Testis-enriched Genes Research Articles

O-137 Functional characterisation of three human male infertility associated X-linked genes using Drosophila melanogaster as model organism

Abstract Study question What is the functional relevance of GLUD2, FAM47A, and TAF7L in spermatogenesis and male infertility? Summary answer Knockdown of orthologues leads to infertility in male flies substantiating GLUD2, FAM47A, and TAF7L as candidate genes for human male infertility. What is known already Non-obstructive azoospermia (NOA) is clinically the most severe form of spermatogenic failure and male infertility. Despite advances in the genetic diagnostics, the precise cause and molecular diagnosis remains elusive for the majority of affected men. GLUD2, FAM47A, and TAF7L are X-linked and testis-enriched genes, with increasing evidence of their relevance in male infertility. However, so far, no functional study or animal model supporting their contribution to the disease exist. Study design, size, duration We queried exome/genome data of more than 2, 300 infertile men with severe quantitative spermatogenic failure from the Male Reproductive Genomics (MERGE) cohort for variants in GLUD2, FAM47A, and TAF7L. To assess the impact of these three genes on male infertility we performed functional studies using Drosophila melanogaster as a model organism. Participants/materials, setting, methods Exome/genome data was filtered for rare (MAF <0.001, gnomAD), hemizygous loss-of-function (LoF) and potentially pathogenic missense variants (CADD score >20) in GLUD2 (NM_012084.4), FAM47A (NM_203408.3), and TAF7L (NM_024885.4). Depending on sample availability, the X-linked inheritance pattern was confirmed by segregation analysis. To investigate the relevance of these genes in male infertility, we generated and functionally characterised testis-specific knockdown (KD) of orthologues in Drosophila melanogaster using GAL4-UAS system. Main results and the role of chance In GLUD2, we identified four missense and one LoF variant in five azoospermic men. All variant carriers revealed impaired spermatogenesis with predominant meiotic arrest (MeiA). In FAM47A, we identified three missense and one LoF variant in five azoospermic men, four of whom consistently exhibited a testicular phenotype of Sertoli cell-only (SCO). In TAF7L, we identified four missense variants in three azoospermic men. One of them was a carrier of two variants. Notably, all the identified variants were clustered in the mutation hotspot region within exons 9, 10, and 13. Testis-specific KD of GLUD2, FAM47A, and TAF7Lorthologues consistently resulted in complete sterility in male flies. Compared to controls, Glud2-KD flies had normal testicular morphology but revealed small seminal vesicles without mature sperm. Testicular squash analysis showed germ cell maturation arrest at the elongating spermatid stage, leading to individualisation complex ablation. Immunofluorescence staining revealed a lack of cystic bulges and waste bags, indicating disrupted spermatogenesis. In contrast, Fam47a-KD and Taf7l-KD flies were infertile due to total germ cell loss in the testis, resulting in an extremely small testis size compared with controls, consistent with the SCO testicular phenotype observed in infertile men with FAM47A and TAF7L variants. Limitations, reasons for caution The main limitation is that the study does not analyse a gene-specific genotype-phenotype correlation of the missense variants identified. Furthermore, extensive research is required to elucidate the underlying molecular mechanisms by which GLUD2, FAM47A, and TAF7L regulate male fertility in humans. Wider implications of the findings This study highlights the utility of Drosophila for studying the role of human male fertility genes, underlining the importance of validating genetic findings. Additionally, it contributes significantly to advancing our understanding of the genetic basis of azoospermia, offering potential targets for research aimed at addressing male infertility. Trial registration number not applicable

Individual disruption of 12 testis-enriched genes via the CRISPR/Cas9 system does not affect the fertility of male mice

More than 1200 genes have been shown in the database to be expressed predominantly in the mouse testes. Advances in genome editing technologies such as the CRISPR/Cas9 system have made it possible to create genetically engineered mice more rapidly and efficiently than with conventional methods, which can be utilized to screen genes essential for male fertility by knocking out testis-enriched genes. Finding such genes related to male fertility would not only help us understand the etiology of human infertility but also lead to the development of male contraceptives. In this study, we generated knockout mice for 12 genes (Acrv1, Adgrf3, Atp8b5, Cfap90, Cfap276, Fbxw5, Gm17266, Lrrd1, Mroh7, Nemp1, Spata45, and Trim36) that are expressed predominantly in the testis and examined the appearance and histological morphology of testes, sperm motility, and male fertility. Mating tests revealed that none of these genes is essential for male fertility at least individually. Notably, knockout mice for Gm17266 showed smaller testis size than the wild-type but did not exhibit reduced male fertility. Since 12 genes were not individually essential for male fertilization, it is unlikely that these genes could be the cause of infertility or contraceptive targets. It is better to focus on other essential genes because complementary genes to these 12 genes may exist.

CFAP58 is involved in the sperm head shaping and flagellogenesis of cattle and mice.

CFAP58 is a testis-enriched gene that plays an important role in the sperm flagellogenesis of humans and mice. However, the effect of CFAP58 on bull semen quality and the underlying molecular mechanisms involved in spermatogenesis remain unknown. Here, we identified two single-nucleotide polymorphisms (rs110610797, A>G and rs133760846, G>T) and one indel (g.-1811_ g.-1810 ins147bp) in the promoter of CFAP58 that were significantly associated with semen quality of bulls, including sperm deformity rate and ejaculate volume. Moreover, by generating gene knockout mice, we found for the first time that the loss of Cfap58 not only causes severe defects in the sperm tail, but also affects the manchette structure, resulting in abnormal sperm head shaping. Cfap58 deficiency causes an increase in spermatozoa apoptosis. Further experiments confirmed that CFAP58 interacts with IFT88 and CCDC42. Moreover, it may be a transported cargo protein that plays a role in stabilizing other cargo proteins, such as CCDC42, in the intra-manchette transport/intra-flagellar transport pathway. Collectively, our findings reveal that CFAP58 is required for spermatogenesis and provide genetic markers for evaluating semen quality in cattle.

Spem2, a novel testis-enriched gene, is required for spermiogenesis and fertilization in mice

Spermiogenesis is considered to be crucial for the production of haploid spermatozoa with normal morphology, structure and function, but the mechanisms underlying this process remain largely unclear. Here, we demonstrate that SPEM family member 2 (Spem2), as a novel testis-enriched gene, is essential for spermiogenesis and male fertility. Spem2 is predominantly expressed in the haploid male germ cells and is highly conserved across mammals. Mice deficient for Spem2 develop male infertility associated with spermiogenesis impairment. Specifically, the insufficient sperm individualization, failure of excess cytoplasm shedding, and defects in acrosome formation are evident in Spem2-null sperm. Sperm counts and motility are also significantly reduced compared to controls. In vivo fertilization assays have shown that Spem2-null sperm are unable to fertilize oocytes, possibly due to their impaired ability to migrate from the uterus into the oviduct. However, the infertility of Spem2−/− males cannot be rescued by in vitro fertilization, suggesting that defective sperm–egg interaction may also be a contributing factor. Furthermore, SPEM2 is detected to interact with ZPBP, PRSS21, PRSS54, PRSS55, ADAM2 and ADAM3 and is also required for their processing and maturation in epididymal sperm. Our findings establish SPEM2 as an essential regulator of spermiogenesis and fertilization in mice, possibly in mammals including humans. Understanding the molecular role of SPEM2 could provide new insights into future therapeutic treatment of human male infertility and development of non-hormonal male contraceptives.

SL2 - Testis-enriched genes and their functions for spermatogenesis and fertilization

SL2 - Testis-enriched genes and their functions for spermatogenesis and fertilization

Bi-allelic human TEKT3 mutations cause male infertility with oligoasthenoteratozoospermia owing to acrosomal hypoplasia and reduced progressive motility.

Oligoasthenoteratozoospermia (OAT) can result in male infertility owing to reduced sperm motility and abnormal spermatozoan morphology. The Tektins are a family of highly conserved filamentous proteins expressed in the axoneme and associated structures in many different metazoan species. Earlier studies on mice identified Tektin3 (Tekt3) as a testis-enriched gene, and knockout of Tekt3 resulted in asthenozoospermia in the mice. Here, whole-exome sequencing of 100 males with asthenozoospermia from unrelated families was performed, followed by Sanger sequencing, leading to the identification of TEKT3 as a candidate gene in two of these patients and their associated family members. In total, three mutations in the TEKT3 gene were identified in both these patients, including one homozygous deletion-insertion mutation (c.543_547delinsTTGAT: p.Glu182*) and one compound heterozygous mutation (c.[548G > A]; [752A > C], p.[Arg183Gln]; [Gln251Pro]). Both of these mutations resulted in the complete loss of TEKT3 expression. The patients were both found to produce sperm that, although those showed no apparent defects in the flagellar structure, had reduced progressive motility. In contrast to mice, most sperm from these two patients exhibited acrosomal hypoplasia, although this did not prevent the use of the sperm for in vitro fertilization through an ICSI approach. TEKT3 was found to bind to other TEKT proteins, suggesting that these proteins form a complex within human spermatozoa. Overall, these results suggest that a loss of TEKT3 function can contribute to OAT incidence in humans. TEKT3 deficiencies can reduce sperm motility and contribute to severe acrosomal hypoplasia in spermatozoa, compromising their normal function.

The testis-, epididymis-, or seminal vesicle-enriched genes Aldoart2, Serpina16, Aoc1l3, and Pate14 are not essential for male fertility in mice.

Spermatozoa released from the testis acquire fertilizing ability by translocating thorough the epididymis. Further, accessory gland secretions ejaculated into the female reproductive tract along with spermatozoa are also required to ensure male fecundity, such as the maintenance of proper sperm count and inhibition of premature sperm capacitation in the uterus. Here, we focus on a testis-enriched gene "Aldoart2", an epididymis-enriched gene "Serpina16", and seminal vesicle-enriched genes "Aoc1l3" and "Pate14" which were thought to be important for male fertility based on the previous studies. We independently deleted almost the entire protein-coding sequence of these genes in mice using CRISPR/Cas9. There were no overt defects in the histology and the sperm morphology and motility of any knockout (KO) mice. Further, Aoc1l3 and Pate14 KO males were able to form copulatory plugs. Finally, female mice that mated with these KO males delivered pups at a comparable level with the control males. Given our data, we demonstrated that the four genes predominantly expressed in the testis, epididymis, or seminal vesicle are independently dispensable for male fertility.

OR16-3 Analysis of Testis-Enriched Genes in Estrogen-Dependent Genome Regulation in Breast Cancer

Abstract Emerging studies show that testis-enriched genes play critical roles in several biological processes. The expression of these genes is tightly regulated and limited to immune-privileged organs; however, many of them escape regulation and are aberrantly expressed in several diseases such as cancer. Intriguingly, our genomic analysis revealed that many of them are transcribed from previously considered gene deserts. We also found that a significant number of them are robustly regulated by estrogen and other peptide hormones such as Tumor Necrosis Factor-alpha (TNF alpha). However, their direct role in estrogen-dependent transcriptional regulation and biological outcomes remains unstudied. In this regard, our integrated genomic approach, including RNA with 4-thiouridine (4sU) coupled with RNA-sequencing and CRISPR-based genetic screening, has identified key estrogen-regulated testis-enriched genes that may modulate estrogen-signaling. Through a series of molecular and phenotypic assays, we selected a potential but understudied transcript, which is predominantly localized to the nucleus. Furthermore, in the current study, we have interrogated the functional mechanisms by which selected transcript controls estrogen-dependent gene regulation in estrogen receptor-positive (ER+) breast cancer. We found that the transcript controls estrogen-dependent cell and tumor growth. Additionally, we have manipulated the levels of the transcript in estrogen-receptor-positive (ER+) breast cancer cells employing gain- and loss-of-function studies and performed 4sU-RNA-seq. Interestingly, the transcript modulates the expression of estrogen-driven transcripts that are transcribed from ER-alpha (ERa) binding sites. Mechanistically, the transcript is localized in the nucleus and interacts with chromatin-associated proteins. In addition, our integrated genomic analyses comprising 'Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq)' and 'chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing ChIP-seq' (H3K27ac, H3K27me3) identified a critical role for the transcript in controlling the epigenetic landscape at target gene promoters and enhancers in ER+ breast cancer. Collectively, our data support a functional role for testis-enriched genes in enhancer transcription and chromatin organization. This study sheds light on the molecular functions of testis-enriched genes and presents an opportunity to identify new diagnostic and develop treatment strategies in ER+ breast cancer. S.S.G. is supported by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT; RR170020). S.S.G. is also supported by a grant from Lizanell and Colbert Coldwell foundation. Presentation: Sunday, June 12, 2022 11:30 a.m. - 11:45 a.m.

Murine fertility and spermatogenesis are independent of the testis-specific Spdye4a gene

BackgroundWhile many testis-enriched genes have been identified as important regulators of the spermatogenic process, the specific roles played by several of these genes and their functional importance has yet to be fully clarified. MethodsWe employed a CRISPR/Cas9 approach to introduce a 5 bp in-frame deletion within the Spdye4a gene (Exon 2) of C57BL/6 mice (Spdye4a−/−). Fertility and sperm counts were evaluated. Testes tissues and cell suspensions were analyzed via histological and immunofluorescence staining. mRNA and protein levels of candidate genes were assessed through qPCR and Western blotting. In vitro fertilization was used to assess the ability of sperm cells to bind to egg cells. ResultsSpdye4a−/− mice did not exhibit any reduction in fertility, and exhibited comparable sperm counts, morphology and motility to those of wildtype littermates. Functionally, Spdye4a−/− sperm exhibited normal sperm-egg binding activity in vitro. Furthermore, the testes of Spdye4a−/− mice exhibited a full range of germ cells from spermatogonia to mature spermatozoa. No differences in the progression of meiotic prophase I were observed when comparing Spdye4a−/− and wildtype mice, indicating that the loss of Spdye4a had no adverse effect on spermatogenesis. DiscussionSpdye4a is dispensable in the context of mice fertility and spermatogenesis. This study will prevent other laboratories from expending repeated efforts to generate similar knockout mice.

A novel testis-enriched gene, Samd4a, regulates spermatogenesis as a spermatid-specific factor.

Spermatogenesis is the highly orchestrated process involving expression of a series of testicular genes. Testis-enriched genes are critical for cellular processes during spermatogenesis whose disruption leads to impaired spermatogenesis and male infertility. Nevertheless, among poorly investigated testicular genes are the mouse Samd4a and human SAMD4A which were identified in the current study as novel testis-enriched genes through transcriptomic analyses. In particular, as orthologous alternative splicing isoforms, mouse Samd4a E-form and human SAMD4AC-form containing the SAM domain were specific to testes. Western blot analyses revealed that the murine SAMD4AE-form was predominantly found in the testis. Analyses on GEO2R and single-cell RNA-seq datasets revealed that the Samd4a/SAMD4A expression was enriched in spermatids among various types of cells in adult testes. To investigate in vivo functions of Samd4a, Samd4a knockout mice were generated using the CRISPR/Cas9 system. The Samd4a deficiency resulted in lower testis weight, absence of elongated spermatids, and an increased number of apoptotic cells. Profiling of gene expression in human testis samples revealed that the SAMD4A expression was comparable between obstructive azoospermia patients and normal controls, but significantly lowered in nonobstructive azoospermia (NOA) patients. Among three subgroups of NOA, pre-meiotic arrest (NOA-pre), meiotic arrest (NOA-mei), and post-meiotic arrest (NOA-post), expression level of SAMD4A was higher in the NOA-post than the NOA-mei, but there was no difference between the NOA-pre and NOA-mei. The current studies demonstrated spermatid stage-specific expression of Samd4a/SAMD4A, and impairment of the late stages of spermatogenesis by disruption of the mouse Samd4a gene. These data suggest that Samd4a/SAMD4A plays an essential role in normal spermatogenesis, and SAMD4A, as a spermatid specific marker, can be used for subcategorizing NOA patients. Further understanding the molecular role of SAMD4A will advance our knowledge on genetic regulations in male infertility.

Stonewall prevents expression of ectopic genes in the ovary and accumulates at insulator elements in D. melanogaster.

Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. In Drosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell—daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. We performed RNA-Seq on stwl mutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. We also discovered inappropriate expression of multiple classes of genes. Most prominent are testis-enriched genes, including the male germline sex-determination switch Phf7, the differentiation factor bgcn, and a large testis-specific gene cluster on chromosome 2, all of which are upregulated or ectopically expressed in stwl mutant ovaries. Surprisingly, we also found that RNAi knockdown of stwl in somatic S2 cells results in ectopic expression of these testis genes. Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, we discovered that Stwl localizes upstream of transcription start sites and at heterochromatic sequences including repetitive sequences associated with telomeres. Stwl is also enriched at bgcn, suggesting that it directly regulates this essential differentiation factor. Finally, we identify Stwl binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation, including repression of male transcripts in the female germline, by binding insulators and establishing chromatin boundaries.

Commentary on "CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice".

Commentary on "CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice".

AXDND1, a novel testis-enriched gene, is required for spermiogenesis and male fertility

Spermiogenesis is a complex process depending on the sophisticated coordination of a myriad of testis-enriched gene regulations. The regulatory pathways that coordinate this process are not well understood, and we demonstrate here that AXDND1, as a novel testis-enriched gene is essential for spermiogenesis and male fertility. AXDND1 is exclusively expressed in the round and elongating spermatids in humans and mice. We identified two potentially deleterious mutations of AXDND1 unique to non‐obstructive azoospermia (NOA) patients through selected exonic sequencing. Importantly, Axdnd1 knockout males are sterile with reduced testis size caused by increased germ cell apoptosis and sloughing, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Axdnd1 mutated late spermatids showed head deformation, outer doublet microtubules deficiency in the axoneme, and loss of corresponding accessory structures, including outer dense fiber (ODF) and mitochondria sheath. These phenotypes were probably due to the perturbed behavior of the manchette, a dynamic structure where AXDND1 was localized. Our findings establish AXDND1 as a novel testis-enrich gene essential for spermiogenesis and male fertility probably by regulating the manchette dynamics, spermatid head shaping, sperm flagellum assembly.

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice.

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules and some attached projections. Radial spokes (RSs) facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 radial spoke proteins (RSP1-RSP23), although the roles of some radial spoke proteins remain unknown. Recently, RSP15 has been reported to be bound to the stalk of RS2, but its homolog in mammals has not been identified. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. We found that LRRC23 localizes to the RS complex within murine sperm flagella and interacts with RSPH3A and RSPH3B. The knockout of Lrrc23 resulted in male infertility due to RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was not significantly affected. These data indicate that LRRC23 is a key regulator that underpins the integrity of the RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. This article has an associated First Person interview with the first author of the paper.

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice.

Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.

Testis-expressed protein 33 is not essential for spermiogenesis and fertility in mice.

Gene expression analyses have revealed that there are >2,300 testis-enriched genes in mice, and gene knockout models have shown that a number of them are responsible for male fertility. However, the functions of numerous genes have yet to be clarified. The aim of the present study was to identify the expression pattern of testis-expressed protein 33 (TEX33) in mice and explore the role of TEX33 in male reproduction. Reverse transcription-polymerase chain reaction and western blot assays were used to investigate the mRNA and protein levels of TEX33 in mouse testes during the first wave of spermatogenesis. Immunofluorescence analysis was also performed to identify the cellular and structural localization of TEX33 protein in the testes. Tex33 knockout mice were generated by CRISPR/Cas9 gene-editing. Histological analysis with hematoxylin and eosin or periodic acid-Schiff (PAS) staining, computer-assisted sperm analysis (CASA) and fertility testing, were also carried out to evaluate the effect of TEX33 on mouse spermiogenesis and male reproduction. The results showed that Tex33 mRNA and protein were exclusively expressed in mouse testes and were first detected on postnatal days 21–28 (spermiogenesis phase); their expression then remained into adulthood. Immunofluorescence analysis revealed that TEX33 protein was located in the spermatids and sperm within the seminiferous tubules of the mouse testes, and exhibited specific localization to the acrosome, flagellum and manchette during spermiogenesis. These results suggested that TEX33 may play a role in mouse spermiogenesis. However, Tex33 knockout mice presented no detectable difference in testis-to-body weight ratios when compared with wild-type mice. PAS staining and CASA revealed that spermatogenesis and sperm quality were normal in mice lacking Tex33. In addition, fertility testing suggested that the Tex33 knockout mice had normal reproductive functions. In summary, the findings of the present study indicate that TEX33 is associated with spermiogenesis but is not essential for sperm development and male fertility.

ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity.

As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

Sperm-oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm-oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm-oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm-oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm-oocyte fusion.

Population-specific, recent positive directional selection suggests adaptation of human male reproductive genes to different environmental conditions

BackgroundRecent human transcriptomic analyses revealed a very large number of testis-enriched genes, many of which are involved in spermatogenesis. This comprehensive transcriptomic data lead us to the question whether positive selection was a decisive force influencing the evolution and variability of testis-enriched genes in humans. We used two methodological approaches to detect different levels of positive selection, namely episodic positive diversifying selection (i.e., past selection) in the human lineage within primate phylogeny, potentially driven by sperm competition, and recent positive directional selection in contemporary human populations, which would indicate adaptation to different environments.ResultsIn the human lineage (after correction for multiple testing) we found that only the gene TULP2, for which no functional data are yet available, is subject to episodic positive diversifying selection. Using less stringent statistical criteria (uncorrected p-values), also the gene SPATA16, which has a pivotal role in male fertility and for which episodes of adaptive evolution have been suggested, also displays a putative signal of diversifying selection in the human branch. At the same time, we found evidence for recent positive directional selection acting on several human testis-enriched genes (MORC1, SLC9B1, ROPN1L, DMRT1, PLCZ1, RNF17, FAM71D and WBP2NL) that play important roles in human spermatogenesis and fertilization. Most of these genes are population-specifically under positive selection.ConclusionEpisodic diversifying selection, possibly driven by sperm competition, was not an important force driving the evolution of testis-enriched genes in the human lineage. Population-specific, recent positive directional selection suggests an adaptation of male reproductive genes to different environmental conditions. Positive selection acts on eQTLS and sQTLs, indicating selective effects on important gene regulatory functions. In particular, the transcriptional diversity regulated by sQTLs in testis-enriched genes may be important for spermatocytes to respond to environmental and physiological stress.