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Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin-eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients' spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

Family with sequence similarity 181 (Fam181) is a gene family with two paralogues (Fam181a and Fam181b) found among vertebrates. Fam181a exhibits dynamic and stage-specific expression during murine embryo development. Furthermore, searching in the National Center for Biotechnology Information database revealed predominant expression of Fam181a in mouse and human testes, implying that it may have essential roles in spermatogenesis. In this study we investigated the in vivo function of Fam181a in mouse spermatogenesis and fertility by generating Fam181a–/– mice using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 genome editing technology. The resulting Fam181a–/– mice exhibited normal growth and development. In addition, the mice were completely fertile, with no obvious differences in the testis-to-bodyweight ratio, epididymal sperm count or sperm motility compared with wild-type mice. Further examination of testicular and epididymal histology of Fam181a–/– mice found an intact seminiferous tubule structure and the presence of all types of germ cells, from spermatogonia to mature spermatozoa, similar to wild-type littermates. Similarly, analysis of meiotic prophase I progression revealed normal populations of each substage of prophase I in Fam181a+/+ and Fam181a–/– testes, suggesting that this gene is dispensable for male fertility. These negative findings will help avoid research overlap, save time and resources and allow researchers to concentrate on genes that are critical for male fertility and spermatogenesis.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a specific type of asthenoteratozoospermia, presenting with multiple morphological anomalies in spermatozoa, such as absent, bent, coiled, short, or irregular caliber flagella. Previous genetic studies revealed pathogenic mutations in genes encoding cilia and flagella-associated proteins (CFAPs; e.g., CFAP43, CFAP44, CFAP65, CFAP69, CFAP70, and CFAP251) responsible for the MMAF phenotype in infertile men from different ethnic groups. However, none of them have been identified in infertile Pakistani males with MMAF. In the current study, two Pakistani families with MMAF patients were recruited. Whole-exome sequencing (WES) of patients and their parents was performed. WES analysis reflected novel biallelic loss-of-function mutations in CFAP43 in both families (Family 1: ENST00000357060.3, p.Arg300Lysfs*22 and p.Thr526Serfs*43 in a compound heterozygous state; Family 2: ENST00000357060.3, p.Thr526Serfs*43 in a homozygous state). Sanger sequencing further confirmed that these mutations were segregated recessively in the families with the MMAF phenotype. Semiquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out to detect the effect of the mutation on mRNA of the affected gene. Previous research demonstrated that biallelic loss-of-function mutations in CFAP43 accounted for the majority of all CFAP43-mutant MMAF patients. To the best of our knowledge, this is the first study to report CFAP43 biallelic loss-of-function mutations in a Pakistani population with the MMAF phenotype. This study will help researchers and clinicians to understand the genetic etiology of MMAF better.

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.

Fascins belong to a family of actin-bundling proteins that are involved in a wide range of biological functions. FSCN3, a newly identified testis-specific actin-bundling protein, is specifically expressed in elongated spermatids. However, its in vivo function in mouse spermiogenesis remains unknown.We generated Fscn3 knockout mice through CRISPR/Cas9 gene-editing technology. Fscn3Our study provides the first evidence that FSCN3 is a testis-specific actin-bundling protein that is not required for mouse spermatogenesis. Our results will help reproductive biologists focus their efforts on genes that are crucial for fertility and avoid research duplication.

Infertility is a multifactorial disorder that affects approximately 12% of couples of childbearing ages worldwide. Few studies have been conducted to understand the genetic causes of infertility in depth. The synaptonemal complex (SC), which is essential for the progression of meiosis, is a conserved tripartite structure that binds homologous chromosomes together and is thus required for fertility. This study investigated genetic causes of infertility in a Pakistani consanguineous family containing two patients suffering from non-obstructive azoospermia (NOA). We performed whole-exome sequencing, followed by Sanger sequencing, and identified a novel pathogenic variant (c.7G A [p.D3N]) in the SC coding gene C14orf39, which was recessively co-segregated with NOA. In silico analysis revealed that charges on wild-type residues were lost, which may result in loss of interactions with other molecules and residues, and a reduction in protein stability occurred, which was caused by the p.D3N mutation. The novel variant generated the mutant protein C14ORF39

Sustaining and maintaining the intricate process of spermatogenesis is liable upon hormones and growth factors acting through endocrine and paracrine pathways. The Sertoli cells (SCs) are the major somatic cells present in the seminiferous tubules and are considered to be the main regulators of spermatogenesis. As each Sertoli cell supports a specific number of germ cells, thus, the final number of Sertoli cells determines the sperm production capacity. Similarly, sex hormones are also major regulators of spermatogenesis and they can determine the proliferation of Sertoli cells. In the present review, we have critically and comprehensively discussed the role of sex hormones and some other factors that are involved in Sertoli cell proliferation, differentiation and maturation. Furthermore, we have also presented a model of Sertoli cell development based upon the recent advancement in the field of reproduction. Hence, our review article provides a general overview regarding the sex hormonal pathways governing Sertoli cell proliferation and development.

Summary Human infertility is a multifactorial disease that affects 8%–12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs∗2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320∗]) and a splicing mutation (c.1180−3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.

Family with sequence similarity 181 (Fam181) is a gene family with two paralogues (Fam181a and Fam181b) found among vertebrates. Fam181a exhibits dynamic and stage-specific expression during murine embryo development. Furthermore, searching in the National Center for Biotechnology Information database revealed predominant expression of Fam181a in mouse and human testes, implying that it may have essential roles in spermatogenesis. In this study we investigated the invivo function of Fam181a in mouse spermatogenesis and fertility by generating Fam181a-/- mice using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 genome editing technology. The resulting Fam181a-/- mice exhibited normal growth and development. In addition, the mice were completely fertile, with no obvious differences in the testis-to-bodyweight ratio, epididymal sperm count or sperm motility compared with wild-type mice. Further examination of testicular and epididymal histology of Fam181a-/- mice found an intact seminiferous tubule structure and the presence of all types of germ cells, from spermatogonia to mature spermatozoa, similar to wild-type littermates. Similarly, analysis of meiotic prophase I progression revealed normal populations of each substage of prophase I in Fam181a+/+ and Fam181a-/- testes, suggesting that this gene is dispensable for male fertility. These negative findings will help avoid research overlap, save time and resources and allow researchers to concentrate on genes that are critical for male fertility and spermatogenesis.

Serine/threonine kinases domain-containing proteins are known to play important functions in sperm flagella and male fertility. However, the roles of these proteins in human reproduction remain poorly understood and whether their variants are associated with human asthenozoospermia have not been reported. Here, we recruited a Pakistani family having four infertile patients diagnosed with idiopathic asthenozoospermia without any ciliary-related symptoms. Whole-exome sequencing identified a novel homozygous frameshift mutation (c.1235del, p.T412Kfs*14) in serine/threonine kinase 33 (STK33), which displays a highly conserved and predominant expression in testis in humans. This variant led to a dramatic reduction of STK33 messenger RNA (mRNA) in the patients. Patients homozygous for the STK33 variant presented reduced sperm motility, frequent morphological abnormalities of sperm flagella and completely disorganized flagellar ultrastructures, which are typical for multiple morphological abnormalities of the flagella (MMAF) phenotypes. Overall, these findings present evidence establishing that STK33 is an MMAF-related gene and provide new insights for understanding the role of serine/threonine kinases domain-containing proteins in human male reproduction.

Several genes have been reported to be involved in spermatogenesis but their functional importance in male fertility is yet needed to be elucidated. Therefore, in current research, we focused to explore the in vivo role of evolutionary conserved and testis-specifically expressed, C4orf46, gene in male mouse fertility and spermatogenesis. The expression profile of C4orf46 is specific to testes and expressed in testes from 7 days of postpartum to onward. Thus, we generated the C4orf46 knockout mice by utilizing CRISPR/Cas9 genome editing technology and examined gene function in spermatogenesis and fertility. Surprisingly, C4orf46 knockout mice were completely fertile, displayed normal testes morphology, however, higher sperm contents were observed in knockout mice compared to wild type (WT) littermates. Subsequently, intact testis histology and architecture of seminiferous tubules were observed in C4orf46 knockout and WT mice. Similarly, sperm morphology and swimming velocity of C4orf46 knockout mice were comparable with the WT littermates. Furthermore, all type of germ cells ranging from spermatogonia to mature spermatozoa were observed in the testes and epididymis sections of C4orf46 knockout mice suggesting that disruption of C4orf46 did not impact spermatogenesis. Moreover, meiotic prophase I progression was normal, and each type of cell population was comparable between knockout and WT mice. Overall, finding from this research indicates that C4orf46 is not an essential gene for fertility in mice. This study will help researchers to avoid the repetition and duplication of efforts, and to explore the genes that are indispensable for spermatogenesis and male fertility.

Family with sequence similarity 114 member A2 (Fam114a2) is sperm binding protein that is highly conserved in mammals with homologs both in fungi and plants. Previous studies have demonstrated that miR-762 and P63 are two crucial players of spermatogenesis, and CricFM114A2 regulates their expression. Thus, the current study was focused on describing the role of Fam114a2 in spermatogenesis by generating Fam114a2 knockout (Fam114a2-/-) mice using CRISPR/Cas9 genome editing techniques. We identified that Fam114a2-/- mouse has normal fertility and normal morphology of sperm. Furthermore, histological investigation of testicular and epididymis tissues showed no subtle difference, and seminiferous tubules comprised of all stages of germ cells, including mature spermatozoa in Fam114a2-/- mice. Moreover, cytological investigation of spermatocytes in the progression of prophase I also did not display any notable difference in Fam114a2-/- mice. Additionally, normal expression of p63 and miR-762 was observed in Fam114a2+/+ and Fam114a2-/- testis indicating that Fam114a2 is not involved in the direct regulation of in mice spermatogenesis. Moreover, the removal of Fam114a2 in mouse did not affect the expression of its paralogue Fam114a1 in multiple tissues. Taken together our data determined that Fam114a2 is not essential for male fertility and spermatogenesis in mice.

Research question Male infertility is a global issue worldwide and multiple morphological abnormalities of the sperm flagella (MMAF) is one of the most severe forms of the qualitative sperm defects with a heterogeneous genetic cause that has not been completely understood. Can whole-exome sequencing (WES) reveal novel genetic causes contributing to MMAF in a consanguineous Pakistani family, comprising three infertile brothers? Design WES and bioinformatic analysis were conducted to screen potential pathogenic variants. The identified variant was validated by Sanger sequencing in all available family members Transmission electron microscopy analyses was carried out to examine the flagella ultrastructure of spermatozoa from patient. Results WES and Sanger sequencing identified a novel homozygous stop-gain mutation (ENST00000392644.4, c.182C>G, p.S61X) in ARMC2, which is expected to lead to loss of protein functions. Transmission electron microscopy analyses revealed that the flagellar ultrastructure of the patient's spermatozoa was disorganized along with a complete absence of central pair complex (CPC), suggesting that ARMC2 is involved in the assembly, stability of the axonemal complex, or both, particularly the CPC. Conclusion We report that a familial stop-gain mutation in ARMC2 is associated with male infertility in humans caused by MMAF accompanied with loss of CPCs and axonemal disorganization. We provide genetic evidence that ARMC2 is essential for human spermatogenesis and its mutation may be pathogenic for MMAF. These findings will improve the knowledge about the genetic basis of MMAF and provide information for genetic counselling of this disease.

Exonuclease 5 (Exo5) belongs to a class of bi-directional, ssDNA-specific exonucleases that mainly involved in the DNA repair pathways. Exo5 has been reported to be crucial for DNA- DNA mismatch repair (MMR) in several human cell lines. However, its in vivo function in mammals still needs to be explored. Thus, to study the in vivo role of Exo5 in spermatogenesis, Exo5 knockout mice were generated using CRISPR/Cas9 technology. Unexpectedly, we found that the knockout mice are fertile despite a slight decrease in sperm count. Furthermore, Exo5-/- mice showed no detectable developmental anomalies, exhibited no remarkable differences in the epididymal histology and testis/body weight ratio. Moreover, cytological investigations on meiocytes revealed non-significant differences in chromosomal synapsis, recombination, and meiotic progression of prophase I, further demonstrating that Exo5 has no essential role in spermatogenesis in mice under normal breeding conditions. Collectively, these data indicate that Exo5 is dispensable for meiotic progression and fertility in mice.