Mammalian Testis Research Articles

The landscape of RNA binding proteins in mammalian spermatogenesis.

Despite continuous expansion of the RNA binding protein (RBP) world, there is a lack of systematic understanding of RBPs in the mammalian testis, which harbors one of the most complex tissue transcriptomes. We adapted RNA interactome capture to mouse male germ cells, building an RBP atlas characterized by multiple layers of dynamics along spermatogenesis. Trapping of RNA-cross-linked peptides showed that the glutamic acid-arginine (ER) patch, a residue-coevolved polyampholytic element present in coiled coils, enhances RNA binding of its host RBPs. Deletion of this element in NONO (non-POU domain-containing octamer-binding protein) led to a defective mitosis-to-meiosis transition due to compromised NONO-RNA interactions. Whole-exome sequencing of over 1000 infertile men revealed a prominent role of RBPs in the human genetic architecture of male infertility and identified risk ER patch variants.

Mechanisms of Germline Stem Cell Competition across Species.

In this review, we introduce the concept of cell competition, which occurs between heterogeneous neighboring cell populations. Cells with higher relative fitness become "winners" that outcompete cells of lower relative fitness ("losers"). We discuss the idea of super-competitors, mutant cells that expand at the expense of wild-type cells. Work on adult stem cells (ASCs) has revealed principles of neutral competition, wherein ASCs can be stochastically lost and replaced, and of biased competition, in which a winning ASC with a competitive advantage replaces its neighbors. Germline stem cells (GSCs) are ASCs that are uniquely endowed with the ability to produce gametes and, therefore, impact the next generation. Mechanisms of GSC competition have been elucidated by studies in Drosophila gonads, tunicates, and the mammalian testis. Competition between ASCs is thought to underlie various forms of cancer, including spermatocytic tumors in the human testis. Paternal age effect (PAE) disorders are caused by de novo mutations in human GSCs that increase their competitive ability and make them more likely to be inherited, leading to skeletal and craniofacial abnormalities in offspring. Given its widespread effects on human health, it is important to study GSC competition to elucidate how cells can become winners or losers.

Adaptive Evolution and Functional Differentiation of Testis-Expressed Genes in Theria.

Gene expression patterns differ in different tissues, and the expression pattern of genes in the mammalian testis is known to be extremely variable in different species. To clarify how the testis transcriptomic pattern has evolved in particular species, we examined the evolution of the adult testis transcriptome in Theria using 10 species: two marsupials (opossum and Tasmanian devil), six eutherian (placental) mammals (human, chimpanzee, bonobo, gorilla, rhesus macaque, and mouse), and two outgroup species (platypus and chicken). We show that 22 testis-expressed genes are marsupial-specific, suggesting their acquisition in the stem lineage of marsupials after the divergence from eutherians. Despite the time length of the eutherian stem lineage being similar to that of the marsupial lineage, acquisition of testis-expressed genes was not found in the stem lineage of eutherians; rather, their expression patterns differed by species, suggesting rapid gene evolution in the eutherian ancestors. Fifteen testis-expressed genes are therian-specific, and for three of these genes, the evolutionary tempo is markedly faster in eutherians than in marsupials. Our phylogenetic analysis of Rho GTPase-activating protein 28 (ARHGAP28) suggests the adaptive evolution of this gene in the eutherians, probably together with the expression pattern differentiation.

MALDI Imaging Mass Spectrometry Reveals Lipid Alterations in Physiological and Sertoli Cell-Only Syndrome Human Testicular Tissue Sections.

Azoospermia, the absence of sperm cells in semen, affects around 15% of infertile males. Sertoli cell-only syndrome (SCOS) is the most common pathological lesion in the background of non-obstructive azoospermia and is characterised by the complete absence of germinal epithelium, with Sertoli cells exclusively present in the seminiferous tubules. Studies have shown a correlation between successful spermatogenesis and male fertility with lipid composition of spermatozoa, semen, seminal plasma or testis. The aim of this research was to discover the correlation between the Johnsen scoring system and phospholipid expressions in testicular cryosections of SCOS patients. MALDI imaging mass spectrometry is used to determine spatial distributions of molecular species, such as phospholipids. Phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and sphingomyelins (SMs) are the most abundant phospholipids in mammalian cells and testis. SMs, the structural components of plasma membranes, are crucial for spermatogenesis and sperm function. Plasmalogens, are unique PCs in testis with strong antioxidative properties. This study, using imaging mass spectrometry, demonstrates the local distribution of phospholipids, particularly SMs, PCs, plasmalogens and PEs in human testicular samples with SCOS for the first time. This study found a strong relationship between the Johnsen scoring system and phospholipid expression levels in human testicular tissues. Future findings could enable routine diagnostic techniques during microTESE procedures for successful sperm extraction.

Temporal maturation of Sertoli cells during the establishment of the cycle of the seminiferous epithelium†.

Sertoli cells, omnipresent, somatic cells within the seminiferous tubules of the mammalian testis are essential to male fertility. Sertoli cells maintain the integrity of the testicular microenvironment, regulate hormone synthesis, and of particular importance, synthesize the active derivative of vitamin A, all trans retinoic acid (atRA), which is required for germ cell differentiation and the commitment of male germ cells to meiosis. Stages VIII-IX, when atRA synthesis occurs in the testis, coincide with multiple germ cell development and testicular restructuring events that rely on Sertoli cell gene products to proceed normally. In this study, we have synchronized and captured the mouse testis at four recurrent points of atRA synthesis to observe transcriptomic changes within Sertoli cells as mice age and the Sertoli cells are exposed to increasingly developed germ cell subtypes. This work provides comprehensive, high-resolution characterization of the timing of induction of functional Sertoli cell genes across the first wave of spermatogenesis, and outlines in silico predictions of germ cell derived signaling mechanisms targeting Sertoli cells. We have found that Sertoli cells adapt to their environment, especially to the needs of the germ cell populations present and establish germ-Sertoli cell and Sertoli-Sertoli cell junctions early but gain many of their known immune-regulatory and protein secretory functions in preparation for spermiogenesis and spermiation. Additionally, we have found unique patterns of germ-Sertoli signaling present at each endogenous pulse of atRA, suggesting individual functions of the various germ cells in germ-Sertoli communication.

PiRNA-disease association prediction based on multi-channel graph variational autoencoder.

Piwi-interacting RNA (piRNA) is a type of non-coding small RNA that is highly expressed in mammalian testis. PiRNA has been implicated in various human diseases, but the experimental validation of piRNA-disease associations is costly and time-consuming. In this article, a novel computational method for predicting piRNA-disease associations using a multi-channel graph variational autoencoder (MC-GVAE) is proposed. This method integrates four types of similarity networks for piRNAs and diseases, which are derived from piRNA sequences, disease semantics, piRNA Gaussian Interaction Profile (GIP) kernel, and disease GIP kernel, respectively. These networks are modeled by a graph VAE framework, which can learn low-dimensional and informative feature representations for piRNAs and diseases. Then, a multi-channel method is used to fuse the feature representations from different networks. Finally, a three-layer neural network classifier is applied to predict the potential associations between piRNAs and diseases. The method was evaluated on a benchmark dataset containing 5,002 experimentally validated associations with 4,350 piRNAs and 21 diseases, constructed from the piRDisease v1.0 database. It achieved state-of-the-art performance, with an average AUC value of 0.9310 and an AUPR value of 0.9247 under five-fold cross-validation. This demonstrates the method's effectiveness and superiority in piRNA-disease association prediction.

Deletion of an evolutionarily conserved TAD boundary compromises spermatogenesis in mice.

Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of protein coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of non-coding mutations remains largely unexplored. As a case study to evaluate the effects of non-coding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain (TAD) boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8 . We then used CRISPR-Cas9 to generate a mouse line where 26kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting increase in the DNA-DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, abnormal testis histology, a 35% drop in the estimated efficiency of spermatogenesis and a 28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that non-coding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a TAD boundary can have a negative impact on sperm production and highlight the importance of considering non-coding mutations in the analysis of patients with male infertility.

P-104 Cytoprotective effect of melatonin in prepubertal Sertoli cells: role in cadmium-induced idiopathic male infertility

Abstract Study question Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. Summary answer MLT produced a potent cytoprotective effect in Sertoli cells (SCs) exposed to Cd-toxicity, reducing its H2O2 generation and reductive stress effects by modulation Nrf2 activity. What is known already MLT a hormonal substance exhibiting strong antioxidative and antiapoptotic properties, has been proved to hold great potential as cytoprotec tive agent in a range of tissues and cell models exposed to Cd toxicity, including the rat bone, ovaries, human MSC, and even mammalian testes. However, cellular and mechanistic aspects of this effect of MLT in testes remain poorly understood. Cd is a pro-oxidant heavy metal reported to interfere with testicular function and fertility. Both the toxicity of Cd and the cytoprotective function of MLT involve redox-dependent mechanisms with the activity of Nrf2 transcription factor as key player on the cellular stress response. Study design, size, duration Porcine pre-pubertal SCs were maintained at 37 °C in a 5% CO2 humidified atmosphere in the absence (unexposed-control group) or presence of 5 or 10µM CdCl2 (Sigma Chemical Co.) and/or 50nM MLT (Sigma-Aldrich) for 48h in HAMF12 (Euroclone) supplemented with 0.166nM retinoic acid (Sigma-Aldrich Co.) and 5/500ml of Insulin-Transferrin-Selenium (ITS) + Premix (Cat. No. 354352; Corning). Participants/materials, setting, methods After treatment the parameters investigated in the study were: a) SCs viability (MTT test); b) SCs functionality parameters AMH and inhibin B. (real-time PCR and ELISA analysis); c) Intracellular and extracellular ROS production ( with Amplex Red and DCFH-DA fluorescent probe respectively); d) Catalase enzyme activity (spectrophotometric assay procedure); e) Nrf2 and other transcription factors (by semi-quantitative microplate confocal microscopy analysis); f) Intracellular and extracellular levels of GSH and other thiols (by HPLC); g) c-Jun and ERK1/2 (Immunoblot). Main results and the role of chance MLT co-treatment significantly prevented the effects of Cd on cell viability and abnormal ROS production. The reduction of H2O2 efflux was very important and combined with an increased CAT activity that was demonstrated upon MLT treatment significantly in SCs exposed to 5μMCd. The co-treatment with MLT compared to Cd treatment, showed a trend toward a significant recovery of AMH and inhibin B gene expression and secretion levels, respectively, demonstrating the cytoprotective effect of MLT in SCs exposed to Cd toxicity at the functional level. In Cd-treated cells, MLT significantly reduced the expression and nuclear translocation of Nrf2 protein, and decreased GSTP induction. However, MLT reduced Cys uptake and stimulated GSH efflux when the cells were treated with 10 µM Cd, suggesting a specific role for the Nrf2-GSTP axis in the cytoprotective response to MLT of Cd-treated SCs. Finally MLT induced MAPK-ERK1-2 pathway and c-Jun transcription factor when the cells were treated with Cd. MLT is a cytoprotective agent in SCs exposed to Cd toxicity capable to restore cell viability and function. Its activity is associated with the modulation of Nrf2 and other stress response genes important in the control of thiols and H2O2 metabolism of SCs, including cJun and MAPKs. Limitations, reasons for caution Other ROS-scavenging genes other than CAT may have a role in the antioxidant response to MLT of SCs, such as superoxide dismutase, glutathione peroxidases, and peroxyredoxins that are worth investigating further. Wider implications of the findings The aspects that emerged from our study and, described for the first time in these cells, warrant further preclinical and clinical investigation holding potential in the treatment of Cd-induced testicular damage and fertility problems. Trial registration number Not applicable

The Role of Retinoic Acid in Spermatogenesis and Its Application in Male Reproduction.

Spermatogenesis in mammalian testes is essential for male fertility, ensuring a continuous supply of mature sperm. The testicular microenvironment finely tunes this process, with retinoic acid, an active metabolite of vitamin A, serving a pivotal role. Retinoic acid is critical for various stages, including the differentiation of spermatogonia, meiosis in spermatogenic cells, and the production of mature spermatozoa. Vitamin A deficiency halts spermatogenesis, leading to the degeneration of numerous germ cells, a condition reversible with retinoic acid supplementation. Although retinoic acid can restore fertility in some males with reproductive disorders, it does not work universally. Furthermore, high doses may adversely affect reproduction. The inconsistent outcomes of retinoid treatments in addressing infertility are linked to the incomplete understanding of the molecular mechanisms through which retinoid signaling governs spermatogenesis. In addition to the treatment of male reproductive disorders, the role of retinoic acid in spermatogenesis also provides new ideas for the development of male non-hormone contraceptives. This paper will explore three facets: the synthesis and breakdown of retinoic acid in the testes, its role in spermatogenesis, and its application in male reproduction. Our discussion aims to provide a comprehensive reference for studying the regulatory effects of retinoic acid signaling on spermatogenesis and offer insights into its use in treating male reproductive issues.

HIV1-Nef perturbs the integrity of blood testis barrier in rat model

ABSTRACT The blood-testis barrier is a specialized feature within the mammalian testis, located in close proximity to the basement membrane of seminiferous tubules. This barrier serves to divide the seminiferous epithelium into distinct basal and adluminal (apical) compartments. The selectivity of the BTB to foreign particles makes it a safe haven for the virus, and the high affinity of HIV for testis might lead to the vertical transmission of the virus. In the present study, recombinant HIV1-Nef (rNef) protein was injected intravenously to examine the effect of rNef on BTB. SD male rats received 250 µg and 500 µg of rNef along with 2% Evans blue dye within 1 ml through the tail vein. After 1 hour of perfusion, the animals were sacrificed for analysis. The dye migration assay and ELISA confirmed a significant impairment in the blood-testis barrier (BTB) and the manifestation of rNef in testes tissues, respectively. Moreover, a decline in the expression of tight junction proteins, including ZO1 and Occludin, was observed during rNef-induced BTB disruption. Overall, our findings demonstrated that rNef induces BTB disruption through various signaling events. At the site of ectoplasmic specialization of the seminiferous epithelium, the localization of cadherins was found to be disrupted, making the testis a vulnerable site. In conclusion, rNef perturbs the integrity of the blood-testis barrier in rat models; hence, it can also serve as a suitable model for studying the dynamics of the blood-testis barrier.

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

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. 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. 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.

A role for TRPC3 in mammalian testis development.

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

Cell-type specific and differential expression of LINC-RSAS long noncoding RNA declines in the testes during ageing of the rat.

Long noncoding RNAs (lncRNAs) have emerged as major regulators of gene expression, chromatin structure, epigenetic changes, post-transcriptional processing of RNAs, translation of mRNAs into proteins as well as contributing to the process of ageing. Ageing is a universal, slow, progressive change in almost all physiological processes of organisms after attaining reproductive maturity and often associated with age-related diseases. Mammalian testes contain various cell-types, vast reservoir of transcriptome complexity, produce haploid male gametes for reproduction and testosterone for development and maintenance of male sexual characters as well as contribute genetic variation to the species. We report age-related decline in expression and cellular localization of Long intergenic noncoding repeat-rich sense-antisense (LINC-RSAS) RNA in the testes and its major cell-types such as primary spermatocytes, Leydig cells and Sertoli cells during ageing of the rat. LINC-RSAS expression in testes increased from immature (4-weeks) to adult (16- and 44-weeks) and declined from adult (44-weeks) to nearly-old (70-weeks) rats. Genomic DNA methylation in the testes showed a similar pattern. Cell-type specific higher expression of LINC-RSAS was observed in primary spermatocytes (pachytene cells), Leydig cells and Sertoli cells of testes of adult rats. Over-expression of LINC-RSAS in cultured human cell lines revealed its possible role in cell-cycle control and apoptosis. We propose that LINC-RSAS expression is involved in molecular physiology of primary spermatocytes, Leydig cells and Sertoli cells of adult testes and its decline is associated with diminishing function of testes during ageing of the rat.

Characterisation of germline progenitor cells in the testes of phylostomid bats: Artibeus jamaicensis and Sturnira lilium.

Context A population of sperm progenitor cells, known as Asingle spermatogonia, has been described in mammalian testes. During division cycles in spermatogenesis, some cells will form part of the Asingle spermatogonia group, while others form primary spermatocytes. Thus, during spermatogenesis, spermatogonia are the progenitor cells of spermatozoa. Aims In this study, we characterise the spermatogonial stem cells (SSCs) in the testicles of Artibeus jamaicensis and Sturnira lilium bats. The knowledge generated from this will contribute to the understanding of the biology of germ cells and the mechanisms of spermatogenesis in mammals, generating information on wildlife species that are important for biodiversity. Methods Testes were analysed by light and electron microscopy. Likewise, the expression of specific factors of stem cells (Oct4 and C-kit), germ cells (Vasa), cell proliferation (pH3 and SCP1) and testicular somatic cells (MIS, 3βHSD and Sox9) was characterised by immunofluorescence and western blot. Key results The histological analysis enabled the location of type Asingle, Apaired and Aaligned spermatogonia in the periphery of the seminiferous tubules adjacent to Sertoli cells. The expression of genes of stem and germ cells made it possible to corroborate the distribution of the SSCs. Conclusions Results indicate that type Asingle spermatogonia were not randomly distributed, since proliferative activity was detected in groups of cells adjacent to the seminiferous tubules membrane, suggesting the localisation of spermatogonial niches in a specific region of testes. Implications This study provides evidence for the existence of SSCs in the testis of chiropterans that contribute to the renewal of germline progenitor cells to maintain the reproduction of the organisms.

A Comparative Transcriptomic Study and Key Gene Targeting of Lamprey Gonadal Immune Response

ABSTRACT The mammalian testis and ovary possess special immunocompetence, which is central to provide protection against pathogens. However, the innate immune responses to immune challenges in lamprey gonads are poorly understood. In this study, we extracted RNA from testis and ovary tissues of lampreys at 0 hour, 8 hours and 17 days after lipopolysaccharides (LPS) stimulation and performed transcriptome sequencing. While the transcriptome profiles of the two tissues were different for the most part, genes LIP, LECT2, LAL2, GRN, ITLN, and C1q were found to be the most significantly up-regulated genes in both. Quantitative Real-time PCR (qRT-PCR) analysis confirmed that these genes were upregulated after stimulation. Furthermore, immunohistochemical staining showed that these genes in lamprey gonads are expressed in high quantities and have a specific distribution. Taken together, our results suggest that these genes could play an essential role in response of the gonads to LPS induction. This research establishes a basis for investigating the immune mechanism of vertebrate gonads and presents a fresh concept for gaining insight into the evolutionary development of jawless vertebrates.

A comparative analysis of genes differentially expressed between rete testis cells and Sertoli cells of the mouse testis

The rete testis (RT) is a region of the mammalian testis that plays an important role in testicular physiology. The RT epithelium consists of cells sharing some well-known gene markers with supporting Sertoli cells (SCs). However, little is known about the differences in gene expression between these two cell populations. Here, we used fluorescence-activated cell sorting (FACS) to obtain pure cultures of neonatal RT cells and SCs and identified differentially expressed genes (DEGs) between these cell types using RNA sequencing (RNA-seq). We then compared our data with the RNA-seq data of other studies that examined RT cells and SCs of mice of different ages and generated a list of DEGs permanently upregulated in RT cells throughout testis development and in culture, which included 86 genes, and a list of 79 DEGs permanently upregulated in SCs. The analysis of studies on DMRT1 function revealed that nearly half of the permanent DEGs could be regulated by this SC upregulated transcription factor. We suggest that useful cell lineage markers and candidate genes for the specification of both RT cells and SCs may be present among these permanent DEGs.

Molecular cloning, expression and cellular localization of two long noncoding RNAs (mLINC-RBE and mLINC-RSAS) in the mouse testis

Long noncoding RNAs (lncRNAs) are transcribed in complex, overlapping, sense- and antisense orientations from intronic and intergenic regions of mammalian genomes. Transcription of genome in mammalian testis is more widespread compared to other organs. LncRNAs are involved in gene expression, chromatin regulation, mRNA stability and translation of proteins during diverse cellular functions. We report molecular cloning of two novel lncRNAs (mLINC-RBE and mLINC-RSAS) and their expression by RT-PCR as well as cellular localization by RNA in-situ hybridization in the mouse testes. mLINC-RBE is an intergenic lncRNA from chromosome 4, with 16.96 % repeat sequences, expressed as a sense transcript with piRNA sequences and its expression is localized into primary spermatocytes. mLINC-RSAS is an intergenic lncRNA from chromosome 2, with 49.7 % repeat sequences, expressed as both sense- and antisense transcripts with miRNA sequences and its expression is localized into different cell types, such as Sertoli cells, primary spermatocytes and round spermatids. The lncRNAs also contain sequences for some short peptides (micropeptides). This suggests that these two repeat sequence containing, intergenic genomic sense- and antisense transcripts expressed as lncRNAs with piRNAs, miRNAs, and showing cell-type specific, differential expression may regulate important functions in mammalian testes. Such functions may be regulated by RNA structures, RNA processing and RNA-protein complexes.

The katanin A-subunits KATNA1 and KATNAL1 act co-operatively in mammalian meiosis and spermiogenesis to achieve male fertility.

Katanins, a class of microtubule-severing enzymes, are potent M-phase regulators in oocytes and somatic cells. How the complex and evolutionarily crucial, male mammalian meiotic spindle is sculpted remains unknown. Here, using multiple single and double gene knockout mice, we reveal that the canonical katanin A-subunit KATNA1 and its close paralogue KATNAL1 together execute multiple aspects of meiosis. We show KATNA1 and KATNAL1 collectively regulate the male meiotic spindle, cytokinesis and midbody abscission, in addition to diverse spermatid remodelling events, including Golgi organisation, and acrosome and manchette formation. We also define KATNAL1-specific roles in sperm flagellum development, manchette regulation and sperm-epithelial disengagement. Finally, using proteomic approaches, we define the KATNA1, KATNAL1 and KATNB1 mammalian testis interactome, which includes a network of cytoskeletal and vesicle trafficking proteins. Collectively, we reveal that the presence of multiple katanin A-subunit paralogs in mammalian spermatogenesis allows for 'customised cutting' via neofunctionalisation and protective buffering via gene redundancy.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1−/− rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1−/− ovaries did not undergo meiosis and folliculogenesis. XX DMRT1−/− adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility.

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.