The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

STUDY QUESTION: Are sperm phospholipase C zeta (PLCζ) profiles linked to the quality of embryogenesis and pregnancy? SUMMARY ANSWER: Sperm PLCζ levels in both mouse and humans correlate with measures of ideal embryogenesis whereby minimal levels seem to be required to result in successful pregnancy. WHAT IS KNOWN ALREADY: While causative factors underlying male infertility are multivariable, cases are increasingly associated with the efficacy of oocyte activation, which in mammals occurs in response to specific profiles of calcium (Ca2+) oscillations driven by sperm-specific PLCζ. Although sperm PLCζ abrogation is extensively linked with human male infertility where oocyte activation is deficient, less is clear as to whether sperm PLCζ levels or localization underlies cases of defective embryogenesis and failed pregnancy following fertility treatment. STUDY DESIGN, SIZE, DURATION: A cohort of 54 couples undergoing fertility treatment were recruited at the assisted reproductive technology laboratory at the King Faisal Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia. The recruitment criteria for males was a minimum sperm concentration of 5×106 sperm/ml, while all female patients had to have at least five oocytes. Sperm PLCζ analysis was performed in research laboratories, while semen assessments were performed, and time-lapse morphokinetic data were obtained, in the fertility clinic as part of routine treatment. The CRISPR/Cas9 system was concurrently used to induce indels and single-nucleotide mutations within the Plcζ gene to generate strains of Plcζ mutant mice. Sperm PLCζ was evaluated using immunofluorescence and immunoblotting with an antibody of confirmed consistent specificity against PLCζ. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated PLCζ profiles in sperm samples from 54 human couples undergoing fertility treatment in the context of time-lapse morphokinetic analysis of resultant embryos, correlating such profiles to pregnancy status. Concurrently, we generated two strains of mutant Plcζ mice using CRISPR/Cas9, and performed IVF with wild type (WT) oocytes and using WT or mutant Plcζ sperm to generate embryos. We also assessed PLCζ status in WT and mutant mice sperm in the context of time-lapse morphokinetic analysis and breeding outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: A significant (P ≤ 0.05) positive relationship was observed between both PLCζ relative fluorescence and relative density with the times taken for both the second cell division (CC2) (r = 0.26 and r = 0.43, respectively) and the third cell division (S2) (r = 0.26). Examination of localization patterns also indicated significant correlations between the presence or absence of sperm PLCζ and CC2 (r = 0.27 and r = -0.27, respectively; P ≤ 0.025). Human sperm PLCζ levels were at their highest in the ideal times of CC2 (8-12 h) compared to time ranges outside the ideal timeframe (<8 and >12 h) where levels of human sperm PLCζ were lower. Following assignment of PLCζ level thresholds, quantification revealed a significantly higher (P ≤ 0.05) rate of successful pregnancy in values larger than the assigned cut-off for both relative fluorescence (19% vs 40%, respectively) and relative density (8% vs 54%, respectively). Immunoblotting indicated a single band for PLCζ at 74 kDa in sperm from WT mice, while a single band was also observed in sperm from heterozygous of Plcζ mutant mouse sperm, but at a diminished intensity. Immunofluorescent analysis indicated the previously reported (Kashir et al., 2021) fluorescence patterns in WT sperm, while sperm from Plcζ mutant mice exhibited a significantly diminished and dispersed pattern at the acrosomal region of the sperm head. Breeding experiments indicated a significantly reduced litter size of mutant Plcζ male mice compared to WT mice, while IVF-generated embryos using sperm from mutant Plcζ mice exhibited high rates of polyspermy, and resulted in significantly reduced numbers of these embryos reaching developmental milestones. LIMITATIONS, REASONS FOR CAUTION: The human population examined was relatively small, and should be expanded to examine a larger multi-centre cohort. Infertility conditions are often multivariable, and it was not possible to evaluate all these in human patients. However, our mutant Plcζ mouse experiments do suggest that PLCζ plays a significant role in early embryo development. WIDER IMPLICATIONS OF THE FINDINGS: We found that minimal levels of PLCζ within a specific range were required for optimal early embryogenesis, correlating with increased pregnancy. Levels of sperm PLCζ below specific thresholds were associated with ineffective embryogenesis and lower pregnancy rates, despite eliciting successful fertilization in both mice and humans. To our knowledge, this represents the first time that PLCζ levels in sperm have been correlated to prognostic measures of embryogenic efficacy and pregnancy rates in humans. Our data suggest for the first time that the clinical utilization of PLCζ may stand to benefit not just a specific population of male infertility where oocyte activation is completely deficient (wherein PLCζ is completely defective/abrogated), but also perhaps the larger population of couples seeking fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): J.K. is supported by a faculty start up grant awarded by Khalifa University (FSU-2023-015). This study was also supported by a Healthcare Research Fellowship Award (HF-14-16) from Health and Care Research Wales (HCRW) to J.K., alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST) for J.K. and A.M.A. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Antigen Unmasking Is Required to Clinically Assess Levels and Localisation Patterns of Phospholipase C Zeta in Human Sperm.

Mammalian oocyte activation is initiated by intracellular calcium (Ca2+) oscillations, driven by the testis-specific phospholipase C zeta (PLCζ). Sperm PLCζ analysis represents a diagnostic measure of sperm fertilisation capacity. The application of antigen unmasking/retrieval (AUM) generally enhanced the visualisation efficacy of PLCζ in mammalian sperm, but differentially affected the PLCζ profiles in sperm from different human males. It is unclear whether AUM affects the diagnosis of PLCζ in human sperm. Herein, we examined whether the application of AUM affected the correlation of PLCζ profiles with sperm parameters and fertilisation capacity. PLCζ fluorescence levels and localisation patterns were examined within the sperm of males undergoing fertility treatment (55 patients aged 29-53) using immunofluorescence in the absence/presence of AUM. The changes in PLCζ profiles following AUM were examined in relation to sperm health and fertilisation outcome. AUM enhanced the observable levels and specific localisation patterns of PLCζ in relation to both optimal sperm parameters and fertilisation outcome, without which significant differences were not observed. The extent of the change in levels and localisation ratios of PLCζ was also affected to a larger degree in terms of the optimal parameters of sperm fertility and fertilisation capacity by AUM. Collectively, AUM was essential to accurately assesses PLCζ in human sperm in both scientific and clinical contexts.

Disruption of male fertility-critical Dcaf17 dysregulates mouse testis transcriptome.

During mammalian spermatogenesis, the ubiquitin proteasome system maintains protein homoeostasis (proteastasis) and spermatogenic cellular functions. DCAF17 is a substrate receptor in the ubiquitin CRL4 E3 Ligase complex, absence of which causes oligoasthenoteratozoospermia in mice resulting in male infertility. To determine the molecular phenomenon underlying the infertility phenotype caused by disrupting Dcaf17, we performed RNA-sequencing-based gene expression profiling of 3-weeks and 8-weeks old Dcaf17 wild type and Dcaf17 disrupted mutant mice testes. At three weeks, 44% and 56% differentially expressed genes (DEGs) were up- and down-regulated, respectively, with 32% and 68% DEGs were up- and down-regulated, respectively at 8 weeks. DEGs include protein coding genes and lncRNAs distributed across all autosomes and the X chromosome. Gene ontology analysis revealed major biological processes including proteolysis, regulation of transcription and chromatin remodelling are affected due to Dcaf17 disruption. We found that Dcaf17 disruption up-regulated several somatic genes, while germline-associated genes were down-regulated. Up to 10% of upregulated, and 12% of downregulated, genes were implicated in male reproductive phenotypes. Moreover, a large proportion of the up-regulated genes were highly expressed in spermatogonia and spermatocytes, while the majority of downregulated genes were predominantly expressed in round spermatids. Collectively, these data show that the Dcaf17 disruption affects directly or indirectly testicular proteastasis and transcriptional signature in mouse.

Dynamic cytosolic foci of DPPA4 in human pluripotent stem cells.

DPPA4 is essential for the pluripotent stem cell state, yet its function is poorly understood. DPPA4 is localized in the nucleus, where it is associated with active chromatin. We now report that it is also present in the cytosol, where it appears as diffused clouds, distinct foci and sometimes as spaghetti-like structures. This cytosolic localization is dynamic and DPPA4 shuttles between the cytosol and the nucleus. Its presence is almost abolished from the nucleus upon differentiation. Co-immunoprecipitation studies highlighted novel protein interactors, many of which are also found in the cytosol and are implicated in mRNA processing and RNA and protein transport between the cytosol and the nucleus. Finally, the depletion of DPPA4 resulted in cytosolic accumulation of vesicles. The cytosolic presence of DPPA4 highlights unexplored research directions that could significantly advance the understanding of DPPA4 in pluripotent stem cells and in cancer.

Subcellular localization of the mouse PRAMEL1 and PRAMEX1 reveals multifaceted roles in the nucleus and cytoplasm of germ cells during spermatogenesis.

BACKGROUND: Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal gametogenic tissues and a variety of tumors. Members of the PRAME gene family encode leucine-rich repeat (LRR) proteins that provide a versatile structural framework for the formation of protein-protein interactions. As a nuclear receptor transcriptional regulator, PRAME has been extensively studied in cancer biology and is believed to play a role in cancer cell proliferation by suppressing retinoic acid (RA) signaling. The role of the PRAME gene family in germline development and spermatogenesis has been recently confirmed by a gene knockout approach. To further understand how PRAME proteins are involved in germ cell development at a subcellular level, we have conducted a systematic immunogold electron microscopy (IEM) analysis on testis sections of adult mice with gene-specific antibodies from two members of the mouse Prame gene family: Pramel1 and Pramex1. Pramel1 is autosomal, while Pramex1 is X-linked, both genes are exclusively expressed in the testis. RESULTS: Our IEM data revealed that both PRAMEL1 and PRAMEX1 proteins were localized in various cell organelles in different development stages of spermatogenic cells, including the nucleus, rER, Golgi, mitochondria, germ granules [intermitochondrial cement (IMC) and chromatoid body (CB)], centrioles, manchette, and flagellum. Unlike other germ cell-specific makers, such as DDX4, whose proteins are evenly distributed in the expressed-organelle(s), both PRAMEL1 and PRAMEX1 proteins tend to aggregate together to form clusters of protein complexes. These complexes were highly enriched in the nucleus and cytoplasm (especially in germ granules) of spermatocytes and spermatids. Furthermore, dynamic distribution of the PRAMEL1 protein complexes were observed in the microtubule-based organelles, such as acroplaxome, manchette, and flagellum, as well as in the nuclear envelope and nuclear pore. Dual staining with PRAMEL1 and KIF17B antibodies further revealed that the PRAMEL1 and KIF17B proteins were co-localized in germ granules. CONCLUSION: Our IEM data suggest that the PRAMEL1 and PRAMEX1 proteins are not only involved in transcriptional regulation in the nucleus, but may also participate in nucleocytoplasmic transport, and in the formation and function of germ cell-specific organelles during spermatogenesis.

Advancing male age differentially alters levels and localization patterns of PLCzeta in sperm and testes from different mouse strains.

Sperm-specific phospholipase C zeta (PLCζ) initiates intracellular calcium (Ca2+) transients which drive a series of concurrent events collectively termed oocyte activation. Numerous investigations have linked abrogation and absence/reduction of PLCζ with forms of male infertility in humans where oocyte activation fails. However, very few studies have examined potential relationships between PLCζ and advancing male age, both of which are increasingly considered to be major effectors of male fertility. Initial efforts in humans may be hindered by inherent PLCζ variability within the human population, alongside a lack of sufficient controllable repeats. Herein, utilizing immunoblotting, immunofluorescence, and quantitative reverse transcription PCR (qRT-PCR) we examined for the first time PLCζ protein levels and localization patterns in sperm, and PLCζ mRNA levels within testes, from mice at 8 weeks, 12 weeks, 24 weeks, and 36 weeks of age, from two separate strains of mice, C57BL/6 (B6; inbred) and CD1 (outbred). Collectively, advancing male age generally diminished levels and variability of PLCζ protein and mRNA in sperm and testes, respectively, when both strains were examined. Furthermore, advancing male age altered the predominant pattern of PLCζ localization in mouse sperm, with younger mice exhibiting predominantly post-acrosomal, and older mice exhibiting both post-acrosomal and acrosomal populations of PLCζ. However, the specific pattern of such decline in levels of protein and mRNA was strain-specific. Collectively, our results demonstrate a negative relationship between advancing male age and PLCζ levels and localization patterns, indicating that aging male mice from different strains may serve as useful models to investigate PLCζ in cases of male infertility and subfertility in humans.

Expression profiling of WD40 family genes including DDB1- and CUL4- associated factor (DCAF) genes in mice and human suggests important regulatory roles in testicular development and spermatogenesis.

BACKGROUND: The WD40-repeat containing proteins, including DDB1-CUL4-associated factors (DCAFs), are abundant and conserved proteins that play important roles in different cellular processes including spermatogenesis. DCAFs are subset of WD40 family proteins that contain WDxR motif and have been proposed to function as substrate receptor for Cullin4-RING-based E3 ubiquitin ligase complexes to recruit diverse proteins for ubiquitination, a vital process in spermatogenesis. Large number of WD40 genes has been identified in different species including mouse and human. However, a systematic expression profiling of WD40 genes in different tissues of mouse and human has not been investigated. We hypothesize that large number of WD40 genes may express highly or specifically in the testis, where their expression is uniquely regulated during testis development and spermatogenesis. Therefore, the objective of this study is to mine and characterize expression patterns of WD40 genes in different tissues of mouse and human with particular emphasis on DCAF genes expressions during mouse testicular development. RESULTS: Publically available RNA sequencing (RNA seq) data mining identified 347 and 349 WD40 genes in mouse and human, respectively. Hierarchical clustering and heat map analyses of RNA seq datasets revealed differential expression patterns of WD40 genes with around 60-73% of the genes were highly or specifically expressed in testis. Similarly, around 74-83% of DCAF genes were predominantly or specifically expressed in testis. Moreover, WD40 genes showed distinct expression patterns during embryonic and postnatal testis development in mice. Finally, different germ cell populations of testis showed specific patterns of WD40 genes expression. Predicted gene ontology analyses revealed more than 80% of these proteins are implicated in cellular, metabolic, biological regulation and cell localization processes. CONCLUSIONS: We have identified large number of WD40 family genes that are highly or specifically expressed in the testes of mouse and human. Moreover, WD40 genes have distinct expression patterns during embryonic and postnatal development of the testis in mice. Further, different germ cell populations within the testis showed specific patterns of WD40 genes expression. These results provide foundation for further research towards understanding the functional genomics and molecular mechanisms of mammalian testis development and spermatogenesis.

Phospholipase C zeta profiles are indicative of optimal sperm parameters and fertilisation success in patients undergoing fertility treatment.

BACKGROUND: Oocyte activation is driven by intracellular calcium (Ca2+ ) oscillations induced by sperm-specific PLCζ, abrogation of which causes oocyte activation deficiency in humans. Clinical PLCζ investigations have been limited to severe male infertility conditions, while PLCζ levels and localisation patterns have yet to be associated with general sperm viability. MATERIALS AND METHODS: PLCζ profiles were examined within a general population of males attending a fertility clinic (65 patients; aged 29-53), examining PLCζ throughout various fractions of sperm viability. 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. Sperm count, motility and semen volume were recorded according to standard WHO reference guidelines and correlated with PLCζ profiles examined via immunoblotting and immunofluorescence. Appropriate fertility treatments were performed following routine clinical standard operating protocols, and fertilisation success determined by successful observation of second polar body extrusion. RESULTS AND DISCUSSION: Four distinct PLCζ patterns were observed at the equatorial, acrosomal + equatorial regions of the sperm head, alongside a dispersed pattern, and a population of spermatozoa without any PLCζ. Acrosomal + equatorial PLCζ correlated most to sperm health, while dispersed PLCζ correlated to decreased sperm viability. Total levels of PLCζ exhibited significant correlations with sperm parameters. PLCζ variance corresponded to reduced sperm health, potentially underlying cases of male sub-fertility and increasing male age. Finally, significantly higher levels of PLCζ were exhibited by cases of fertilisation success, alongside higher proportions of Ac + Eq, and lower levels of dispersed PLCζ. CONCLUSIONS: PLCζ potentially represents a biomarker of sperm health, and fertilisation 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.

Publisher Correction: Deletion of DDB1- and CUL4- associated factor-17 (Dcaf17) gene causes spermatogenesis defects and male infertility in mice.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Deletion of DDB1- and CUL4- associated factor-17 (Dcaf17) gene causes spermatogenesis defects and male infertility in mice.

DDB1- and CUL4-associated factor 17 (Dcaf17) is a member of DCAF family genes that encode substrate receptor proteins for Cullin-RING E3 ubiquitin ligases, which play critical roles in many cellular processes. To unravel the function of DCAF17, we performed expression profiling of Dcaf17 in different tissues of wild type mouse by qRT-PCR and generated Dcaf17 knockout mice by gene targeting. Expression profiling of Dcaf17 showed highest expression in testis. Analyses of Dcaf17 transcripts during post-natal development of testis at different ages displayed gradual increase in Dcaf17 mRNA levels with the age. Although Dcaf17 disruption did not have any effect on female fertility, Dcaf17 deletion led to male infertility due to abnormal sperm development. The Dcaf17 -/- mice produced low number of sperm with abnormal shape and significantly low motility. Histological examination of the Dcaf17 -/- testis revealed impaired spermatogenesis with presence of vacuoles and sloughed cells in the seminiferous tubules. Disruption of Dcaf17 caused asymmetric acrosome capping, impaired nuclear compaction and abnormal round spermatid to elongated spermatid transition. For the first time, these data indicate that DCAF17 is essential for spermiogenesis.

Differential expression of PRAMEL1, a cancer/testis antigen, during spermatogenesis in the mouse.

PRAME belongs to a group of cancer/testis antigens (CTAs) that are characterized by their restricted expression in normal gametogenic tissues and a variety of tumors. The PRAME family is one of the most amplified gene families in the mouse and other mammalian genomes. Members of the PRAME gene family encode leucine-rich repeat (LRR) proteins functioning as transcription regulators in cancer cells. However, the role of PRAME in normal gonads is unknown. The objective of this study is to characterize the temporal and spatial expression of the mouse Pramel1 gene, and to determine the cellular localization of the PRAMEL1 protein during the mouse spermatogenesis. Our results indicated that the mouse Pramel1 was expressed in testis only. The mRNA and protein expression level was low in the newborn testes, and gradually increased from 1- to 3-week-old testes, and then remained constant after three weeks of age. Immunofluorescent staining on testis sections with the mouse PRAMEL1 antibody revealed that PRAMEL1 was localized in the cytoplasm of spermatocytes and the acrosomal region of round, elongating and elongated spermatids. Further analyses on the testis squash preparation and spermatozoa at a subcellular level indicated that the protein localization patterns of PRAMEL1 were coordinated with morphological alterations during acrosome formation in spermatids, and were significantly different in connecting piece, middle piece and principal piece of the flagellum between testicular and epididymal spermatozoa. Collectively, our results suggest that PRAMEL1 may play a role in acrosome biogenesis and sperm motility.

FtsW is a dispensable cell division protein required for Z-ring stabilization during sporulation septation in Streptomyces coelicolor.

The conserved rodA and ftsW genes encode polytopic membrane proteins that are essential for bacterial cell elongation and division, respectively, and each gene is invariably linked with a cognate class B high-molecular-weight penicillin-binding protein (HMW PBP) gene. Filamentous differentiating Streptomyces coelicolor possesses four such gene pairs. Whereas rodA, although not its cognate HMW PBP gene, is essential in these bacteria, mutation of SCO5302 or SCO2607 (sfr) caused no gross changes to growth and septation. In contrast, disruption of either ftsW or the cognate ftsI gene blocked the formation of sporulation septa in aerial hyphae. The inability of spiral polymers of FtsZ to reorganize into rings in aerial hyphae of these mutants indicates an early pivotal role of an FtsW-FtsI complex in cell division. Concerted assembly of the complete divisome was unnecessary for Z-ring stabilization in aerial hyphae as ftsQ mutants were found to be blocked at a later stage in cell division, during septum closure. Complete cross wall formation occurred in vegetative hyphae in all three fts mutants, indicating that the typical bacterial divisome functions specifically during nonessential sporulation septation, providing a unique opportunity to interrogate the function and dependencies of individual components of the divisome in vivo.