Trivial role for NSMCE2 during in vitro proliferation and differentiation of male germline stem cells

Abstract

Spermatogenesis, starting with spermatogonial differentiation, is characterized by ongoing and dramatic alterations in composition and function of chromatin. Failure to maintain proper chromatin dynamics during spermatogenesis may lead to mutations, chromosomal aberrations or aneuploidies. When transmitted to the offspring, these can cause infertility or congenital malformations. The structural maintenance of chromosomes (SMC) 5/6 protein complex has recently been described to function in chromatin modeling and genomic integrity maintenance during spermatogonial differentiation and meiosis. Among the subunits of the SMC5/6 complex, non-SMC element 2 (NSMCE2) is an important small ubiquitin-related modifier (SUMO) ligase. NSMCE2 has been reported to be essential for mouse development, prevention of cancer and aging in adult mice and topological stress relief in human somatic cells. By using cultured primary mouse spermatogonial stem cells (SSCs), referred to as male germline stem (GS) cells, we investigated the function of NSMCE2 during spermatogonial proliferation and differentiation. We first optimized a protocol to generate genetically modified GS cell lines using CRISPR-Cas9 and generated an GS cell line. Using this GS cell line, we found that NSMCE2 was dispensable for proliferation, differentiation and topological stress relief in mouse GS cells. Moreover, RNA sequencing analysis demonstrated that the transcriptome was only minimally affected by the absence of NSMCE2. Only differential expression of appeared highly significant, but with SGSM1 protein levels being unaffected without NSMCE2. Hence, despite the essential roles of NSMCE2 in somatic cells, chromatin integrity maintenance seems differentially regulated in the germline.

Kisspeptin and the regulation of the reproductive axis in domestic animals

“…Further unpublished data cited by Ralph et al (2016) showed that stress-like levels of cortisol increased pre-prodynorphin mRNA levels in the arcuate nucleus of ewes, compared with control animals. Lipopolysaccharide injection reduced the expression of Fos protein in kisspeptin cells in the arcuate nucleus observed at the time of the preovulatory LH surge (Fergani et al 2017), indicating reduced activation of the kisspeptin neurons at this time due to the stress of the toxin. The role of stress in the regulation of preoptic kisspeptin neurons in domestic animals does not appear to have been tested.…”

The influence of retinoic acid-induced differentiation on the radiation response of male germline stem cells

“…A mouse GS cell line was established as previously reported [24,28]. Briefly, testes were harvested from neonatal DBA/2 J male mice, and after removing the tunica albuginea, testicular tissues were mechanically dissociated and subjected to a collagenase-trypsin dissociation to obtain a single-cell suspension.…”

In Vitro Meiosis of Male Germline Stem Cells

“…To visualize the progress of spermatogenesis in vitro, cytospin slides were used. After the cells were cytospun, they (cytospins) were fixed, permeabilized, and blocked as previously described (Zheng et al, 2017), followed by overnight incubation at 4 C with primary antibodies (Table S1). For negative controls, primary antibodies were replaced with complementary immunoglobulin G. On the next day, the cytospins were washed and incubated with the corresponding host-specific secondary antibodies (Table S1) and counterstained with DAPI.…”