Edward M. Eddy

Chen and Liu (1) agree with our main conclusion that glial cell line-derived neurotrophic factor (GDNF) produced by peritubular myoid (PM) cells signals to undifferentiated spermatogonia and regulates their maintenance (2), but argue that a PM cell-specific gene knockout is impossible because the myosin heavy polypeptide 11 smooth muscle-Cre (Myh11-Cre) is not expressed specifically in PM cells. Chen and Liu (1) note that in previous studies the Myh11-Cre transgene was expressed in vascular smooth muscle cells in a variety of tissues (3). However, what is important is whether the transgene and the …

Significance: Glial cell line-derived neurotrophic factor (GDNF) growth factor induces spermatogonial stem cells to proliferate in culture to produce progenitor spermatogonia. Sertoli cell GDNF was thought responsible for this in the testis. We showed that testosterone triggers GDNF secretion by peritubular myoid (PM) cells in culture and that undifferentiated spermatogonia (spermatogonial stem cells and progenitors) co-cultured with testosterone-treated PM cells and transplanted to sterile mice restored spermatogenesis. To determine if PM cell GDNF is essential to maintain the undifferentiated spermatogonial population, we made mice lacking theGdnfgene in PM cells. The number of undifferentiated spermatogonia was severely depleted in 2-wk-old mice and adults were infertile. This is the first study, to our knowledge, to show that the undifferentiated spermatogonial pool cannot be maintained without GDNF from PM cells.

While cyclin dependent kinase 1 (CDK1) has a critical role in controlling resumption of meiosis in oocytes, its role has not been investigated directly in spermatocytes. Unique aspects of male meiosis led us to hypothesize that its role is different in male meiosis than in female meiosis. We generated a conditional knockout (cKO) of the Cdk1 gene in mouse spermatocytes to test this hypothesis. We found that CDK1-null spermatocytes undergo synapsis, chiasmata formation, and desynapsis as is seen in oocytes. Additionally, CDK1-null spermatocytes relocalize SYCP3 to centromeric foci, express H3pSer10, and initiate chromosome condensation. However, CDK1-null spermatocytes fail to form condensed bivalent chromosomes in prophase of meiosis I and instead are arrested at prometaphase. Thus, CDK1 has an essential role in male meiosis that is consistent with what is known about the role of CDK1 in female meiosis, where it is required for formation of condensed bivalent metaphase chromosomes and progression to the first meiotic division. We found that cKO spermatocytes formed fully condensed bivalent chromosomes in the presence of okadaic acid, suggesting that cKO chromosomes are competent to condense, although they do not do so in vivo. Additionally, arrested cKO spermatocytes exhibited irregular cell shape, irregular large nuclei, and large distinctive nucleoli. These cells persist in the seminiferous epithelium through the next seminiferous epithelial cycle with a lack of stage XII checkpoint-associated cell death. This indicates that CDK1 is required upstream of a checkpoint-associated cell death as well as meiotic metaphase progression in mouse spermatocytes.

Peritubular myoid (PM) cells surround the seminiferous tubule and together with Sertoli cells form the cellular boundary of the spermatogonial stem cell (SSC) niche. However, it remains unclear what role PM cells have in determining the microenvironment in the niche required for maintenance of the ability of SSCs to undergo self-renewal and differentiation into spermatogonia. Mice with a targeted disruption of the androgen receptor gene (Ar) in PM cells experienced a progressive loss of spermatogonia, suggesting that PM cells require testosterone (T) action to produce factors influencing SSC maintenance in the niche. Other studies showed that glial cell line-derived neurotrophic factor (GDNF) is required for SSC self-renewal and differentiation of SSCs in vitro and in vivo. This led us to hypothesize that T-regulated GDNF expression by PM cells contributes to the maintenance of SSCs. This hypothesis was tested using an adult mouse PM cell primary culture system and germ cell transplantation. We found that T induced GDNF expression at the mRNA and protein levels in PM cells. Furthermore, when thymus cell antigen 1-positive spermatogonia isolated from neonatal mice were cocultured with PM cells with or without T and transplanted to the testes of germ cell-depleted mice, the number and length of transplant-derived colonies was increased considerably by in vitro T treatment. These results support the novel hypothesis that T-dependent regulation of GDNF expression in PM cells has a significant influence on the microenvironment of the niche and SSC maintenance.

Summary: Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co‐localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post‐meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2‐GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post‐meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage‐specific spermatogenic cell proteins and developmental events. genesis 52:976–984, 2014.

In this issue of Biology of Reproduction, de Rooij and van Beek describe a computer model simulating the population dynamics and interactions of SSCs and their descendants in and around the spermatogonial niche.

We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable red

Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the f

Wedemonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sper

Disruption of the Esr1 gene encoding estrogen receptor α (ERα) by insertion of a neomycin resistance gene (neo) into exon 2 (αERKO mice) was shown previously to cause infertility in male mice. While full-length ERα protein was not expressed in αERKO mice, alternative splicing resulted in the low-level expression of a truncated form lacking the N-terminus A/B domain and containing the DNA- and ligand-binding domains. Thus, it was unclear whether the reproductive phenotype in αERKO males was only due to the lack of full-length ERα or was affected by the presence of the variant ERα isoform. The present study examined male mice with deletion of exon 3 of Esr1 gene, lacking the DNA-binding domain, and null for ERα (Ex3αERKO). Dilation of some seminiferous tubules was apparent in male Ex3αERKO mice as early as postnatal day 10 and was pronounced in all tubules from day 20 onward. At 6 weeks of age, sperm numbers and sperm motility were lower in Ex3αERKO mice than in wild-type (WT) mice, and the rete testis and efferent ductules were dilated. Mating studies determined that adult Ex3αERKO males were infertile and failed to produce copulatory plugs. Serum testosterone levels and Hsd17b3 and Cyp17a1 transcript levels were significantly higher, but serum estradiol, progesterone, LH, and FSH levels and Cyp19a1 transcript levels were not significantly different from those in WT mice. These results confirm and extend those seen in other studies on male mice with deletion of exon 3 of Esr1 gene. In addition, the reproductive phenotype of male Ex3αERKO mice recapitulated the phenotype of αERKO mice, strongly suggesting that the αERKO male infertility was not due to the presence of the DNA-binding domain in the truncated form of ERα and that full-length ERα is essential for maintenance of male fertility.