Male Germ Cell Development Research Articles

Generation of B6-Ddx4em1(CreERT2)Utr , a novel CreERT2 knock-in line, for germ cell lineage by CRISPR/Cas9.

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock-out mouse studies. In this study, we established a novel knock-in mouse line, B6-Ddx4 em1(CreERT2)Utr , which expresses CreERT2 recombinase under the control of the endogenous DEAD-box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen-treated B6-Ddx4 em1(CreERT2)Utr ::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6-Ddx4 em1(CreERT2)Utr is beneficial for studying female and male germ cell development.

The Interlocking Lives of LARP7: Fine-Tuning Transcription, RNA Modification, and Splicing through Multiple Non-coding RNAs

Elegant studies by Hasler etal. (2020) and Wang etal. (2020) uncover a novel role of LARP7 in facilitating the 2'-O-methylation of the spliceosomal U6 snRNA, which is functionally required for fidelity of pre-mRNA splicing and development of male germ cells.

Unraveling epigenomic abnormality in azoospermic human males by WGBS, RNA-Seq, and transcriptome profiling analyses.

To determine associations between genomic DNA methylation in testicular cells and azoospermiain human males. This was a case-control study investigating the differences and conservations in DNA methylation, genome-wide DNA methylation, and bulk RNA-Seq for transcriptome profiling using testicular biopsy tissues from NOA and OA patients. Differential methylation and different conserved methylation regions associated with azoospermia were identified by comparing genomic DNA methylation of testicular seminiferous cells derived from NOA and OA patients. The genome methylation modification of testicular cells from NOA patients was disordered, and the reproductive-related gene expression was significantly different. Our findings not only provide valuable knowledge of human spermatogenesis but also paved the way for the identification ofgenes/proteins involved in male germ cell development. The approach presented in this report provides a powerful tool to identify responsible biomolecules, and/or cellular changes (e.g., epigenetic abnormality) thatinduce male reproductive dysfunction such as OA and NOA.

Gene expression patterns and protein cellular localization suggest a novel role for DAZL in developing Tibetan sheep testes

Deleted in azoospermia-like (DAZL) is essential for mammalian spermatogenesis as it regulates proliferation, development, maturation and functional maintenance of male germ cells. Its expression and regulation vary with different species or in the same animal at different developmental stages, and despite its importance, very little is known about its roles in sheep, especially Tibetan sheep. To investigate the expression patterns and regulatory roles of DZAL in Tibetan sheep testis, testicular tissue was isolated from sheep at three crucial development stages: 3 months old, 1 year old and 3 years old. Using quantitative real-time PCR and Western blot, we found that DAZL mRNA first decreased and then increased with advancing age, while DAZL protein exhibited an opposite expression pattern, with first increased and subsequently decreased levels. Immunohistochemistry and immunofluorescence revealed that DAZL protein was located predominantly in the cytoplasm of Leydig cells and in both the cytoplasm and nucleus of spermatids. ELISA indicated that testosterone content within developing testes was first enhanced and then declined. Our results, taken together, demonstrate, for the first time, that DAZL gene is involved in Tibetan sheep spermatogenesis by regulating the development of spermatids in post-pubertal rams, along with a novel role in functional maintenance of Leydig cells in postnatal rams.

Characterisation, expression and possible functions of prohibitin during spermatogenesis in the silver pomfret Pampus argenteus.

Mitochondria play an important role in spermatogenesis, and some mitochondrial proteins are specifically related to this process. In this study we investigated the cytological characteristics of spermatogenic cells, including mitochondrial dynamics, during spermatogenesis in Pampus argenteus. In addition, we characterised the mitochondria-related protein prohibitin (PHB), which has been reported to play roles in mitochondrial dynamics and animal fertility. The full-length cDNA of the P. argenteus phb gene (Pa-phb) is 1687bp, including a 102-bp 5'-untranslated region (UTR), a 772-bp 3'-UTR and an 813-bp open reading frame encoding 271 amino acids. The predicted P. argenteus PHB protein (Pa-PHB) contains three functional domains (a transmembrane domain, an SPFH domain (the conserved region of stomatins, prohibitins, flotillins and HflK/C) and a coiled-coil domain) and exhibits high similarity with its homologue in other animals. The Pa-phb gene was widely expressed in all tissues examined, especially the liver and heart. We primarily focused on Pa-phb expression during spermatogenesis after observing the cytological features of male germ cells, and found that Pa-phb transcripts were detected throughout the course of development of male germ cells. Notably, we observed colocalised signals of Pa-PHB and mitochondria, which were distributed in the cytoplasm around the nucleus in spermatogonia, spermatocytes and early spermatids, tended to move to one side of the cell in middle spermatids and, finally, were colocalised in the sperm midpiece. These observations indicate that Pa-PHB is primarily localised in mitochondria during spermatogenesis, indicating that it has a role in mitochondria. Based on the results of this and previous studies regarding the essential roles of PHB in mitochondria and spermatogenesis in animals, we propose a functional model for PHB during spermatogenesis, including possible roles in the proliferation of spermatogonia and in the regulation of mitochondrial morphology and function in spermatogenic cells.

Cannabinoid Receptors Signaling in the Development, Epigenetics, and Tumours of Male Germ Cells.

Endocannabinoids are natural lipid molecules whose levels are regulated by specific biosynthetic and degradative enzymes. They bind to and activate two main cannabinoid receptors type 1 (CB1) and type 2 (CB2), and together with their metabolizing enzymes form the “endocannabinoid system” (ECS). In the last years, the relevance of endocannabinoids (eCBs) as critical modulators in various aspects of male reproduction has been pointed out. Mammalian male germ cells, from mitotic to haploid stage, have a complete ECS which is modulated during spermatogenesis. Compelling evidence indicate that in the testis an appropriate “eCBs tone”, associated to a balanced CB receptors signaling, is critical for spermatogenesis and for the formation of mature and fertilizing spermatozoa. Any alteration of this system negatively affects male reproduction, from germ cell differentiation to sperm functions, and might have also an impact on testicular tumours. Indeed, most of testicular tumours develop during early germ-cell development in which a maturation arrest is thought to be the first key event leading to malignant transformation. Considering the ever-growing number and complexity of the data on ECS, this review focuses on the role of cannabinoid receptors CB1 and CB2 signaling in male germ cells development from gonocyte up to mature spermatozoa and in the induction of epigenetic alterations in these cells which might be transmitted to the progeny. Furthermore, we present new evidence on their relevance in testicular cancer.

Deletion of Ck2β gene causes germ cell development arrest and azoospermia in male mice.

ObjectivesIn humans, non‐obstructive azoospermia (NOA) is a major cause of male infertility. However, the aetiology of NOA is largely unknown. Previous studies reported that protein CK2β was abundantly and broadly expressed in spermatogenic cells. Here, we investigate whether protein CK2β participates in spermatogenesis.Materials and MethodsIn this study, we separated spermatogenic cells using STA‐PUT velocity sedimentation, analysed the expression pattern of protein CK2β by immunoblotting, specifically deleted Ck2β gene in early‐stage spermatogenic cells by crossing Ck2βfl mice with Stra8‐Cre+ mice and validated the knockout efficiency by quantitative RT‐PCR and immunoblotting. The phenotypes of Ck2βfl/Δ;SCre+ mice were studied by immunohistochemistry and immunofluorescence. The molecular mechanisms of male germ cell development arrest were elucidated by immunoblotting and TUNEL assay.ResultsAblation of Ck2β gene triggered excessive germ cell apoptosis, germ cell development arrest, azoospermia and male infertility. Inactivation of Ck2β gene caused distinctly reduced expression of Ck2α′ gene and CK2α′ protein.Conclusions Ck2β is a vital gene for germ cell survival and male fertility in mice.

Organelle fusion in fertility

Reproduction The fertility of organisms as diverse as male fruit flies and mice requires a mitochondrial fusion gene. But what for? Mitochondria cluster and fuse during early sperm development with the help of guanosine triphosphatases called mitofusins. When mitochondrial fusion fails, oxidative phosphorylation decreases, and mitochondrial transport and degradation are also affected. Varuzhanyan et al. tested how reliant the success of male germ cell development in mice is on mitofusins. Mitofusins are needed to trigger a metabolic shift during meiosis. When mitofusins are eliminated, germ cells display damage to the cristae ultrastructure, increased apoptosis, reduced oxidative phosphorylation, and a loss of mitochondrial ribosomes. Thus, mitofusins are vital for maintaining translation of respiratory chain and mitochondrial proteins to enable male fertility. eLife 8 , e51601 (2019).

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro.

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated onday 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.

Diverse roles for CDK-associated activity during spermatogenesis.

The primary function of cyclin‐dependent kinases (CDKs) in complex with their activating cyclin partners is to promote mitotic division in somatic cells. This canonical cell cycle‐associated activity is also crucial for fertility as it allows the proliferation and differentiation of stem cells within the reproductive organs to generate meiotically competent cells. Intriguingly, several CDKs exhibit meiosis‐specific functions and are essential for the completion of the two reductional meiotic divisions required to generate haploid gametes. These meiosis‐specific functions are mediated by both known CDK/cyclin complexes and meiosis‐specific CDK‐regulators and are important for a variety of processes during meiotic prophase. The majority of meiotic defects observed upon deletion of these proteins occur during the extended prophase I of the first meiotic division. Importantly a lack of redundancy is seen within the meiotic arrest phenotypes described for many of these proteins, suggesting intricate layers of cell cycle control are required for normal meiotic progression. Using the process of male germ cell development (spermatogenesis) as a reference, this review seeks to highlight the diverse roles of selected CDKs their activators, and their regulators during gametogenesis.

Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis.

The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development, whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction. We previously demonstrated that in mice, the makorin-2 (Mkrn2) gene is expressed exclusively in the testis and its deletion leads to male infertility. To understand the potential molecular mechanism, in this study, we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn2. To identify specific gene(s) involved, we performed digital gene expression profiling (DGE) and pathway analysis via gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22 (PERP) expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels. GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility. Further, Gene Expression Omnibus (GEO) dataset analysis showed that p53, upstream of PERP, was upregulated in oligoasthenoteratozoospermia (OAT). These observations suggest that Mkrn2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility.

Comprehensive Analysis of Mouse Cancer/Testis Antigen Functions in Cancer Cells and Roles of TEKT5 in Cancer Cells and Testicular Germ Cells.

The cancer/testis antigen (CTA) genes were identified as human genes preferentially expressed in cancer cells and testis, but the contribution of CTAs to cancer and male germ cell development is unclear. In this study, we comprehensively examined mouse CTA functions and found that the majority of CTAs are involved in growth and/or survival of cancer cells. We focused on one mouse CTA gene, Tekt5, for its detailed functional analysis. Tekt5 knockdown (KD) in ovarian cancer cells caused G1 arrest and apoptosis, and p27kip1 was concomitantly upregulated. Tekt5 KD also resulted in decreased levels of acetylated α-tubulin and subsequent fragmentation of β-III-tubulin, upregulation of HDAC6 that deacetylates α-tubulin, and nuclear accumulation of SMAD3 that induces p27kip1 expression. Because depolymerization of tubulin is known to cause translocation of SMAD3 to the nucleus, these results together suggested that TEKT5 negatively regulates Hdac6 expression and consequently maintains cell cycle via stabilization of tubulin. We also found that the number of spermatids was significantly decreased and acetylated α-tubulin levels were decreased in vivo by KD of Tekt5 in testis. Because acetylated α-tubulin is required for sperm morphogenesis, these results suggest that TEKT5 is necessary for spermiogenesis via maintenance of acetylated α-tubulin levels.

Histone hyperacetylation and DNA methylation interplay during murine spermatogenesis.

Male germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves the incorporation of testis-specific histone variants, mitotic and meiotic divisions, haploid gene expression, histone-protamine transitions and massive epigenetic reprogramming. Understanding the protein players and the epigenetic mark network involved in the setting of the epigenetic programme in spermatogenesis is an exciting new clue in the field of reproductive biology with translational outcomes. As information in the existing literature regarding cross-talk between DNA methylation and histone hyperacetylation in the advanced stages of murine spermatogenesis is still scarce and controversial we have investigated the effect of a DNA-methyltransferase inhibitor, 5-aza-2'-deoxycytidine, at the cytological and molecular level (by transmission electron microscopy, immunocytochemistry and immunoprecipitation methods). Our results revealed an important role for regulation of DNA methylation in controlling histone hyperacetylation and chromatin remodelling during spermatogenesis.

Functions of genes related to testicular germ cell tumour development

Testicular germ cell tumour (TGCT) is a malignancy with a high heritable component. The inherited risk is polygenic, and around 50 susceptibility genes are identified. The functional role of the gene products for TGCT development is not well understood. The focus of this review is functional studies of genetic risk factors for TGCT derived from GCNIS and the signalling pathways involved in the pathogenesis. Genome-wide association studies have identified new risk loci for TGCT and confirmed previously identified susceptibility genes. Many of these risk genes are related to male germ cell development, sex determination and genomic integrity. Gain- and loss-of-function studies in animal models and TGCT cell lines, as well as gene and protein expression studies in TGCT patient samples, have contributed to the understanding of TGCT development. KITLG-KIT signalling is of crucial importance, but several other signal transduction pathways may also play a role. Many of the risk loci are in non-coding regions, and studies have revealed that non-coding RNAs may act as oncogenes or tumour suppressors in TGCT development. The risk of TGCT is polygenic, and the underlying molecular mechanisms are complex. Several signalling pathways are related to TGCT development, and both proteins and non-coding RNAs may act as oncogenes or tumour suppressors. Epigenetic studies are of importance to get further knowledge about how the signalling pathways are regulated.

Abstract 2684: Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility

Abstract Testicular germ cell tumors (TGCT) are ideally suited to agnostic genome-wide association methods because of their known high heritability and homogeneous cell of origin. Here we report the most updated results from the international Testicular Cancer Consortium (TECAC) that extends published findings from our recent meta-analysis, which included 3,558 cases and 13,970 controls from five centers. To these existing samples, we added summary statistics from 300 cases and 151,991 controls provided by deCODE combined with imputed genotypes from an additional 5,602 cases and 5,006 controls assembled from 15 TECAC centers, almost all (97%) of which were centrally genotyped using the Illumina Human Core array. A total of 9,458 TGCT cases were included in our current meta-analysis, representing a 66% increase since our last publication. We identified 14 new and confirmed 41 previously reported loci that surpassed genome-wide significance (5 × 10-8). Fifteen previously reported loci did not reach this threshold, although one did so in subgroup analysis. In addition, 57 new loci attained nominal significance (5 × 10-8 ≤ p ≤ 1 × 10-5). As expected, a substantial proportion of top hits continue to map to genetic regions belonging to biological pathways essential to male germ cell development, sex determination, and chromosomal segregation. For example, newly identified genes include anaphase promoting complex subunit 2 (ANAPC2), BCL2 like 11 (BCL211), anti-Mullerian hormone receptor type 2 (AMHR2), and DEP domain containing mTOR interacting protein (DEPTOR). Our findings also further implicate genes located on the X chromosome, specifically the androgen receptor (AR). In analyses stratified by tumor subtype, we identified GATA-binding protein 4 (GATA4) as a susceptibility locus among men diagnosed with seminoma. Despite a similar effect size observed among men diagnosed solely with non-seminoma (i.e. excluding cases with a mixture of both seminoma and non-seminoma tumors), this marker did not surpass genome-wide significance in this subtype likely because of the smaller case sample size. GATA4 previously has been associated with overall risk of TGCT (Litchfield et al., Nat Genet. 2017). Biological mechanisms for all genome-wide significant loci are currently being explored using publicly available data sets, ATAC-seq of four TGCT cell lines, and SPATIaL-Seq (a novel high-resolution chromatin conformation capture (3C) assay) of the NTERA2 pluripotent human embryonal carcinoma cell line. Additional genotyping of top hits in an independent sample set of 1,103 TGCT cases and 1,210 controls is currently ongoing. Citation Format: Louise C. Pyle, John Pluta, Kevin T. Nead, Nandita Mitra, Javier Benitez, D. Timothy Bishop, Victoria Cortessis, Alberto Ferlin, Jourik Gietema, Mark Greene, Tom Grotmol, Ramneek Gupta, Rob Hamilton, Michelle A. Hildebrandt, Trine B. Haugen, Lambertus Kiemeney, Christian Kubisch, Davor Lessel, Paloma Martin, Thorunn Rafnar, Lorenzo Richiardi, Rolf Skotheim, Clare Turnbull, Fredrik Wiklund, Tongzhang Zheng, Ewa Rajpert-De Meyts, Stephen M. Schwartz, Katherine A. McGlynn, Peter A. Kanetsky, Katherine L. Nathanson, Testicular Cancer Consortium (TECAC). Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2684.

Overexpression of MicroRNA-10a in Germ Cells Causes Male Infertility by Targeting Rad51 in Mouse and Human.

Spermatogenesis is a complicated process including spermatogonial stem cells self-renewal and differentiates into mature spermatozoa. MicroRNAs (miRNAs) as a class of small non-coding RNAs play a crucial role during the process of spermatogenesis. However, the function of a plenty of miRNAs on spermatogenesis and the potential mechanisms remain largely unknown. Here, we show that genetically conditional overexpressed miR-10a in germ cells caused complete male sterility, characterized by meiotic arrested in germ cells. Analysis of miR-10a overexpression mouse testes reveals that failure of double strand break (DSB) repairs and aberrant spermatogonial differentiation. Furthermore, we identified Rad51 as a key target of miR-10a in germ cell by bioinformatics prediction and luciferase assay, which may be responsible for the infertility of the miR-10a overexpressed mice and germ cell arrested patients. Our data show that miR-10a dependent genetic regulation of meiotic process is crucial for male germ cell development and spermatogenesis in both mouse and human. These findings facilitate our understanding of the roles of miRNA-10a in spermatogenesis and male fertility.

Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses.

Fishes exhibit remarkably diverse, and plastic, patterns of sexual development, most striking of which is sequential hermaphroditism, where individuals readily reverse sex in adulthood. How this stunning example of phenotypic plasticity is controlled at a genetic level remains poorly understood. Several genes have been implicated in regulating sex change, yet the degree to which a conserved genetic machinery orchestrates this process has not yet been addressed. Using captive and in-the-field social manipulations to initiate sex change, combined with a comparative qPCR approach, we compared expression patterns of four candidate regulatory genes among three species of wrasses (Labridae)—a large and diverse teleost family where female-to-male sex change is pervasive, socially-cued, and likely ancestral. Expression in brain and gonadal tissues were compared among the iconic tropical bluehead wrasse (Thalassoma bifasciatum) and the temperate spotty (Notolabrus celidotus) and kyusen (Parajulus poecilepterus) wrasses. In all three species, gonadal sex change was preceded by downregulation of cyp19a1a (encoding gonadal aromatase that converts androgens to oestrogens) and accompanied by upregulation of amh (encoding anti-müllerian hormone that primarily regulates male germ cell development), and these genes may act concurrently to orchestrate ovary-testis transformation. In the brain, our data argue against a role for brain aromatase (cyp19a1b) in initiating behavioural sex change, as its expression trailed behavioural changes. However, we find that isotocin (it, that regulates teleost socio-sexual behaviours) expression correlated with dominant male-specific behaviours in the bluehead wrasse, suggesting it upregulation mediates the rapid behavioural sex change characteristic of blueheads and other tropical wrasses. However, it expression was not sex-biased in temperate spotty and kyusen wrasses, where sex change is more protracted and social groups may be less tightly-structured. Together, these findings suggest that while key components of the molecular machinery controlling gonadal sex change are phylogenetically conserved among wrasses, neural pathways governing behavioural sex change may be more variable.

Upregulated lncRNA Gm2044 inhibits male germ cell development by acting as miR-202 host gene

ABSTRACTLong non-coding RNAs (lncRNAs) have been found to participate in the regulation of human spermatogenic cell development. However, little is known about the abnormal expression of lncRNAs associated with spermatogenic failure and their molecular mechanisms. Using lncRNA microarray of testicular tissue for male infertility and bioinformatics methods, we identified the relatively conserved lncRNA Gm2044 which may play important roles in non-obstructive azoospermia. The UCSC Genome Browser showed that lncRNA Gm2044 is the miR-202 host gene. This study revealed that lncRNA Gm2044 and miR-202 were significantly increased in non-obstructive azoospermia of spermatogonial arrest. The mRNA and protein levels of Rbfox2, a known direct target gene of miR-202, were regulated by lncRNA Gm2044. Furthermore, the miR-202-Rbfox2 signalling pathway was shown to mediate the suppressive effects of lncRNA Gm2044 on the proliferation of human testicular embryonic carcinoma cells. Understanding of the molecular signalling pathways for lncRNA-regulated spermatogenesis will provide new clues into the pathogenesis and treatment of patients with male infertility.

The intramembrane protease SPPL2c promotes male germ cell development by cleavingphospholamban.

Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c-/- mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c-/- mice.

Efficient GFP-labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry.

SummarySpermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue‐specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre‐lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.