Gdnf, a germ cell-derived factor, regulates zebrafish germ cell stemness through the creation of new spermatogonial niches (germ and Sertoli cells) and inhibition of spermatogonial differentiation in an autocrine and paracrine manners

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1 - GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in the mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testis. Considering this background, this study aimed to understand the roles of Gdnf-Gfrα1 signaling pathway in the zebrafish testis by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibited two paralogs of Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in agreement with the teleost-specific third round (3R) of whole genome duplication. Expression analysis further revealed that gdnfa and gfrα1a were the most expressed copies in the zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating proliferation of both type Aund spermatogonia and their surrounding Sertoli cells, but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation as shown by the decrease of type B spermatogonia and down-regulation of dazl in the co-treatment with Fsh. Altogether, our data revealed for the first time that a germ cell-derived factor is associated with maintaining germ cell stemness through the creation of new available niches, supporting development of differentiating spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine and paracrine manners.

Can antioxidants protect against chemotherapy in rat spermatogonial stem cell line?

LAY ABSTRACT: Boys administered chemotherapy to treat childhood cancer are at risk of damage to their healthy testicular tissue, which can lead to infertility in adulthood. Researchers are therefore investigating treatments to protect the testis during cancer treatment. Here, cells originating from the testicles of rats were cultured for four days and exposed to chemotherapy drugs with or without antioxidants for the final two days. Antioxidants can reduce cellular damage by inactivating toxic compounds. Here, the antioxidants melatonin or n-acetylcysteine were tested against chemotherapy agents’ cisplatin, doxorubicin, or vincristine. Cultures were repeated four times, with cell survival measured at the end of culture. The antioxidants themselves were not damaging to the cells and partially protected against cisplatin, although not against doxorubicin. Surprisingly, n-acetylcysteine was found to enhance the damage induced by vincristine. The results suggest that using antioxidants to try to protect the testis could have either beneficial or harmful effects when given alongside different chemotherapy drugs: this is important, considering that patients are often treated with multiple drugs which could react differently to protectants.

A novel posttranslational modification of histone, H3 S-sulfhydration, is down-regulated in asthenozoospermic sperm

Oxidative stress is one of the major causes leading to male infertility including asthenozoospermia. Hydrogen sulfide (H2S) has been widely recognized to be a potent antioxidant whose role is partially implemented by protein S-sulfhydration. However, protein S-sulfhydration has not been reported in germ cells. Therefore, we investigated whether asthenozoospermia could be associated with sperm protein S-sulfhydration. S-sulfhydrated proteins in human sperm were enriched via biotin-switch assay and analyzed using LC-MS/MS spectrometry. Two hundred forty-four S-sulfhydrated proteins were identified. Importantly, we validated that sperm histones H3.1 and H3.3 were the S-sulfhydrated proteins. Their S-sulfhydrated amino acid residue was Cysteine111. Abundances of S-sulfhydrated H3 (sH3) and S-sulfhydrated H3.3 (sH3.3) were significantly down-regulated in asthenozoospermic sperm, compared with the fertile controls, and were significantly correlated with progressive motility. Retinoic acid (RA) up-regulated level of sH3.3 in primary round spermatids and the C18-4 cells (a mouse spermatogonial stem cell line). Overexpression of the mutant H3.3 (Cysteine111 was replaced with serine) affected expression of 759 genes and raised growth rate of C18-4 cells. For the first time, S-sulfhydration H3 and H3.3 were demonstrated in the present study. Our results highlight that aberrant S-sulfhydration of H3 is a new pathophysiological basis in male infertility.

Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model

Preserving the spermatogonial stem cells (SSCs) in long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium. Testicular torsion - a mouse model for long-term infertility- was used to transplant fresh SSCs (n = 6), fresh SSCs treated with PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6) and cryopreserved SSCs treated with PTX (n = 6). Eight weeks after germ cell transplantation, samples were assessed for proliferation, through evaluation of Ddx4 and Id4 markers, and differentiation via evaluation of C-Kit and Sycp3, Tnp1, Tnp2, and Prm1 markers. According to morphological and flow cytometry results, SSCs are able to form colonies and express Gfra1, ID4, α6‐integrin and β1‐integrin markers. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice. In the recipient testis, donor SSCs formed spermatogenic colonies and sperm. Respecting these data, adding pentoxifylline is a practical way to precisely cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup. However, more studies are needed to ensure its safety in the long term.