PFN4 is required for manchette development and acrosome biogenesis during mouse spermiogenesis.

Profilin 4 (Pfn4) is expressed during spermiogenesis and localizes to the acrosome-acroplaxome-manchette complex. Here, we generated PFN4-deficient mice, with sperm displaying severe impairment in manchette formation. Interestingly, HOOK1 staining suggests that the perinuclear ring is established; however, ARL3 staining is disrupted, suggesting that lack of PFN4 does not interfere with the formation of the perinuclear ring and initial localization of HOOK1, but impedes microtubular organization of the manchette. Furthermore, amorphous head shape and flagellar defects were detected, resulting in reduced sperm motility. Disrupted cis- and trans-Golgi networks and aberrant production of proacrosomal vesicles caused impaired acrosome biogenesis. Proteomic analysis showed that the proteins ARF3, SPECC1L and FKBP1, which are involved in Golgi membrane trafficking and PI3K/AKT pathway, are more abundant in Pfn4-/- testes. Levels of PI3K, AKT and mTOR were elevated, whereas AMPK level was reduced, consistent with inhibition of autophagy. This seems to result in blockage of autophagic flux, which could explain the failure in acrosome formation. In vitro fertilization demonstrated that PFN4-deficient sperm is capable of fertilizing zona-free oocytes, suggesting a potential treatment for PFN4-related human infertility.

WDR38, a novel equatorial segment protein, interacts with the GTPase protein RAB19 and Golgi protein GM130 to play roles in acrosome biogenesis.

The WD40-repeat containing (WDR) proteins are enriched in the testis and play important roles in spermatogenesis. In the present study, we investigate the expression profile of WDR38, a novel member of the WDR protein family, in humans and mice. RT-qPCR (reverse transcription-quantitative polymerase chain reaction) results demonstrate that WDR38 mRNA is abundantly expressed in both the human and mouse testis. The expression of mouse Wdr38 is strictly regulated during development. Further immunofluorescence staining results show that WDR38 is located in the equatorial segment of the acrosome in human and mouse mature spermatozoa and is involved in acrosome biogenesis. Subcellular localization analysis reveals that the mouse Wdr38 protein is distributed in the perinuclear cytoplasm of transfected cells and colocalizes with the GTPase protein Rab19 and Golgi protein GM130. Coimmunoprecipitation (co-IP) assays demonstrate that Wdr38, Rab19 and GM130 interact with each other in the mouse testis and in HEK293T cells. In acrosome biogenesis, Wdr38, Rab19 and GM130 aggregate at the nuclear membrane to form large vesicles, and GM130 then detaches and moves towards the caudal region of the nucleus, whereas the Wdr38/Rab19 complex spreads along the dorsal nuclear edge and finally docks to the equatorial segment. These results indicate that WDR38 is a novel equatorial segment protein that interacts with the GTPase protein RAB19 and Golgi protein GM130 to play roles in acrosome biogenesis.

Testis-specific serine protease PRSS54 regulates acrosomal granule localization and sperm head morphogenesis in mice†.

Serine proteases (PRSS) constitute nearly one-third of all proteases, and many of them have been identified to be testis-specific and play significant roles during sperm development and male reproduction. PRSS54 is one of the testis-specific PRSS in mouse and human but its physiological function remains largely unclear. In the present study, we demonstrate in detail that PRSS54 exists not only in testis but also in mature sperm, exhibiting a change in protein size from 50 kDa in testis to 42 kDa in sperm. Loss of PRSS54 in mice results in male subfertility, acrosome deformation, defective sperm-zona penetration, and phenotypes of male subfertility and acrosome deformation can be rescued by Prss54 transgene. Ultrastructure analyses by transmission electronic microscopy further reveal various morphological abnormalities of Prss54-/- spermatids during spermiogenesis, including unfused vacuoles in acrosome, detachment and eccentrical localization of the acrosomal granules, and asymmetrical elongation of the nucleus. Subcellular localization of PRSS54 display that it appears in the acrosomal granule at the early phase of acrosome biogenesis, then extends along the inner acrosomal membrane, and ultimately presents in the acrosome region of the mature sperm. PRSS54 interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R, and loss of PRSS54 affects the distribution of these proteins in testis and sperm, although their protein levels are largely unaffected. Moreover, Prss54-/- sperm are more sensitive to acrosome reaction inducers.

Loss of SPACA1 function causes autosomal recessive globozoospermia by damaging the acrosome-acroplaxome complex.

STUDY QUESTION: Is the sperm acrosome membrane-associated protein 1 (SPACA1) gene critical to human globozoospermia? SUMMARY ANSWER: The biallelic loss-of-function (variant of SPACA1) causes globozoospermia as a result of acrosome-acroplaxome complex damage. WHAT IS KNOWN ALREADY: SPACA1 expression decreases in patients with globozoospermia. Spaca1 gene-disrupted mice have abnormally shaped sperm heads that resemble those of human globozoospermia. STUDY DESIGN, SIZE, DURATION: We recruited a consanguineous family with two brothers affected by infertility as a consequence of globozoospermia. The semen analysis data and ART outcomes were collected. Exome sequencing (ES) was used to identify potential pathogenic variants. Protein-protein interaction (PPI) technologies and proteomic analysis were utilized to explore the pathogenic mechanism. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two globozoospermic brothers and their consanguineous parents were recruited to identify the potential pathogenic variant through ES. A homozygous nonsense variant in the SPACA1 gene in both brothers inherited from the heterozygous parents was identified. Twenty normal fertile males were recruited as controls. Sperm ultrastructure was observed with transmission electron microscopy. Western blotting was performed to measure SPACA1 expression level in the sperm from the patients. Mass spectrometry (MS) analyses were used to identify differentially expressed proteins and to investigate proteins that interact with SPACA1. Co-immunoprecipitation (co-IP), yeast two-hybrid (Y2H) and immunofluorescence colocalization assays were used to confirm the PPI. MAIN RESULTS AND THE ROLE OF CHANCE: A nonsense variant (NM_030960.2: c.53G>A; p. Trp18*) in the SPACA1 gene was identified as the pathogenic variant in a family with globozoospermia. Patient IV:1 and Patient IV:2 had a phenotype very similar to that of Spaca1 gene-disrupted mice. The nonsense variant in SPACA1 led to premature transcriptional termination in the signal peptide, which was confirmed by western blotting. MS-based proteomics analysis showed that eight interactors of SPACA1 were differentially expressed in the patients' sperm, including actin-like Protein 7A (ACTL7A), an important component of the acrosome-acroplaxome complex. The PPI of SPACA1 and ACTL7A was confirmed via co-IP and Y2H assays. Immunofluorescence showed that SPACA1 and ACTL7A colocalized in mature sperm, revealing that these proteins were coexpressed spatially. LIMITATIONS, REASONS FOR CAUTION: Given the rarity of globozoospermia, only two patients from one family harbouring the SPACA1 variant were found. Future studies should evaluate SPACA1 variants in larger cohorts to corroborate this finding. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that the SPACA1 gene was critical for globozoospermia, which expanded the spectrum of causative genes for globozoospermia. This study also provided evidence for ICSI clinical outcomes for patients with SPACA1-deficient globozoospermia, which may guide clinical treatment strategies. Furthermore, this study explored the pathogenesis of globozoospermia caused by SPACA1 deficiency. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Precision Medical Research of National Key Research and Development Program (2018YFC1002400), National Natural Science Foundation of China (81873724), and Natural Science Foundation of Shanghai (20ZR1472700). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

IZUMO family member 3, IZUMO3, is involved in male fertility through the acrosome formation.

Many factors are involved in acrosome biogenesis in order for appropriate acrosome formation to occur. Here, we demonstrate that IZUMO family member 3, IZUMO3, plays an important role in acrosome biogenesis, as proven by gene disruption experiments. A loss of IZUMO3 in round spermatids affects acrosomal granule positioning due to lack of acrosomal granule contact with the inner acrosomal membrane, leading to the formation of grossly malformed spermatozoa associated with male subfertility. Thus, we suggest that mammalian spermiogenesis needs an elaborate acrosome biogenesis through IZUMO3 involvement.

Destruction or Reconstruction: A Subtle Liaison between the Proteolytic and Signaling Role of Protein Ubiquitination in Spermatogenesis.

Ubiquitination is one of the most diverse forms of protein post-translational modification that changes the function of the landscape of substrate proteins in response to stimuli, without the need for "de novo" protein synthesis. Ubiquitination is involved in almost all aspects of eukaryotic cell biology, from the best-studied role in promoting the removal of faulty or unnecessary proteins by the way of the ubiquitin proteasome system and autophagy-lysosome pathway to the recruitment of proteins in specific non-proteolytic signaling pathways, as emerged by the more recent discoveries about the protein signature with peculiar types of ubiquitin chains. Spermatogenesis, on its own, is a complex cellular developmental process in which mitosis, meiosis, and cell differentiation coexist so to result in the continuous formation of haploid spermatozoa. Successful spermatogenesis is thus at the same time a mixed result of the precise expression and correct intracellular destination of structural proteins and enzymes, from one hand, and the fine removal by targeted degradation of unfolded or damaged proteins as well as of obsolete, outlived proteins, from the other hand. In this minireview, I will focus on the importance of the ubiquitin system all over the spermatogenic process, discussing both proteolytic and non-proteolytic functions of protein ubiquitination. Alterations in the ubiquitin system have been in fact implicated in pathologies leading to male infertility. Notwithstanding several aspects of the multifaceted world of the ubiquitin system have been clarified, the physiological meaning of the so-called ubiquitin code remains still partially elusive. The studies reviewed in this chapter provide information that could aid the investigators to pursue new promising discoveries in the understanding of human and animal reproductive potential.

Sirt1 regulates acrosome biogenesis by modulating autophagic flux during spermiogenesis in mice.

Sirt1 is a member of the sirtuin family of proteins and has important roles in numerous biological processes. Sirt1-/- mice display an increased frequency of abnormal spermatozoa, but the mechanism of Sirt1 in spermiogenesis remains largely unknown. Here, we report that Sirt1 might be directly involved in spermiogenesis in germ cells but not in steroidogenic cells. Germ cell-specific Sirt1 knockout mice were almost completely infertile; the early mitotic and meiotic progression of germ cells in spermatogenesis were not obviously affected after Sirt1 depletion, but subsequent spermiogenesis was disrupted by a defect in acrosome biogenesis, which resulted in a phenotype similar to that observed in human globozoospermia. In addition, LC3 and Atg7 deacetylation was disrupted in spermatids after knocking out Sirt1, which affected the redistribution of LC3 from the nucleus to the cytoplasm and the activation of autophagy. Furthermore, Sirt1 depletion resulted in the failure of LC3 to be recruited to Golgi apparatus-derived vesicles and in the failure of GOPC and PICK1 to be recruited to nucleus-associated acrosomal vesicles. Taken together, these findings reveal that Sirt1 has a novel physiological function in acrosome biogenesis.

The histone methyltransferase SETD2 is required for expression of acrosin-binding protein 1 and protamines and essential for spermiogenesis in mice.

Spermatogenesis is precisely controlled by complex gene expression programs and involves epigenetic reprogramming, including histone modification and DNA methylation. SET domain-containing 2 (SETD2) is the predominant histone methyltransferase catalyzing the trimethylation of histone H3 lysine 36 (H3K36me3) and plays key roles in embryonic stem cell differentiation and somatic cell development. However, its role in male germ cell development remains elusive. Here, we demonstrate an essential role of Setd2 for spermiogenesis, the final stage of spermatogenesis. Using RNA-seq, we found that, in postnatal mouse testes, Setd2 mRNA levels dramatically increase in 14-day-old mice. Using a germ cell-specific Setd2 knockout mouse model, we also found that targeted Setd2 knockout in germ cells causes aberrant spermiogenesis with acrosomal malformation before step 8 of the round-spermatid stage, resulting in complete infertility. Furthermore, we noted that the Setd2 deficiency results in complete loss of H3K36me3 and significantly decreases expression of thousands of genes, including those encoding acrosin-binding protein 1 (Acrbp1) and protamines, required for spermatogenesis. Our findings thus reveal a previously unappreciated role of the SETD2-dependent H3K36me3 modification in spermiogenesis and provide clues to the molecular mechanisms in epigenetic disorders underlying male infertility.

Loss of the Na+/H+ exchanger NHE8 causes male infertility in mice by disrupting acrosome formation.

Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na+/H+ exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, Nhe8-/- mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from Nhe8-/- mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution, and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific Nhe8 disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na+/H+ exchanger. It points to NHE8 as a candidate gene for human globozoospermia and a possible drug target for male contraception.

Dynamic Changes in Equatorial Segment Protein 1 (SPESP1) Glycosylation During Mouse Spermiogenesis.

ESP1/SPESP1 is a testis-specific, postmeiotic gene expressed in round spermatids that encodes equatorial segment protein 1, an intra-acrosomal protein found in the acrosomal matrix and on the luminal surface of the inner and outer acrosomal membranes within the equatorial segment domain of mature spermatozoa. A comparison of testicular protein extracts with caput, corpus, and caudal epididymal sperm proteins revealed striking differences in the apparent masses of SPESP1 isoforms. The predominant isoforms of SPESP1 in the testis were 77 and 67 kDa, with 47-kDa forms present to a minor degree. In contrast, SPESP1 isoforms of 47 and 43 kDa were found in caput, corpus, and caudal sperm, indicating that SPESP1 undergoes noticeable mass changes during spermiogenesis and/or subsequent transport to the epididymis. On two-dimensional (2D) SDS-PAGE, testicular SPESP1 isoforms resolved as a train of pI values from 4.9 to 5.2. Immunoprecipitated 77-kDa SPESP1 from testis reacted with the glycoprofile stain after one-dimensional and 2D gel electrophoresis, indicating that the 77-kDa testicular isoform was highly glycosylated. One charge variant of the 67-kDa isoform was also glycoprofile positive after 2D gel resolution. The 47- and 43-kDa isoforms of SPESP1 from epididymal sperm did not stain with glycoprofile, suggesting an absence of, or few, glycoprofile-sensitive glycoconjugates in epididymal SPESP1. Treatment of testicular extracts with a variety of glycosidases resulted in mass shifts in immunoreactive SPESP1, indicating that testicular SPESP1 was glycosylated and that terminal sialic acid, N- and O-glycans were present. A mixture of deglycosidase enzymes (including PNGase-F, neuraminidase, beta1-4 galactosidase, endo-alpha-N-acetylgalactosaminidase, and beta N-acetyl-glucosaminidase) completely eliminated the 77- and 67-kDa SPESP1 bands and resulted in the appearance of 75-, 60-, 55-, 50-, 47-, and 43-kDa forms, confirming that both the 77- and 67-kDa testicular forms of SPESP1 contain complex carbohydrate residues. Treatment of caudal epididymal sperm with PNGase-F enzymes showed a faint deglycosylated band at 30 kDa, but neuraminidase did not result in any molecular shift, indicating that epididymal sperm SPESP1 did not contain sialic acid/N-acetylglucosamine residues. These findings are consistent with the hypothesis that SPSPESP1 undergoes significant glycosylation in the testis and that the majority of these glycoconjugates are removed by the time sperm reach the caput epididymis. Studies of the fate of SPESP1 after the acrosome reaction localized SPESP1 to the equatorial segment region in both noncapacitated and capacitated, acrosome-reacted sperm. During capacitation, SPESP1 underwent proteolysis, resulting in a 27-kDa fragment. Zona-free oocytes incubated with recSPESP1 protein showed complementary binding sites on the microvillar oolemmal domain. Both recSPESP1 and anti-recSPESP1 antibody inhibited in vitro fertilization.

Unraveling the Impact of the PROCA1 Mutation in Male Infertility: Incorporating Whole Exome Sequencing in Teratozoospermia Patients and Analyzing Proca1 Knockout Mice.

In the world, about 15% of couples are infertile, and nearly half of all infertility was caused by men. A large number of genetic mutations are thought to affect spermatogenesis by regulating acrosome formation. Here, we identified three patients harbouring the protein interacting with cyclin A1 (PROCA1) mutation by whole exome sequencing (WES) and Sanger sequencing among patients with predominantly acrosome-deficient teratozoospermia. However, the expression and roles of PROCA1 in infertile men remain unclear. We found that PROCA1 is predominantly expressed in the testis, where it is specifically localized to the acrosome of normal human sperm. Proca1 knockout (KO) mice were subsequently generated using CRISPR-Cas9 technology. However, Proca1 KO adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type (WT) mice. Testicular tissue or sperm morphology were not significantly different in Proca1 KO mice compared to WT mice. Expression of the acrosome markers PNA and SP56 in the acrosome was comparable between Proca1 KO and WT mice. In summary, these findings suggested that the PROCA1 mutation identified in humans does not affect acrosome biogenesis in mice.

TMC7 deficiency causes acrosome biogenesis defects and male infertility in mice.

Transmembrane channel-like (TMC) proteins are a highly conserved ion channel family consisting of eight members (TMC1-TMC8) in mammals. TMC1/2 are components of the mechanotransduction channel in hair cells, and mutations of TMC1/2 cause deafness in humans and mice. However, the physiological roles of other TMC proteins remain largely unknown. Here, we show that Tmc7 is specifically expressed in the testis and that it is required for acrosome biogenesis during spermatogenesis. Tmc7-/- mice exhibited abnormal sperm head, disorganized mitochondrial sheaths, and reduced number of elongating spermatids, similar to human oligo-astheno-teratozoospermia. We further demonstrate that TMC7 is colocalized with GM130 at the cis-Golgi region in round spermatids. TMC7 deficiency leads to aberrant Golgi morphology and impaired fusion of Golgi-derived vesicles to the developing acrosome. Moreover, upon loss of TMC7 intracellular ion homeostasis is impaired and ROS levels are increased, which in turn causes Golgi and endoplasmic reticulum stress. Taken together, these results suggest that TMC7 is required to maintain pH and ion homeostasis, which is needed for acrosome biogenesis. Our findings unveil a novel role for TMC7 in acrosome biogenesis during spermiogenesis.

The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice.

The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles toward nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knockout (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.

Repression of autophagy leads to acrosome biogenesis disruption caused by a sub-chronic oral administration of polystyrene nanoparticles.

As a new widespread contaminant, nanoplastics (NPs) pose a potential risk to human health. Nevertheless, the adverse effects of NPs on the male reproductive system are poorly understood. In this study, we aimed to determine the effects of polystyrene nanoplastics (PS-NPs) (50 nm) on sperm quality, with a focus on the acrosome defects. After 35 days of intragastric administration, sperm quality was decreased and testicular structures were impaired in mice exposed to PS-NPs in both the medium (1.0 mg/kg) and high dose (10 mg/kg) groups. No significant changes were observed in the low dose (0.2 mg/kg) group. Meanwhile, acrosome parameters including acrosome integrity and acrosome reaction were decreased after the administration of PS-NPs. These findings were consistent with the disruption of acrosome biogenesis, as identified by the changed testicular ultrastructure. Additionally, the findings were further validated using seven marker genes (Gba2, Pick1, Gopc, Hrb, Zpbp1, Spaca1 and Dpy19l2) essential for acrosome formation, which showed that two of these genes (Gopc and Dpy19l2) were significantly down-regulated. Moreover, repressed autophagy was observed in the testes of PS-NPs-exposed mice based on autophagy-related protein expression. This phenomenon was further verified in GC-2spd cells treated with PS-NPs (50 µg/mL, 100 µg/mL, 200 µg/mL for 24 h). The potential role of autophagy in such acrosome defects was explored by using the autophagy inhibitor 3-methyladenine (3-MA), autophagy activator rapamycin or beclin-1 siRNA. The results showed that Golgi-associated vesicle disorganization was exacerbated with the 3-MA and beclin-1 siRNA pretreatments, but decreased with the rapamycin pretreatment, and the expression of GOPC and DPY19L2 was also altered. These results indicated that autophagy might be involved in the PS-NPs-induced acrosome lesions based on the regulation of two key acrosome-formation proteins, GOPC and DPY19L2. Altogether, our results will provide new insights into the PS-NPs-induced male reproductive impairment.

cAMP-specific phosphodiesterase 8A and 8B isoforms are differentially expressed in human testis and Leydig cell tumor.

Cyclic adenosine monophosphate/Protein kinase A (cAMP/PKA) signaling pathway is the master regulator of endocrine tissue function. The level, compartmentalization and amplitude of cAMP response are finely regulated by phosphodiesterases (PDEs). PDE8 is responsible of cAMP hydrolysis and its expression has been characterized in all steroidogenic cell types in rodents including adrenal and Leydig cells in rodents however scarce data are currently available in humans. Here we demonstrate that human Leydig cells express both PDE8A and PDE8B isoforms. Interestingly, we found that the expression of PDE8B but not of PDE8A is increased in transformed Leydig cells (Leydig cell tumors-LCTs) compared to non-tumoral cells. Immunofluorescence analyses further reveals that PDE8A is also highly expressed in specific spermatogenic stages. While the protein is not detected in spermatogonia it accumulates nearby the forming acrosome, in the trans-Golgi apparatus of spermatocytes and spermatids and it follows the fate of this organelle in the later stages translocating to the caudal part of the cell. Taken together our findings suggest that 1) a specific pool(s) of cAMP is/are regulated by PDE8A during spermiogenesis pointing out a possible new role of this PDE8 isoform in key events governing the differentiation and maturation of human sperm and 2) PDE8B can be involved in Leydig cell transformation.

Autophagy: a multifaceted player in the fate of sperm.

BACKGROUND: Autophagy is an intracellular catabolic process of degrading and recycling proteins and organelles to modulate various physiological and pathological events, including cell differentiation and development. Emerging data indicate that autophagy is closely associated with male reproduction, especially the biosynthetic and catabolic processes of sperm. Throughout the fate of sperm, a series of highly specialized cellular events occur, involving pre-testicular, testicular and post-testicular events. Nonetheless, the most fundamental question of whether autophagy plays a protective or harmful role in male reproduction, especially in sperm, remains unclear. OBJECTIVE AND RATIONALE: We summarize the functional roles of autophagy in the pre-testicular (hypothalamic-pituitary-testis (HPG) axis), testicular (spermatocytogenesis, spermatidogenesis, spermiogenesis, spermiation) and post-testicular (sperm maturation and fertilization) processes according to the timeline of sperm fate. Additionally, critical mechanisms of the action and clinical impacts of autophagy on sperm are identified, laying the foundation for the treatment of male infertility. SEARCH METHODS: In this narrative review, the PubMed database was used to search peer-reviewed publications for summarizing the functional roles of autophagy in the fate of sperm using the following terms: 'autophagy', 'sperm', 'hypothalamic-pituitary-testis axis', 'spermatogenesis', 'spermatocytogenesis', 'spermatidogenesis', 'spermiogenesis', 'spermiation', 'sperm maturation', 'fertilization', 'capacitation' and 'acrosome' in combination with autophagy-related proteins. We also performed a bibliographic search for the clinical impact of the autophagy process using the keywords of autophagy inhibitors such as 'bafilomycin A1', 'chloroquine', 'hydroxychloroquine', '3-Methyl Adenine (3-MA)', 'lucanthone', 'wortmannin' and autophagy activators such as 'rapamycin', 'perifosine', 'metformin' in combination with 'disease', 'treatment', 'therapy', 'male infertility' and equivalent terms. In addition, reference lists of primary and review articles were reviewed for additional relevant publications. All relevant publications until August 2021 were critically evaluated and discussed on the basis of relevance, quality and timelines. OUTCOMES: (i) In pre-testicular processes, autophagy-related genes are involved in the regulation of the HPG axis; and (ii) in testicular processes, mTORC1, the main gate to autophagy, is crucial for spermatogonia stem cell (SCCs) proliferation, differentiation, meiotic progression, inactivation of sex chromosomes and spermiogenesis. During spermatidogenesis, autophagy maintains haploid round spermatid chromatoid body homeostasis for differentiation. During spermiogenesis, autophagy participates in acrosome biogenesis, flagella assembly, head shaping and the removal of cytoplasm from elongating spermatid. After spermatogenesis, through PDLIM1, autophagy orchestrates apical ectoplasmic specialization and basal ectoplasmic specialization to handle cytoskeleton assembly, governing spermatid movement and release during spermiation. In post-testicular processes, there is no direct evidence that autophagy participates in the process of capacitation. However, autophagy modulates the acrosome reaction, paternal mitochondria elimination and clearance of membranous organelles during fertilization. WIDER IMPLICATIONS: Deciphering the roles of autophagy in the entire fate of sperm will provide valuable insights into therapies for diseases, especially male infertility.

Loss of Profilin3 Impairs Spermiogenesis by Affecting Acrosome Biogenesis, Autophagy, Manchette Development and Mitochondrial Organization.

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome-manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3-/- males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3-/- sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3-/- sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3-/- testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3-ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3-/- testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

TDRD7 participates in lens development and spermiogenesis by mediating autophagosome maturation.

In humans, TDRD7 (tudor domain containing 7) mutations lead to a syndrome combining congenital cataracts (CCs) and non-obstructive azoospermia (NOA), characterized by abnormal lens development and spermiogenesis. However, the molecular mechanism underlying TDRD7's functions in eye and testicular development are still largely unknown. Here, we show that the depletion of this gene in mice and humans resulted in the accumulation of autophagosomes and the disruption of macroautophagic/autophagic flux. The disrupted autophagic flux in tdrd7-deficient mouse embryonic fibroblasts (MEFs) was caused by a failure of autophagosome fusion with lysosomes. Furthermore, transcriptome analysis and biochemical assays showed that TDRD7 might directly bind to Tbc1d20 mRNAs and downregulate its expression, which is a key regulator of autophagosome maturation, resulting in the disruption of autophagosome maturation. In addition, we provide evidence to show that TDRD7-mediated autophagosome maturation maintains lens transparency by facilitating the removal of damaged proteins and organelles from lens fiber cells and the biogenesis of acrosome. Altogether, our results showed that TDRD7 plays an essential role in the maturation of autophagosomes and that tdrd7 deletion results in eye defects and testicular abnormalities in mice, implicating disrupted autophagy might be the mechanism that contributes to lens development and spermiogenesis defects in human.Abbreviations: CB: chromatoid bodies; CC: congenital cataract; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; LAMP1: lysosomal-associated membrane protein 1; LECs: lens epithelial cells; MAP1LC3/LC3/Atg8: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; NOA: non-obstructive azoospermia; OFZ: organelle-free zone; RG: RNA granules; SQSTM1/p62: sequestosome 1; TBC1D20: TBC1 domain family member 20; TDRD7: tudor domain containing 7; TEM: transmission electron microscopy; WT: wild type.

Mechanism of Acrosome Biogenesis in Mammals.

During sexual reproduction, two haploid gametes fuse to form the zygote, and the acrosome is essential to this fusion process (fertilization) in animals. The acrosome is a special kind of organelle with a cap-like structure that covers the anterior portion of the head of the spermatozoon. The acrosome is derived from the Golgi apparatus and contains digestive enzymes. With the progress of our understanding of acrosome biogenesis, a number of models have been proposed to address the origin of the acrosome. The acrosome has been regarded as a lysosome-related organelle, and it has been proposed to have originated from the lysosome or the autolysosome. Our review will provide a brief historical overview and highlight recent findings on acrosome biogenesis in mammals.

Flotillin-2 is an acrosome-related protein involved in mouse spermiogenesis.

SPERMATOGENESIS IS A COMPLEX PROCESS OF TERMINAL DIFFERENTIATION BY WHICH MATURE SPERMS ARE GENERATED, AND IT CAN BE DIVIDED INTO THREE PHASES: mitosis, meiosis and spermiogenesis. In a previous study, we established a series of proteomic profiles for spermatogenesis to understand the regulation of male fertility and infertility. Here, we further investigated the localization and the role of flotillin-2 in spermiogenesis. Flotillin-2 expression was investigated in the testis of male CD1 mice at various developmental stages of spermatogenesis by using Western blotting, immunohistochemistry and immunofluorescence. Flotillin-2 was knocked down in vivo in three-week-old male mice using intratesticular injection of small inhibitory RNA (siRNA), and sperm abnormalities were assessed three weeks later. Flotillin-2 was expressed at high levels in male germ cells during spermatogenesis. Flotillin-2 immunoreactivity was observed in pachytene spermatocytes as a strong dot-shaped signal and in round spermatids as a sickle-shaped distribution ahead of the acrosome. Immunofluorescence confirmed flotillin-2 was localized in front of the acrosome in round spermatids, indicating that flotillin-2 was localized to the Golgi apparatus. Knockdown of flotillin-2 in vivo led to a significant increase in head sperm abnormalities isolated from the cauda epididymis, compared with control siRNA-injected testes. This study indicates that flotillin-2 is a novel Golgi-related protein involved in sperm acrosome biogenesis.