RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.This work was supported by grants from the National Health and Medical Research Council (NHMRC) to DJ (#606503); the Australian Research Council (ARC) to MKO and CJO; the New South Wales Cancer Council, Cancer Institute New South Wales, Banque Nationale de Paris-Paribas Australia and New Zealand, RT Hall Trust, and the National Breast Cancer Foundation to CJO. DJ was an NHMRC Peter Doherty Postdoctoral Fellow (#384297). MKO and CJO are NHMRC Senior Research Fellows (#545805, #481310). CCG is an NHMRC Australia Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

An Essential Role for Katanin p80 and Microtubule Severing in Male Gamete Production

Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development

Mechanisms of spermiogenesis and spermiation and how they are disturbed

Haploid round spermatids undergo a remarkable transformation during spermiogenesis. The nucleus polarizes to one side of the cell as the nucleus condenses and elongates, and the microtubule-based manchette sculpts the nucleus into its species-specific head shape. The assembly of the central component of the sperm flagellum, known as the axoneme, begins early in spermiogenesis, and is followed by the assembly of secondary structures needed for normal flagella. The final remodelling of the mature elongated spermatid occurs during spermiation, when the spermatids line up along the luminal edge, shed their residual cytoplasm and are ultimately released into the lumen. Defects in spermiogenesis and spermiation are manifested as low sperm number, abnormal sperm morphology and poor motility and are commonly observed during reproductive toxicant administration, as well as in genetically modified mouse models of male infertility. This chapter summarizes the major physiological processes and the most commonly observed defects in spermiogenesis and spermiation, to aid in the diagnosis of the potential mechanisms that could be perturbed by experimental manipulation such as reproductive toxicant administration

Localisation of the Chromatin Remodelling Protein, ATRX in the Adult Testis

Mutations in ATRX (alpha-thalassaemia and mental retardation on the X-chromosome) can give rise to ambiguous or female genitalia in XY males, implying a role for ATRX in testicular development. Studies on ATRX have mainly focused on its crucial role in brain development and α-globin regulation; however, little is known about its function in sexual differentiation and its expression in the adult testis. Here we show that the ATRX protein is present in adult human and rat testis and is expressed in the somatic cells; Sertoli, Leydig, and peritubular myoid cells, and also in germ cells; spermatogonia and early meiotic spermatocytes. The granular pattern of ATRX staining is consistent with that observed in other cell-types and suggests a role in chromatin regulation. The findings suggest that ATRX in humans may play a role in adult spermatogenesis as well as in testicular development

Variability in sperm suppression during testosterone administration to adult monkeys is related to follicle stimulating hormone suppression and not to intratesticular androgens

Sex steroid-based male contraceptive regimens do not induce consistent azoospermia. The reason for this variable response is obscure. We used normal adult male monkeys, Macaca fascicularis (n = 9) as a model of testosterone (T)-induced gonadotropin suppression to understand the basis for variability in spermatogenic suppression during hormonal contraception. As observed in men, T administration to these monkeys induced azoospermia in some animals and variable degrees of spermatogenic suppression in others. Based on their sperm counts, we divided these animals into two groups: azoospermic (azoo; n = 4) and nonazoospermic (nonazoo; n = 5) groups. Sperm density, testis volumes, and serum T, bioassayable LH (bioLH), immunoassayable FSH (immunoFSH), bioassayable FSH (bioFSH), and inhibin B were examined every 2 wk during the control period, 20 wk of T administration using SILASTIC brand (Dow Corning Corp.) implants, and recovery. Testes were biopsied for estimation of intratesticular T, dihydrotestosterone, and 5alpha-androstane-3alpha,17beta-diol. Serum T levels increased 1.5- to 2-fold, leading to decreased bioLH levels (48% of control) and intratesticular T levels (15% of control); neither LH nor intratesticular T levels differed between the azoo and nonazoo groups. In contrast, serum levels of FSH, by both bio- and immunoassay, during T administration were significantly lower in the azoo than in the nonazoo group. These results suggest that the degree of suppression of spermatogenesis is closely related to the degree of suppression of FSH levels and not to the levels of intratesticular androgens or to serum LH. These results imply that FSH plays a key role in supporting spermatogenesis in monkeys in this experimental regimen and suggest that maximal suppression of FSH may be essential to ensure consistent azoospermia in men during hormonal contraceptio

Impairment of Spermatogonial Development and Spermiation after Testosterone-Induced Gonadotropin Suppression in Adult Monkeys (Macaca fascicularis)

Human male hormonal contraceptive regimens do not consistently induce azoospermia, and the basis of this variable response is unclear. This study used nine adult macaque monkeys (Macaca fascicularis) given testosterone (T) implants for 20 weeks to study changes in germ cell populations in relation to sperm output. Germ cell numbers were determined using the optical disector stereological method. Four animals achieved consistent azoospermia (azoo group), whereas five animals did not (nonazoo group). T-induced gonadotropin suppression in all animals decreased A pale (Ap) spermatogonia to 45% of baseline within 2 weeks, leading to decreased B spermatogonia (32--38%) and later germ cells (20--30%) after 14 and 20 weeks. Though the reduction in later germ cell types could be primarily attributed to the loss of spermatogonia, the data suggested that some cells were lost during the spermatocyte and spermatid phase of development. B spermatogonial number was more markedly suppressed in azoospermic animals, compared with the nonazoo group, as was the conversion ratio between Ap and B spermatogonia. Abnormal retention of elongated spermatids (failed spermiation) was also prominent in some animals after long-term T administration. We conclude that: 1) the variable suppression of sperm output is attributed to the degree of inhibition of germ cell development from type B spermatogonia onwards, and this is related to the degree of FSH suppression; and 2) inhibition of Ap and B spermatogonial development and of spermiation are the major defects caused by long-term T administration to monkeys

You can't have one without the other: Transactions between education and wellbeing for Indigenous peoples.

Casuarina, NT, Australi

RBM5 is a male germ cell splicing factor and is required for spermatid differentiation and male fertility

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing