Differentiation Disorders of Chara vulgaris Spermatids following Treatment with Propyzamide.

Microtubules are cytoskeletal cell elements that also build flagella and cilia. Moreover, these structures participate in spermatogenesis and form a microtubular manchette during spermiogenesis. The present study aims to assess the influence of propyzamide, a microtubule-disrupting agent, on alga Chara vulgaris spermatids during their differentiation by means of immunofluorescent and electron microscopy methods. Propyzamide blocks the functioning of the ß-tubulin microtubule subunit, which results in the creation of a distorted shape of a sperm nucleus at some stages. Present ultrastructural studies confirm these changes. In nuclei, an altered chromatin arrangement and nuclear envelope fragmentation were observed in the research as a result of incorrect nucleus-cytoplasm transport behavior that disturbed the action of proteolytic enzymes and the chromatin remodeling process. In the cytoplasm, large autolytic vacuoles and the dilated endoplasmic reticulum (ER) system, as well as mitochondria, were revealed in the studies. In some spermatids, the arrangement of microtubules present in the manchette was disturbed and the structure was also fragmented. The observations made in the research at present show that, despite some differences in the manchette between Chara and mammals, and probably also in the alga under study, microtubules participate in the intramanchette transport (IMT) process, which is essential during spermatid differentiation. In the present study, the effect of propyzamide on Chara spermiogenesis is also presented for the first time; however, the role of microtubule-associated proteins in this process still needs to be elucidated in the literature.

Generation and Characterization of a Transgenic Mouse That Specifically Expresses the Cre Recombinase in Spermatids.

Spermiogenesis is the step during which post-meiotic cells, called spermatids, undergo numerous morphological changes and differentiate into spermatozoa. Thousands of genes have been described to be expressed at this stage and could contribute to spermatid differentiation. Genetically-engineered mouse models using Cre/LoxP or CrispR/Cas9 are the favored approaches to characterize gene function and better understand the genetic basis of male infertility. In the present study, we produced a new spermatid-specific Cre transgenic mouse line, in which the improved iCre recombinase is expressed under the control of the acrosomal vesicle protein 1 gene promoter (Acrv1-iCre). We show that Cre protein expression is restricted to the testis and only detected in round spermatids of stage V to VIII seminiferous tubules. The Acrv1-iCre line can conditionally knockout a gene during spermiogenesis with a > 95% efficiency. Therefore, it could be useful to unravel the function of genes during the late stage of spermatogenesis, but it can also be used to produce an embryo with a paternally deleted allele without causing early spermatogenesis defects.

DOT1L promotes spermatid differentiation by regulating expression of genes required for histone-to-protamine replacement.

Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is histone-to-protamine exchange, which must be executed correctly to avoid sperm DNA damage, embryonic lethality and male sterility. Here, we define an essential role for the histone methyltransferase DOT1L in the histone-to-protamine transition. We show that DOT1L is abundantly expressed in mouse meiotic and postmeiotic germ cells, and that methylation of histone H3 lysine 79 (H3K79), the modification catalyzed by DOT1L, is enriched in developing spermatids in the initial stages of histone replacement. Elongating spermatids lacking DOT1L fail to fully replace histones and exhibit aberrant protamine recruitment, resulting in deformed sperm heads and male sterility. Loss of DOT1L results in transcriptional dysregulation coinciding with the onset of histone replacement and affecting genes required for histone-to-protamine exchange. DOT1L also deposits H3K79me2 and promotes accumulation of elongating RNA Polymerase II at the testis-specific bromodomain gene Brdt. Together, our results indicate that DOT1L is an important mediator of transcription during spermatid differentiation and an indispensable regulator of male fertility.

Disrupted intercellular bridges and spermatogenesis in fatty acyl-CoA reductase 1 knockout mice: A new model of ether lipid deficiency.

Peroxisomal fatty acyl-CoA reductase 1 (FAR1) is a rate-limiting enzyme for ether lipid (EL) synthesis. Gene mutations in FAR1 cause a rare human disease. Furthermore, altered EL homeostasis has also been associated with various prevalent human diseases. Despite their importance in human health, the exact cellular functions of FAR1 and EL are not well-understood. Here, we report the generation and initial characterization of the first Far1 knockout (KO) mouse model. Far1 KO mice were subviable and displayed growth retardation. The adult KO male mice had smaller testes and were infertile. H&E and immunofluorescent staining showed fewer germ cells in seminiferous tubules. Round spermatids were present but no elongated spermatids or spermatozoa were observed, suggesting a spermatogenesis arrest at this stage. Large multi-nucleated giant cells (MGC) were found lining the lumen of seminiferous tubules with many of them undergoing apoptosis. The immunofluorescent signal of TEX14, an essential component of intercellular bridges (ICB) between developing germ cells, was greatly reduced and mislocalized in KO testis, suggesting the disrupted ICBs as an underlying cause of MGC formation. Integrative analysis of our total testis RNA-sequencing results and published single-cell RNA-sequencing data unveiled cell type-specific molecular alterations underlying the spermatogenesis arrest. Many genes essential for late germ cell development showed dramatic downregulation, whereas genes essential for extracellular matrix dynamics and cell-cell interactions were among the most upregulated genes. Together, this work identified the cell type-specific requirement of ELs in spermatogenesis and suggested a critical role of Far1/ELs in the formation/maintenance of ICB during meiosis.

DNALI1 interacts with the MEIG1/PACRG complex within the manchette and is required for proper sperm flagellum assembly in mice.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

Small RNA shuffling between murine sperm and their cytoplasmic droplets during epididymal maturation.

Reports that mouse sperm gain small RNAs from the epididymosomes secreted by epididymal epithelial cells and that these "foreign" small RNAs act as an epigenetic information carrier mediating the transmission of acquired paternal traits have drawn great attention because the findings suggest that heritable information can flow from soma to germ line, thus invalidating the long-standing Weismann's barrier theory on heritable information flow. Using small RNA sequencing (sRNA-seq), northern blots, sRNA in situ hybridization, and immunofluorescence, we detected substantial changes in the small RNA profile in murine caput epididymal sperm (sperm in the head of the epididymis), and we further determined that the changes resulted from sperm exchanging small RNAs, mainly tsRNAs and rsRNAs, with cytoplasmic droplets rather than the epididymosomes. Moreover, the murine sperm-borne small RNAs were mainly derived from the nuclear small RNAs in late spermatids. Thus, caution is needed regarding sperm gaining foreign small RNAs as an underlying mechanism of epigenetic inheritance.

LCRMP-1 is required for spermatogenesis and stabilises spermatid F-actin organization via the PI3K-Akt pathway.

Long-form collapsin response mediator protein-1 (LCRMP-1) belongs to the CRMP family which comprises brain-enriched proteins responsible for axon guidance. However, its role in spermatogenesis remains unclear. Here we find that LCRMP-1 is abundantly expressed in the testis. To characterize its physiological function, we generate LCRMP-1-deficient mice (Lcrmp-1-/-). These mice exhibit aberrant spermiation with apoptotic spermatids, oligospermia, and accumulation of immature testicular cells, contributing to reduced fertility. In the seminiferous epithelial cycle, LCRMP-1 expression pattern varies in a stage-dependent manner. LCRMP-1 is highly expressed in spermatids during spermatogenesis and especially localized to the spermiation machinery during spermiation. Mechanistically, LCRMP-1 deficiency causes disorganized F-actin due to unbalanced signaling of F-actin dynamics through upregulated PI3K-Akt-mTOR signaling. In conclusion, LCRMP-1 maintains spermatogenesis homeostasis by modulating cytoskeleton remodeling for spermatozoa release.

In vitro spermatogenesis in isolated seminiferous tubules of immature mice.

Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.

TSKS localizes to nuage in spermatids and regulates cytoplasmic elimination during spermiation.

Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding sequence of testis-specific serine kinase substrate (TSKS) in mice and demonstrate that TSKS is essential for male fertility through the formation of both RB and CR, prominent sites of TSKS localization. Due to the lack of TSKS-derived nuage (TDN), the cytoplasmic contents cannot be eliminated from spermatid cytoplasm in Tsks knockout mice, resulting in excess residual cytoplasm with an abundance of cytoplasmic materials and inducing an apoptotic response. In addition, ectopic expression of TSKS in cells results in formation of amorphous nuage-like structures; dephosphorylation of TSKS helps to induce nuage, while phosphorylation of TSKS blocks the formation. Our results indicate that TSKS and TDN are essential for spermiation and male fertility by eliminating cytoplasmic contents from the spermatid cytoplasm.

An ancient testis-specific IQ motif-containing H gene regulates specific transcript isoform expression during spermatogenesis.

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications - principally increased intron retention - resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biology.

Single-cell RNA-sequencing reveals the transcriptional landscape of ND-42 mediated spermatid elongation via mitochondrial derivative maintenance in Drosophila testes.

During spermatogenesis, mitochondria extend along the whole length of spermatid tail and offer a structural platform for microtubule reorganization and synchronized spermatid individualization, that eventually helps to generate mature sperm in Drosophila. However, the regulatory mechanism of spermatid mitochondria during elongation remains largely unknown. Herein, we demonstrated that NADH dehydrogenase (ubiquinone) 42 kDa subunit (ND-42) was essential for male fertility and spermatid elongation in Drosophila. Moreover, ND-42 depletion led to mitochondrial disorders in Drosophila testes. Based on single-cell RNA-sequencing (scRNA-seq), we identified 15 distinct cell clusters, including several unanticipated transitional subpopulations or differentiative stages for testicular germ cell complexity in Drosophila testes. Enrichments of the transcriptional regulatory network in the late-stage cell populations revealed key roles of ND-42 in mitochondria and its related biological processes during spermatid elongation. Notably, we demonstrated that ND-42 depletion led to maintenance defects of the major mitochondrial derivative and the minor mitochondrial derivative by affecting mitochondrial membrane potential and mitochondrial-encoded genes. Our study proposes a novel regulatory mechanism of ND-42 for spermatid mitochondrial derivative maintenance, contributing to a better understanding of spermatid elongation.

miRNA Expression Profiles of Mouse Round Spermatids in GRTH/DDX25-Mediated Spermiogenesis: mRNA-miRNA Network Analysis.

GRTH/DDX25 is a testis-specific DEAD-box family of RNA helicase, which plays an essential role in spermatogenesis and male fertility. There are two forms of GRTH, a 56 kDa non-phosphorylated form and a 61 kDa phosphorylated form (pGRTH). GRTH-KO and GRTH Knock-In (KI) mice with R242H mutation (lack pGRTH) are sterile with a spermatogenic arrest at step 8 of spermiogenesis due to failure of round spermatids (RS) to elongate. We performed mRNA-seq and miRNA-seq analysis on RS of WT, KI, and KO to identify crucial microRNAs (miRNAs) and mRNAs during RS development by establishing a miRNA-mRNA network. We identified increased levels of miRNAs such as miR146, miR122a, miR26a, miR27a, miR150, miR196a, and miR328 that are relevant to spermatogenesis. mRNA-miRNA target analysis on these DE-miRNAs and DE-mRNAs revealed miRNA target genes involved in ubiquitination process (Ube2k, Rnf138, Spata3), RS differentiation, and chromatin remodeling/compaction (Tnp1/2, Prm1/2/3, Tssk3/6), reversible protein phosphorylation (Pim1, Hipk1, Csnk1g2, Prkcq, Ppp2r5a), and acrosome stability (Pdzd8). Post-transcriptional and translational regulation of some of these germ-cell-specific mRNAs by miRNA-regulated translation arrest and/or decay may lead to spermatogenic arrest in KO and KI mice. Our studies demonstrate the importance of pGRTH in the chromatin compaction and remodeling process, which mediates the differentiation of RS into elongated spermatids through miRNA-mRNA interactions.

[Short-term exposure to gossypol causes reversible reproductive toxicity and nephrotoxicity in mice].

OBJECTIVE: To study the toxic effects of short-term exposure to gossypol on the testis and kidney in mice and whether these effects are reversible. METHODS: Twenty 7 to 8-week-old male mice were randomized into blank control group, solvent control group, gossypol treatment group and drug withdrawal group. In the former 3 groups, the mice were subjected to daily intragastric administration of 0.3 mL of purified water, 1% sodium carboxymethylcellulose solution, and 30 mg/mL gossypol solution for 14 days, respectively; In the drug withdrawal group, the mice were treated with gossypol solution in the same manner for 14 days followed by treatment with purified water for another 14 days. After the last administration, the mice were euthanized and tissue samples were collected. The testicular tissue was weighed and observed microscopically with HE and PAS staining; the kidney tissue was stained with HE and examined for mitochondrial ATPase activity. RESULTS: Compared with those in the control group, the mice with gossypol exposure showed reduced testicular seminiferous epithelial cells with rounded seminiferous tubules, enlarged space between the seminiferous tubules, interstitium atrophy of the testis, and incomplete differentiation of the spermatogonia. The gossypol-treated mice also presented with complete, non-elongated spermatids, a large number of cells in the state of round spermatids, and negativity for acrosome PAS reaction; diffuse renal mesangial cell hyperplasia, increased mesangial matrix, and adhesion of the mesangium to the wall of the renal capsule were observed, with significantly shrinkage or even absence of the lumens of the renal capsules and reduced kidney mitochondrial ATPase activity. Compared with the gossypol-treated mice, the mice in the drug withdrawal group showed obvious recovery of morphologies of the testis and the kidney, acrosome PAS reaction and mitochondrial ATPase activity. CONCLUSIONS: Shortterm treatment with gossypol can cause reproductive toxicity and nephrotoxicity in mice, but these toxic effects can be reversed after drug withdrawal.

SUN4 is a spermatid type II inner nuclear membrane protein that forms heteromeric assemblies with SUN3 and interacts with lamin B3.

SUN domain proteins are conserved proteins of the nuclear envelope and key components of the LINC complexes (for 'linkers of the nucleoskeleton and the cytoskeleton'). Previous studies have demonstrated that the testis-specific SUN domain protein SUN4 (also known as SPAG4) is a vital player in the directed shaping of the spermatid nucleus. However, its molecular properties relating to this crucial function have remained largely unknown, and controversial data for the organization and orientation of SUN4 within the spermatid nuclear envelope have been presented so far. Here, we have re-evaluated this issue in detail and show robust evidence that SUN4 is integral to the inner nuclear membrane, sharing a classical SUN domain protein topology. The C-terminal SUN domain of SUN4 localizes to the perinuclear space, whereas the N-terminus is directed to the nucleoplasm, interacting with the spermiogenesis-specific lamin B3. We found that SUN4 forms heteromeric assemblies with SUN3 in vivo and regulates SUN3 expression. Together, our results contribute to a better understanding of the specific function of SUN4 at the spermatid nucleo-cytoplasmic junction and the process of sperm-head formation.

Localization and expression of Orexin B (OXB) and its type 2 receptor (OX2R) in mouse testis during postnatal development.

The orexins (OXs) were first reported in hypothalamus of rat, and they play an important role in diverse physiological actions. The OXs consist of orexin A (OXA) and orexin B (OXB) peptides and their actions are mediated via two G-protein-coupled receptors, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R), respectively. Presence of OXA and OX1R has been also reported in peripheral organs like reproductive tissues. These findings, therefore, highlight a possible role of OXs and their receptors in male reproductive health. Though, expression and localization of OXB and OX2R in the testis and their role in spermatogenesis are not finally clarified. Herein, we elucidated the localization and the patterns of expression of OXB and OX2R in Parkes mice testes during postnatal development. Results suggest that the precursor prepro-orexin (PPO), OXB and OX2R are expressed at the transcript and protein levels in mouse testis throughout the postnatal development. Immunostaining further showed the localization of OXB and OX2R both in interstitium and tubular compartments of the testis. On 7 day postpartum (7 dpp), only spermatogonia showed immunoreactivity of OXB and OX2R, while at 14, 28, 42 and 90 dpp, immunolocalization of OXB and OX2R were noted in the seminiferous tubules, especially in leptotene, pachytene spermatocytes, round and elongating spermatids, and in Leydig cells and Sertoli cells. The immunoreactivity of OXB and OX2R appeared to be stage-specific in adult mouse testis. The results suggest the expression of OXB and OX2R in mouse testis and their possible regulatory role in spermatogenesis and steroidogenesis.

Differentiation-linked changes in the biosynthesis and turnover of sphingomyelins in rat male germ cells: Genes involved and effects of testosterone.

In rodents, sphingomyelins (SMs) species with very-long-chain polyunsaturated fatty acid (VLCPUFA) are required for normal spermatogenesis. Data on the expression of enzymes with roles in their biosynthesis and turnover during germ cell differentiation and on possible effects on such expression of testosterone (Tes), known to promote this biological process, were lacking. Here we quantified, in isolated pachytene spermatocytes (PtS), round spermatids (RS), and later spermatids (LS), the mRNA levels from genes encoding ceramide (Cer), glucosylceramide (GlcCer), and SM synthases (Cers3, Gcs, Sms1, and Sms2) and sphingomyelinases (aSmase, nSmase) and assessed products of their activity in cells in culture using nitrobenzoxadiazole (NBD)-labeled substrates and [3H]palmitate as precursor. Transcript levels from Cers3 and Gcs were maximal in PtS. While mRNA levels from Sms1 increased with differentiation in the direction PtS→RS→LS, those from Sms2 increased between PtS and RS but decreased in LS. In turn, the nSmase transcript increased in the PtS→RS→LS order. During incubations with NBD-Cer, spermatocytes produced more GlcCer and SM than did spermatids. In total germ cells cultured for up to 25 h with NBD-SM, not only abundant NBD-Cer but also NBD-GlcCer were formed, demonstrating SM→Cer turnover and Cer recycling. After 20 h with [3H]palmitate, PtS produced [3H]SM and RS formed [3H]SM and [3H]Cer, all containing VLCPUFA, and Tes increased their labeling. In total germ cells, Tes augmented in 5 h the expression of genes with roles in VLCPUFA synthesis, decreased the mRNA from Sms2, and increased that from nSmase. Thus, Tes enhanced or accelerated the metabolic changes occurring to VLCPUFA-SM during germ cell differentiation.

A shared 'vulnerability code' underpins varying sources of DNA damage throughout paternal germline transmission in mouse.

During mammalian spermatogenesis, the paternal genome is extensively remodelled via replacement of histones with protamines forming the highly compact mature sperm nucleus. Compaction occurs in post-meiotic spermatids and is accompanied by extensive double strand break (DSB) formation. We investigate the epigenomic and genomic context of mouse spermatid DSBs, identifying primary sequence motifs, secondary DNA structures and chromatin contexts associated with this damage. Consistent with previously published results we find spermatid DSBs positively associated with short tandem repeats and LINE elements. We further show spermatid DSBs preferentially occur in association with (CA)n, (NA)n and (RY)n repeats, in predicted Z-DNA, are not associated with G-quadruplexes, are preferentially found in regions of low histone mark coverage and engage the remodelling/NHEJ factor BRD4. Locations incurring DSBs in spermatids also show distinct epigenetic profiles throughout later developmental stages: regions retaining histones in mature sperm, regions susceptible to oxidative damage in mature sperm, and fragile two-cell like embryonic stem cell regions bound by ZSCAN4 all co-localise with spermatid DSBs and with each other. Our results point to a common 'vulnerability code' unifying several types of DNA damage occurring on the paternal genome during reproduction, potentially underpinned by torsional changes during sperm chromatin remodelling.

Single-Cell Transcriptomic Profiling of the Mouse Testicular Germ Cells Reveals Important Role of Phosphorylated GRTH/DDX25 in Round Spermatid Differentiation and Acrosome Biogenesis during Spermiogenesis.

Gonadotropin-regulated testicular RNA helicase (GRTH)/DDX25 is a member of DEAD-box family of RNA helicase essential for the completion of spermatogenesis and male fertility, as evident from GRTH-knockout (KO) mice. In germ cells of male mice, there are two species of GRTH, a 56 kDa non-phosphorylated form and 61 kDa phosphorylated form (pGRTH). GRTH Knock-In (KI) mice with R242H mutation abolished pGRTH and its absence leads to infertility. To understand the role of the GRTH in germ cell development at different stages during spermatogenesis, we performed single-cell RNA-seq analysis of testicular cells from adult WT, KO and KI mice and studied the dynamic changes in gene expression. Pseudotime analysis revealed a continuous developmental trajectory of germ cells from spermatogonia to elongated spermatids in WT mice, while in both KO and KI mice the trajectory was halted at round spermatid stage indicating incomplete spermatogenesis process. The transcriptional profiles of KO and KI mice were significantly altered during round spermatid development. Genes involved in spermatid differentiation, translation process and acrosome vesicle formation were significantly downregulated in the round spermatids of KO and KI mice. Ultrastructure of round spermatids of KO and KI mice revealed several abnormalities in acrosome formation that includes failure of pro-acrosome vesicles to fuse to form a single acrosome vesicle, and fragmentation of acrosome structure. Our findings highlight the crucial role of pGRTH in differentiation of round spermatids into elongated spermatids, acrosome biogenesis and its structural integrity.

es-Arp3 and es-Eps8 regulate spermatogenesis via microfilaments in the seminiferous tubule of Eriocheir sinensis.

Spermatogenesis is a complicated process that includes spermatogonia differentiation, spermatocytes meiosis, spermatids spermiogenesis and final release of spermatozoa. Actin-related protein 3 (Arp3) and epidermal growth factor receptor pathway substrate 8 (Eps8) are two actin binding proteins that regulate cell adhesion in seminiferous tubules during mammalian spermatogenesis. However, the functions of these two proteins during spermatogenesis in nonmammalian species, especially Crustacea, are still unknown. Here, we cloned es-Arp3 and es-Eps8 from the testis of Chinese mitten crab Eriocheir sinensis. es-Arp3 and es-Eps8 were located in spermatocytes, spermatids and spermatozoa. Knockdown of es-Arp3 and es-Eps8 in vivo caused morphological changes to seminiferous tubules including delayed spermatozoa release, shedding of germ cells and vacuoles. Filamentous-actin (F-actin) filaments network was disorganized due to deficiency of es-Arp3 and es-Eps8. Accompanying this, four junctional proteins (α-catenin, ß-catenin, pinin and ZO1) displayed abnormal expression levels as well as penetrating biotin signals in seminiferous tubules. We also used the Arp2/3 complex inhibitor CK666 to block es-Arp3 activity and supported es-Arp3 knockdown results. In summary, our study demonstrated for the first time that es-Arp3 and es-Eps8 are important for spermatogenesis via regulating microfilament-mediated cell adhesion in Eriocheir sinensis.

Actin-related protein ACTL7B ablation leads to OAT with multiple morphological abnormalities of the flagellum and male infertility in mice†.

The formation of fertilisation-competent sperm requires spermatid morphogenesis (spermiogenesis), a poorly understood program that involves complex coordinated restructuring and specialised cytoskeletal structures. A major class of cytoskeletal regulators are the actin-related proteins (ARPs), which include conventional actin variants, and related proteins that play essential roles in complexes regulating actin dynamics, intracellular transport, and chromatin remodeling. Multiple testis-specific ARPs are well conserved among mammals, but their functional roles are unknown. One of these is actin-like 7b (Actl7b) that encodes an orphan ARP highly similar to the ubiquitously expressed beta actin (ACTB). Here we report ACTL7B is expressed in human and mouse spermatids through the elongation phase of spermatid development. In mice, ACTL7B specifically localises to the developing acrosome, within the nucleus of early spermatids, and to the flagellum connecting region. Based on this localisation pattern and high level of sequence conservation in mice, humans, and other mammals, we examined the requirement for ACTL7B in spermiogenesis by generating and characterising the reproductive phenotype of male Actl7b KO mice. KO mice were infertile, with severe and variable oligoteratozoospermia (OAT) and multiple morphological abnormalities of the flagellum (MMAF) and sperm head. These defects phenocopy human OAT and MMAF, which are leading causes of idiopathic male infertility. In conclusion, this work identifies ACTL7B as a key regulator of spermiogenesis that is required for male fertility.