SciCombinator

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Concept: Spermatogenesis

185

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Concepts: Testicle, Genetics, Spermatogenesis, DNA, Gene, Spermatozoon, Sertoli cell, Mutation

59

Zika virus (ZIKV) infection of pregnant women can cause congenital malformations including microcephaly, which has focused global attention on this emerging pathogen(1). In addition to transmission by mosquitoes, ZIKV can be detected in the seminal fluid of affected males for extended periods of time and transmitted sexually(2). Here, using a mouse-adapted African ZIKV strain (Dakar 41519), we evaluated the consequences of infection in the male reproductive tract of mice. We observed persistence of ZIKV, but not the closely related Dengue virus (DENV), in the testis and epididymis of male mice, and this was associated with tissue injury that caused diminished testosterone and inhibin B levels, and oligospermia. ZIKV preferentially infected spermatogonia, primary spermatocytes, and Sertoli cells in the testis, resulting in cell death and destruction of the seminiferous tubules. Less damage was observed with a contemporary Asian ZIKV strain (H/PF/2013), in part because this virus replicates less efficiently in mice. The extent to which these observations in mice translate to humans remains unclear, but longitudinal studies of sperm function and viability in ZIKV-infected humans seem warranted.

Concepts: Epididymis, Sperm, Spermatogenesis, Reproductive system, Male reproductive system, Reproduction, Spermatozoon, Sertoli cell

35

Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells.

Concepts: Spermatogenesis, Reproductive system, Gamete, Oogenesis, Gene, Germ cells, Germ cell, Meiosis

27

One of the most dramatic forms of chromatin reorganization occurs during spermatogenesis, when the paternal genome is repackaged from a nucleosomal to a protamine-based structure. We assessed the role of the canonical histone chaperone CAF1 in Drosophila spermatogenesis. In this process, CAF1 does not behave as a complex, but its subunits display distinct chromatin dynamics. During histone-to-protamine replacement, CAF1-p180 dissociates from the DNA while CAF1-p75 binds and stays on as a component of sperm chromatin. Association of CAF1-p75 with the paternal genome depends on CAF1-p180 and protamines. Conversely, CAF1-p75 binds protamines and is required for their incorporation into sperm chromatin. Histone removal, however, occurs independently of CAF1 or protamines. Thus, CAF1-p180 and CAF1-p75 function in a temporal hierarchy during sperm chromatin assembly, with CAF1-p75 acting as a protamine-loading factor. These results show that CAF1 subunits mediate the assembly of two fundamentally different forms of chromatin.

Concepts: Gene, Genetics, Nucleosome, Chromatin, Histone, Cell nucleus, Spermatogenesis, DNA

27

This study was designed to determine the effects of Compound A on spermatogenesis including assessment of inhibin B levels and on fertility in the male rat over a 15 to 19 weeks treatment and a 19 weeks treatment-free period in control and 30, 60, and 180 mg/kg dose groups (n = 22/group). Compound A in a dose-dependent manner induced various degrees of spermatogenic alterations compatible with Sertoli cells being the primary target, for example, inter- and intracellular Sertoli cell vacuolization and altered cellular morphology followed by germ cell degeneration and marked reduction of epididymidal sperm numbers. Blood-testis barrier remained intact (electron microscopy and hyperosmotic fixation test) until germ cells disappeared. Mating behavior and weights of androgen-dependent prostate and seminal vesicles remained unaffected. Inhibin B levels correlated only with moderate to severe spermatogenic alterations. Ten animals with inhibin B levels below detection limit were encountered and five of these animals were fertile in week 19 but following 19 weeks without treatment, another five animals were rendered infertile and inhibin B levels remained undetectable. In the rat, inhibin B only reflects major spermatogenic alterations and markedly reduced inhibin B levels might indicate irreversibility of these alterations and even infertility.

Concepts: Sperm, Blood-testis barrier, Meiosis, Sertoli cell, Fertility, Testosterone, Spermatogenesis, Spermatozoon

24

The purpose of this review is to describe the endocrine and local testicular factors that contribute to the regulation of the blood-testis barrier (BTB), using information gained from in vivo and in vitro models of BTB formation during/after puberty, and from the maintenance of BTB function during adulthood. In vivo the BTB, in part comprised of tight junctions between adjacent somatic Sertoli cells, compartmentalizes meiotic spermatocytes and post-meiotic spermatids away from the vasculature, and therefore prevents autoantibody production by the immune system against these immunogenic germ cells. This adluminal compartment also features a unique biochemical milieu required for the completion of germ cell development. During the normal process of spermatogenesis, earlier germ cells continually cross into the adluminal compartment, but the regulatory mechanisms and changes in junctional proteins that allow this translocation step without causing a ‘leak’ remain poorly understood. Recent data describing the roles of FSH and androgen on the regulation of Sertoli cell tight junctions and tight junction proteins will be discussed, followed by an examination of the role of paracrine factors, including members of the TGFβ superfamily (TGFβ3, activin A) and retinoid signalling, as potential mediators of junction assembly and disassembly during the translocation process.

Concepts: Puberty, Cell membrane, Immune system, Testicle, Gene, Testosterone, Sertoli cell, Spermatogenesis

23

In this study we analyzed the effect of chronic and low-dose-rate (LDR) radiation on spermatogenic cells of large Japanese field mice ( Apodemus speciosus ) after the Fukushima Daiichi Nuclear Power Plant (FNPP) accident. In March 2014, large Japanese field mice were collected from two sites located in, and one site adjacent to, the FNPP ex-evacuation zone: Tanashio, Murohara and Akogi, respectively. Testes from these animals were analyzed histologically. External dose rate from radiocesium (combined (134)Cs and (137)Cs) in these animals at the sampling sites exhibited 21 μGy/day in Tanashio, 304-365 μGy/day in Murohara and 407-447 μGy/day in Akogi. In the Akogi group, the numbers of spermatogenic cells and proliferating cell nuclear antigen (PCNA)-positive cells per seminiferous tubule were significantly higher compared to the Tanashio and Murohara groups, respectively. TUNEL-positive apoptotic cells tended to be detected at a lower level in the Murohara and Akogi groups compared to the Tanashio group. These results suggest that enhanced spermatogenesis occurred in large Japanese field mice living in and around the FNPP ex-evacuation zone. It remains to be elucidated whether this phenomenon, attributed to chronic exposure to LDR radiation, will benefit or adversely affect large Japanese field mice.

Concepts: Electricity generation, Spermatogenesis, Apodemus, Sertoli cell, Nuclear power, Nuclear safety, Meiosis, Chernobyl disaster

6

Until now, complete ex-vivo spermatogenesis has been reported only in the mouse. In this species the duration of spermatogenesis is 35 days, whereas it is 54 days in the rat and 74 days in the human. We performed long term (until 60 days) cultures of fresh or frozen rat or human seminiferous tubule segments in a bioreactor, made of a hollow cylinder of chitosan hydrogel. Testicular tissues were obtained from 8 or 20 day old male rats, or from adult human subjects having undergone hormonal treatments leading to a near complete regression of their spermatogenesis, before bilateral orchiectomy for gender reassignment. The progression of spermatogenesis was assessed by cytological analyses of the cultures; it was related with a dramatic increase in the levels of mRNAs specifically expressed by round spermatids: Transition protein 1, Transition protein 2 and Protamine 3 in rat cultures. Two to 3.8% of cells were found haploid cells by FISH analysis of human cultures. In this bioreactor, long term cultures of seminiferous tubule segments from (pre)-pubertal rats, or from adult men, allowed the completion of the spermatogenic process, leading to morphologically mature spermatozoa. Further studies will have to address the way of optimizing the yield of every step of spermatogenesis, by adjusting the composition of the culture medium, the geometry and material properties of the chitosan hydrogel bioreactors. Another essential requirement is to assess the quality of the gametes produced ex-vivo by showing their ability to produce normal offspring (rat) or their biochemical normality (human).

Concepts: Spermatogenesis, Sperm, Protein, Spermatozoon, Meiosis, Mouse, Rat, Sertoli cell

4

Type 2 cannabinoid receptor (CB2) has been proposed to play a pivotal role in meiotic entry of male germ cells, similar to retinoic acid (RA). In this study, we showed that activation of CB2 with the specific agonist JWH133 (IC50 10(-6) M) mimics epigenetic events induced by RA (IC50 10(-7) M) in spermatogonia. Both JWH133 and RA treatments stimulate the expression of the meiotic genes c-Kit and Stra8, by up-regulating H3K4me3 and down-regulating H3K9me2 levels in genomic regions flanking the transcription start site. Moreover, both agents increase the expression of Prdm9, the gene encoding a meiosis-specific histone H3K4me3 methyltransferase, which marks hotspots of recombination in prophase I, thus resulting in a global increase in H3K4me3. Notably, prolonged administration of JWH133 to immature 7 dpp CD-1 mice induced an acceleration of the onset of spermatogenesis, whereas the specific CB2 antagonist delayed germ cell differentiation. Thus, both hyper- and hypostimulation of CB2 disrupted the temporal dynamics of the spermatogenic cycle. These findings highlight the importance of proper CB2 signaling for the maintenance of a correct temporal progression of spermatogenesis and suggest a possible adverse effect of cannabis in deregulating this process.-Di Giacomo, D., De Domenico, E., Sette, C., Geremia, R., Grimaldi, P. Type 2 cannabinoid receptor contributes to the physiological regulation of spermatogenesis.

Concepts: Cannabinoid receptor, Spermatogenesis, Gene expression, Meiosis, Cellular differentiation, DNA, Germ cell, Gene

3

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades and many of the long standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP inhibiting the hydrolysis of intra-oocyte cAMP and that the preovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle; research over the past 2 decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practiced clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared to clinical IVF. This review focuses in particular on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

Concepts: DNA, Spermatogenesis, Polar body, Oocyte, In vitro fertilisation, Oogenesis, Folliculogenesis, Meiosis