nazarian H, Ghaffari Novin M, et al. Phospholipase C zeta
parameters in sperm from polymorphic teratozoospermic men. Ann
Anat. 2018;215:63-70.
25. Azad N, Nazarian H, Ghaffari Novin M, et al.
Oligoasthenoteratozoospermic (OAT) men display altered phospho-
lipase C ζ (PLCζ) localization and a lower percentage of sperm cells
expressing PLCζ and post-acrosomal sheath WW domain-binding
protein (PAWP). Bosn J Basic Med Sci. 2018;18:178–184.
26. Tavalaee M, Nomikos M, Lai F, Nasr-Esfahani M. Expression of
sperm PLCζ and clinical outcomes of ICSI-AOA in men affected by
globozoospermia due to DPY19L2 deletion. Reprod BioMed Online.
2018;36:348-355.
27. Park JH, Kim SK, Kim J, et al. Relationship between phospholipase
C zeta immunoreactivity and DNA fragmentation and oxidation in
human sperm. Obst Gynecol Sci. 2015;58:232.
28. Tavalaee M, Kiani-Esfahani A, Nasr-Esfahani M. Relationship be-
tween phospholipase C-zeta, semen parameters, and chromatin
status. Syst Biol Reprod Med. 2017;63:259-268.
29. Nikiforaki D, Vanden Meerschaut F, De Gheselle S, et al. Sperm in-
volved in recurrent partial hydatidiform moles cannot induce the
normal pattern of calcium oscillations. Fertil Steril. 2014;102:581-
588.e1.
3 0. Yeste M, Jones C, Amdani SN, et al. Does advancing male age influ-
ence the expression levels and localisation patterns of phospholi-
pase C zeta (PLCζ) in human sperm? Sci Rep. 2016;6:27543.
31. Kashir J, Buntwal L, Nomikos M, et al. Antigen unmasking enhances
visualization efficacy of the oocyte activation factor, phospholipase
C zeta, in mammalian sperm. Mol Hum Reprod. 2017;23:54-67.
32. WHO. WHO laboratory manual for the examination and processing
of human semen. World Health Organization (2019). https://www.
who.int/repro​ducti​vehea​lth/publi​c atio​ns/infer​t ilit​y/97892​41547​
789/en/
33. Nomikos M, Sanders JR, Parthimos D, et al. Essential role of the
EF-hand domain in targeting sperm phospholipase Cζ to mem-
brane phosphatidylinositol 4,5-Bisphosphate (PIP2). J Biol Chem.
2015;290:29519-29530.
3 4. Nomikos M, Yu Y, Elgmati K, et al. Phospholipase Cζ rescues failed
oocyte activation in a prototype of male factor infertility. Fertil
Steril. 2013;99:76-85.
35. Nomikos M, Theodoridou M, Elgmati K, et al. Human PLC exhib-
its superior fertilization potency over mouse PLC in triggering the
Ca2+ oscillations required for mammalian oocyte activation. Mol
Hum Reprod. 2014;20:489-498.
36. Nomikos M, Stamatiadis P, Sanders JR, et al. Male infertility-linked
point mutation reveals a vital binding role for the C2 domain of
sperm PLCζ. Biochem J. 2017;474:1003-1016.
37. Benjamini Y, Krieger A, Yekutieli D. Adaptive linear step-up
procedures that control the false discovery rate. Biometrika.
2006;93:491-507.
38. Kamali-Dolat Abadi M, Tavalaee M, Shahverdi A, Nasr-Esfahani
MH. Evaluation of PLCζ and PAWP expression in globozoospermic
individuals. Cell J. 2016;18:438-445.
39. Janghorban-Laricheh E, Ghazavi-Khorasgani N, Tavalaee
M, Zohrabi D, Abbasi H, Nasr- Esfahani MH. An association

KASHIR et al.
between sperm PLCζ levels and varicocele? J Assist Reprod Genet.
2016;33:1649-1655.
4 0. Nazarian H, Azad N, Nazari L, et al. Effect of artificial oocyte activa-
tion on intra-cytoplasmic sperm injection outcomes in patients with
lower percentage of sperm containing phospholipase Cζ: a random-
ized clinical trial. J Reprod Infert. 2019;20:3-9.
41. Khakpour S, Sadeghi E, Tavalaee M, Bahadorani M, Nasr-Esfahani
M. Zeta method: a noninvasive method based on membrane charge
for selecting spermatozoa expressing high level of phospholipa-
seCζ. Andrologia. 2019;51:e13249.
42. Grasa P, Coward K, Young C, Parrington J. The pattern of localiza-
tion of the putative oocyte activation factor, phospholipase C, in
uncapacitated, capacitated, and ionophore-treated human sperma-
tozoa. Hum Reprod. 2008;23:2513-2522.
43. Kashir J, Heynen A, Jones C, et al. Effects of cryopreservation and
density-gradient washing on phospholipase C zeta concentrations
in human spermatozoa. Reprod BioMed Online. 2011;23:263-267.
4 4. Kashir J, Nomikos M, Lai F, Swann K. Sperm-induced Ca2+ release
during egg activation in mammals. Biochem Biophys Res Comm.
2014;450:1204-1211.
45. Aarabi M, Yu Y, Xu W, et al. The testicular and epididymal expres-
sion profile of PLCζ in mouse and human does not support its role as
a sperm-borne oocyte activating factor. PLoS ONE. 2012;7:e33496.
46. Rahimizadeh P, Topraggaleh TR, Nasr-Esfahani MH, et al. The al-
teration of PLCζ protein expression in unexplained infertile and
asthenoteratozoospermic patients: a potential effect on sperm fer-
tilization ability. Mol Reprod Dev. 2020;87:115-123.
47. Bedford-Guaus SJ, McPartlin LA, Xie J, et al. Molecular cloning and
characterization of phospholipase C zeta in equine sperm and testis
reveals species-specific differences in expression of catalytically
active protein1. Biol Reprod. 2011;85:78-88.
48. Nakai M, Ito J, Sato K-I, et al. Pre-treatment of sperm reduces suc-
cess of ICSI in the pig. Reproduction. 2011;142:285-293.
49. Ramalho-Santos J. A sperm's tail: the importance of getting it right.
Hum Reprod. 2011;26:2590-2591.
50. Bi Y, Xu W-M, Wong HY, et al. NYD-SP27, a novel intrinsic decapac-
itation factor in sperm. Asian J Androl. 2009;11:229-239.
51. Taylor S, Yoon S, Morshedi M, et al. Complete globozoospermia as-
sociated with PLCζ deficiency treated with calcium ionophore and
ICSI results in pregnancy. Reprod BioMed Online. 2010;20:559-564.
|
20472927, 2020, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12796 by Cochrane Saudi Arabia, Wiley Online Library on [07/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
      1159
52. Sermondade N, Hafhouf E, Dupont C, et al. Successful childbirth
after intracytoplasmic morphologically selected sperm injection
without assisted oocyte activation in a patient with globozoosper-
mia. Hum Reprod. 2011;26:2944-2949.
53. Kashir J, Sermondade N, Sifer C, et al. Motile sperm organelle mor-
phology evaluation-selected globozoospermic human sperm with
an acrosomal bud exhibits novel patterns and higher levels of phos-
pholipase C zeta. Hum Reprod. 2012;27:3150-3160.
54. Yamaguchi T, Ito M, Kuroda K, Takeda S, Tanaka A. The establish-
ment of appropriate methods for egg-activation by human PLCZ1
RNA injection into human oocyte. Cell Calcium. 2017;65:22-30.
55. Ducibella T, Huneau D, Angelichio E, et al. Egg-to-embryo transition
is driven by differential responses to Ca2+ oscillation number. Dev
Biol. 2002;250:280-291.
56. Miyazaki S, Ito M. Calcium signals for egg activation in mammals. J
Pharmacol Sci. 2006;100:545-552.
57. Harris ID, Fronczak C, Roth L, Meacham RB. Fertility and the aging
male. Rev Urol. 2011;13:e184-e190.
58. Stone B, Alex A, Werlin L, Marrs R. Age thresholds for changes in
semen parameters in men. Fertil Steril. 2013;100:952-958.
59. Gallo M, Licata E, Meneghini C, et al. Impact of paternal age on
seminal parameters and reproductive outcome of intracytoplas-
matic sperm injection in infertile Italian women. Front Endocrinol.
2019;10:35.
S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Kashir J, Mistry BV, BuSaleh L, et al.
Phospholipase C zeta profiles are indicative of optimal sperm
parameters and fertilisation success in patients undergoing
fertility treatment. Andrology. 2020;8:1143–1159. https://doi.
org/10.1111/andr.12796