Mitochondrial Differentiation during Spermatogenesis: Lessons from Drosophila melanogaster.

Numerous diseases can arise as a consequence of mitochondrial malfunction. Hence, there is a significant focus on studying the role of mitochondria in cancer, ageing, neurodegenerative diseases, and the field of developmental biology. Mitochondria could exist as discrete organelles in the cell; however, they have the ability to fuse, resulting in the formation of interconnected reticular structures. The dynamic changes between these forms correlate with mitochondrial function and mitochondrial health, and consequently, there is a significant scientific interest in uncovering the specific molecular constituents that govern these transitions. Moreover, the specialized mitochondria display a wide array of variable morphologies in their cristae formations. These inner mitochondrial structures are closely associated with the specific functions performed by the mitochondria. In multiple cases, the presence of mitochondrial dysfunction has been linked to male sterility, as it has been observed to cause a range of abnormal spermatogenesis and sperm phenotypes in different species. This review aims to elucidate the dynamic alterations and functions of mitochondria in germ cell development during the spermatogenesis of Drosophila melanogaster.

A Single-Cell Landscape of Spermioteleosis in Mice and Pigs.

(1) Background: Spermatozoa acquired motility and matured in epididymis after production in the testis. However, there is still limited understanding of the specific characteristics of sperm development across different species. In this study, we employed a comprehensive approach to analyze cell compositions in both testicular and epididymal tissues, providing valuable insights into the changes occurring during meiosis and spermiogenesis in mouse and pig models. Additionally, we identified distinct gene expression signatures associated with various spermatogenic cell types. (2) Methods: To investigate the differences in spermatogenesis between mice and pigs, we constructed a single-cell RNA dataset. (3) Results: Our findings revealed notable differences in testicular cell clusters between these two species. Furthermore, distinct gene expression patterns were observed among epithelial cells from different regions of the epididymis. Interestingly, regional gene expression patterns were also identified within principal cell clusters of the mouse epididymis. Moreover, through analysing differentially expressed genes related to the epididymis in both mouse and pig models, we successfully identified potential marker genes associated with sperm development and maturation for each species studied. (4) Conclusions: This research presented a comprehensive single-cell landscape analysis of both testicular and epididymal tissues, shedding light on the intricate processes involved in spermatogenesis and sperm maturation, specifically within mouse and pig models.

Consequences of Exposure to Hypobaric Hypoxia Associated with High Altitude on Spermatogenesis and Seminal Parameters: A Literature Review.

Preclinical research has provided compelling evidence indicating that exposure to hypobaric hypoxia (HH) results in a deterioration of spermatogenesis. This adverse effect extends to the underlying molecular mechanisms, progressively leading to impairments in the seminiferous epithelium and germ cells and alterations in semen parameters. Indeed, several studies have demonstrated that animals exposed to HH, whether in natural high-altitude environments or under simulated hypoxic conditions, exhibit damage to the self-renewal and differentiation of spermatogenesis, an increase in germline cell apoptosis, and structural alterations in the seminiferous tubules. One of the primary mechanisms associated with the inhibition of differentiation and an increase in apoptosis among germ cells is an elevated level of oxidative stress, which has been closely associated with HH exposure. Human studies have shown that individuals exposed to HH, such as mountaineers and alpinists, exhibit decreased sperm count, reduced motility, diminished viability, and increased sperm with abnormal morphology in their semen. This evidence strongly suggests that exposure to HH may be considered a significant risk factor that could elevate the prevalence of male infertility. This literature review aims to provide a comprehensive description and propose potential mechanisms that could elucidate the infertility processes induced by HH. By doing so, it contributes to expanding our understanding of the challenges posed by extreme environments on human physiology, opening new avenues for research in this field.

Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actin.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

Running the gauntlet: challenges to genome integrity in spermiogenesis.

Species' continuity depends on gametogenesis to produce the only cell types that can transmit genetic information across generations. Spermiogenesis, which encompasses post-meiotic, haploid stages of male gametogenesis, is a process that leads to the formation of sperm cells well-known for their motility. Spermiogenesis faces three major challenges. First, after two rounds of meiotic divisions, the genome lacks repair templates (no sister chromatids, no homologous chromosomes), making it incredibly vulnerable to any genomic insults over an extended time (typically days-weeks). Second, the sperm genome becomes transcriptionally silent, making it difficult to respond to new perturbations as spermiogenesis progresses. Third, the histone-to-protamine transition, which is essential to package the sperm genome, counterintuitively involves DNA break formation. How spermiogenesis handles these challenges remains poorly understood. In this review, we discuss each challenge and their intersection with the biology of protamines. Finally, we discuss the implication of protamines in the process of evolution.

Advancements in fertility preservation strategies for pediatric male cancer patients: a review of cryopreservation and transplantation of immature testicular tissue.

Recently, there has been increasing emphasis on the gonadotoxic effects of cancer therapy in prepubertal boys. As advances in oncology treatments continue to enhance survival rates for prepubertal boys, the need for preserving their functional testicular tissue for future reproduction becomes increasingly vital. Therefore, we explore cutting-edge strategies in fertility preservation, focusing on the cryopreservation and transplantation of immature testicular tissue as a promising avenue. The evolution of cryopreservation techniques, from controlled slow freezing to more recent advancements in vitrification, with an assessment of their strengths and limitations was exhibited. Detailed analysis of cryoprotectants, exposure times, and protocols underscores their impact on immature testicular tissue viability. In transplantation strategy, studies have revealed that the scrotal site may be the preferred location for immature testicular tissue grafting in both autotransplantation and xenotransplantation scenarios. Moreover, the use of biomaterial scaffolds during graft transplantation has shown promise in enhancing graft survival and stimulating spermatogenesis in immature testicular tissue over time. This comprehensive review provides a holistic approach to optimize the preservation strategy of human immature testicular tissue in the future.

The rapidly evolving X-linked MIR-506 family fine-tunes spermatogenesis to enhance sperm competition.

Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.

Two heat shock cognate 70 genes involved in spermatogenesis regulate the male fertility of Zeugodacus cucurbitae, as potential targets for pest control.

The melon fly Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agricultural quarantine pest threatening fruit and vegetable production. Heat shock cognate 70 (Hsc70), which is a homolog of the heat shock protein 70 (Hsp70), was first discovered in mice testes and plays an important role in spermatogenesis. In this study, we identified and cloned five Hsc70 genes from melon fly, namely ZcHsc70_1/2/3/4/5. Phylogenetic analysis showed that these proteins are closely related to Hsc70s from other Diptera insects. Spatiotemporal expression analysis showed that ZcHsc70_1 and ZcHsc70_2 are highly expressed in Z. cucurbitae testes. Fluorescence in situ hybridization further demonstrated that ZcHsc70_1 and ZcHsc70_2 are expressed in the transformation and maturation regions of testes, respectively. Moreover, RNA interference-based suppression of ZcHsc70_1 or ZcHsc70_2 resulted in a significant decrease of 74.61% and 63.28% in egg hatchability, respectively. Suppression of ZcHsc70_1 expression delayed the transformation of sperm cells to mature sperms. Meanwhile, suppression of ZcHsc70_2 expression decreased both sperm cells and mature sperms by inhibiting the meiosis of spermatocytes. Our findings show that ZcHsc70_1/2 regulates spermatogenesis and further affects the male fertility in the melon fly, showing potential as targets for pest control in sterile insect technique by genetic manipulation of males.

[Research progress of piRNA as a biomarker for male infertility].

piRNA is a class of small non-coding RNA which specifically binds with PIWI protein. It is mainly expressed in germ cells and involved in the regulation of spermatogenesis. The role of piRNA pathway in the regulation of spermatogenesis mainly includes inhibition of transposons, induction of mRNA translation or degradation, and mediation of degradation of Miwi ubiquitination in late-stage sperm cells. With the detection of piRNA in seminal plasma, more attention has been attracted to whether piRNA can be used as a non-invasive molecular biomarker for the evaluation of spermatogenesis. This paper has reviewed recent studies on the mechanism of piRNA pathways mediating spermatogenesis and potential roles of piRNA disorders in the diagnosis and treatment of male infertility.

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization.

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

Uncovering a multitude of stage-specific splice variants and putative protein isoforms generated along mouse spermatogenesis.

BACKGROUND: Mammalian testis is a highly complex and heterogeneous tissue. This complexity, which mostly derives from spermatogenic cells, is reflected at the transcriptional level, with the largest number of tissue-specific genes and long noncoding RNAs (lncRNAs) compared to other tissues, and one of the highest rates of alternative splicing. Although it is known that adequate alternative-splicing patterns and stage-specific isoforms are critical for successful spermatogenesis, so far only a very limited number of reports have addressed a detailed study of alternative splicing and isoforms along the different spermatogenic stages. RESULTS: In the present work, using highly purified stage-specific testicular cell populations, we detected 33,002 transcripts expressed throughout mouse spermatogenesis not annotated so far. These include both splice variants of already annotated genes, and of hitherto unannotated genes. Using conservative criteria, we uncovered 13,471 spermatogenic lncRNAs, which reflects the still incomplete annotation of lncRNAs. A distinctive feature of lncRNAs was their lower number of splice variants compared to protein-coding ones, adding to the conclusion that lncRNAs are, in general, less complex than mRNAs. Besides, we identified 2,794 unannotated transcripts with high coding potential (including some arising from yet unannotated genes), many of which encode unnoticed putative testis-specific proteins. Some of the most interesting coding splice variants were chosen, and validated through RT-PCR. Remarkably, the largest number of stage-specific unannotated transcripts are expressed during early meiotic prophase stages, whose study has been scarcely addressed in former transcriptomic analyses. CONCLUSIONS: We detected a high number of yet unannotated genes and alternatively spliced transcripts along mouse spermatogenesis, hence showing that the transcriptomic diversity of the testis is considerably higher than previously reported. This is especially prominent for specific, underrepresented stages such as those of early meiotic prophase, and its unveiling may constitute a step towards the understanding of their key events.

In vivo CRISPR screening directly targeting testicular cells.

CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.

Histological differences between the central and peripheral areas of the testes of busulfan-administered mice.

Busulfan is an anticancer drug known to cause serious damage to seminiferous tubules in the testes and deplete germ cells in human and animal models. The testicular artery is anastomosed with deferential and cremasteric arteries and is divided into capsular arteries, which give rise to the centripetal arteries and then recurrent arteries. The arterial blood in the testicular tissue is supplied by such a consequent system of arterial vessels, in order from the peripheral to the central area. As anticancer drugs are generally distributed throughout the whole body via the bloodstream and the running and distribution of arteries differ among the testicular areas, we hypothesized that the efficacy of busulfan differs in different testicular areas, particularly between the central and peripheral areas. In this study, busulfan was intraperitoneally injected at 40 mg/kg body weight into C57BL/6J male mice. After 28 days, in busulfan-treated mice, the diameters of seminiferous tubules were significantly higher in the central than in the peripheral area of the testes. The seminiferous tubular areas also significantly decreased in the peripheral areas compared with the central areas. The number of germ cells per seminiferous tubule was significantly higher in the central than in the peripheral area. Sertoli cell nuclei were detached into the lumen in the peripheral area. The number of Leydig cells was significantly lower in the peripheral areas. These data suggest that the effects of busulfan differ between the central and peripheral areas of the testis at 4 weeks after busulfan administration.

scRNA-seq Reveals Novel Genetic Pathways and Sex Chromosome Regulation in Tribolium Spermatogenesis.

Spermatogenesis is critical to sexual reproduction yet evolves rapidly in many organisms. High-throughput single-cell transcriptomics promises unparalleled insight into this important process but understanding can be impeded in nonmodel systems by a lack of known genes that can reliably demarcate biologically meaningful cell populations. Tribolium castaneum, the red flour beetle, lacks known markers for spermatogenesis found in insect species like Drosophila melanogaster. Using single-cell sequencing data collected from adult beetle testes, we implement a strategy for elucidating biologically meaningful cell populations by using transient expression stage identification markers, weighted principal component clustering, and SNP-based haploid/diploid phasing. We identify populations that correspond to observable points in sperm differentiation and find species specific markers for each stage. Our results indicate that molecular pathways underlying spermatogenesis in Coleoptera are substantially diverged from those in Diptera. We also show that most genes on the X chromosome experience meiotic sex chromosome inactivation. Temporal expression of Drosophila MSL complex homologs coupled with spatial analysis of potential chromatin entry sites further suggests that the dosage compensation machinery may mediate escape from meiotic sex chromosome inactivation and postmeiotic reactivation of the X chromosome.

Production of a Monoclonal Antibody for Histone H2b Isoform H2b3b.

H2b3b is one of the histone H2b isoforms that differs from canonical H2b by five to six amino acids. Previously, we identified H3t as the testis-specific histone H3 variant located in histone cluster 3, which is also the site of H2b3b. In this study, we produced monoclonal antibodies against H2b3b, using the iliac rat lymph node method for rat antibody and the immunochamber method for rabbit antibody. Immunoblot analysis confirmed that our antibodies could specifically discriminate between H2b3b and canonical H2b. Moreover, immunostaining revealed colocalization with a testicular stem cell marker, Plzf, but not with a meiotic marker, Sycp. This indicated that H2b3b is expressed in spermatogenic cells before meiosis. Our monoclonal antibodies enable further studies to reveal specific functions of H2b3b during spermatogenesis. We also hope that the established method will lead to the production of antibodies that can identify other H2b isoforms.

Restoration of fertility in nonablated recipient mice after spermatogonial stem cell transplantation.

Spermatogonial stem cell (SSC) transplantation is a valuable tool for studying stem cell-niche interaction. However, the conventional approach requires the removal of endogenous SSCs, causing damage to the niche. Here we introduce WIN18,446, an ALDH1A2 inhibitor, to enhance SSC colonization in nonablated recipients. Pre-transplantation treatment with WIN18,446 induced abnormal claudin protein expression, which comprises the blood-testis barrier and impedes SSC colonization. Consequently, WIN18,446 increased colonization efficiency by 4.6-fold compared with untreated host. WIN18,446-treated testes remained small despite the cessation of WIN18,446, suggesting its irreversible effect. Offspring were born by microinsemination using donor-derived sperm. While WIN18,446 was lethal to busulfan-treated mice, cyclophosphamide- or radiation-treated animals survived after WIN18,446 treatment. Although WIN18,446 is not applicable to humans due to toxicity, similar ALDH1A2 inhibitors may be useful for SSC transplantation into nonablated testes, shedding light on the role of retinoid metabolism on SSC-niche interactions and advancing SSC research in animal models and humans.

BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

Dissecting the dynamic cellular transcriptional atlas of adult teleost testis development throughout the annual reproductive cycle.

Teleost testis development during the annual cycle involves dramatic changes in cellular compositions and molecular events. In this study, the testicular cells derived from adult black rockfish at distinct stages - regressed, regenerating and differentiating - were meticulously dissected via single-cell transcriptome sequencing. A continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, was delineated, elucidating the molecular events involved in spermatogenesis. Subsequently, the dynamic regulation of gene expression associated with spermatogonia proliferation and differentiation was observed across spermatogonia subgroups and developmental stages. A bioenergetic transition from glycolysis to mitochondrial respiration of spermatogonia during the annual developmental cycle was demonstrated, and a deeper level of heterogeneity and molecular characteristics was revealed by re-clustering analysis. Additionally, the developmental trajectory of Sertoli cells was delineated, alongside the divergence of Leydig cells and macrophages. Moreover, the interaction network between testicular micro-environment somatic cells and spermatogenic cells was established. Overall, our study provides detailed information on both germ and somatic cells within teleost testes during the annual reproductive cycle, which lays the foundation for spermatogenesis regulation and germplasm preservation of endangered species.

piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis.

The intermitochondrial cement (IMC) and chromatoid body (CB) are posited as central sites for piRNA activity in mice, with MIWI initially assembling in the IMC for piRNA processing before translocating to the CB for functional deployment. The regulatory mechanism underpinning MIWI translocation, however, has remained elusive. We unveil that piRNA loading is the trigger for MIWI translocation from the IMC to CB. Mechanistically, piRNA loading facilitates MIWI release from the IMC by weakening its ties with the mitochondria-anchored TDRKH. This, in turn, enables arginine methylation of MIWI, augmenting its binding affinity for TDRD6 and ensuring its integration within the CB. Notably, loss of piRNA-loading ability causes MIWI entrapment in the IMC and its destabilization in male germ cells, leading to defective spermatogenesis and male infertility in mice. Collectively, our findings establish the critical role of piRNA loading in MIWI translocation during spermatogenesis, offering new insights into piRNA biology in mammals.