Sawaied Alaa, Levy Bat-El, Arazi Eden, Lunenfeld Eitan, Shi Qinghua, Huleihel Mahmoud
The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
Int J Mol Sci. 2025 Mar 17;26(6):2702. doi: 10.3390/ijms26062702.
Spermatogenesis is a process of self-renewal of spermatogonial stem cells and their proliferation and differentiation to generate mature sperm. This process involves interactions between testicular somatic (mainly Sertoli cells) and spermatogonial cells at their different stages of development. The functionality of Sertoli cells is regulated by hormones and testicular autocrine/paracrine factors. In this study, we investigated the effects of follicle-stimulating hormone (FSH) and testosterone addition on Sertoli cell cultures that undergo hypotonic shock, with a primary focus on Sertoli cell activity. Cells were enzymatically isolated from testicular seminiferous tubules of 7-day-old mice. These cells were cultured in vitro for 3 days. Thereafter, some cultures were treated with hypotonic shock to remove germ cells. After overnight, fresh media without (control; CT) or with FSH, testosterone (Tes), or FSH+T were added to the hypotonic shock-treated or untreated (CT) cultures for 24 h. The morphology of the cultures and the presence of Sertoli cells and germ cells were examined. The expression of growth factors (CSF-1, LIF, SCF, GDNF) or other specific Sertoli cell factors [transferrin, inhibin b, androgen receptor (AR), androgen binding protein (ABP), FSH receptor (FSHR)] was examined by qPCR. Our immunofluorescence staining showed depletion/major reduction in VASA-positive germ cells in Sertoli cell cultures following hypotonic shock (HYP) treatment compared to untreated cultures (WO). Furthermore, the expression of the examined growth factors and other factors was significantly increased in HYP cultures compared to WO (in the CT). However, the addition of hormones significantly decreased the expression levels of the growth factors in HYP cultures compared to WO cultures under the same treatment. In addition, the expression of all other examined Sertoli cell factors significantly changed following HYP treatment compared to WO and following treatment with FSH and or T. However, the expression levels of some factors remained normal following the treatment of Sertoli cell cultures with one or both hormones (transferrin, Fsh-r, Abp, Ar). Thus, our results demonstrate the crucial role of germ cells in the functionality of Sertoli cells and the possible role of FSH and T in maintaining, at least partially, the normal activity of Sertoli cells following germ cell depletion in vitro by hypotonic shock treatment.
精子发生是精原干细胞自我更新、增殖并分化产生成熟精子的过程。该过程涉及睾丸体细胞（主要是支持细胞）与处于不同发育阶段的精原细胞之间的相互作用。支持细胞的功能受激素及睾丸自分泌/旁分泌因子调控。在本研究中，我们探究了添加促卵泡激素（FSH）和睾酮对经历低渗休克的支持细胞培养物的影响，主要聚焦于支持细胞活性。细胞通过酶解法从小鼠7日龄睾丸生精小管中分离出来。这些细胞在体外培养3天。之后，部分培养物经低渗休克处理以去除生殖细胞。过夜后，向经低渗休克处理或未处理（对照；CT）的培养物中添加不含（对照；CT）或含有FSH、睾酮（Tes）或FSH + T的新鲜培养基，培养24小时。检查培养物的形态以及支持细胞和生殖细胞的存在情况。通过qPCR检测生长因子（CSF - 1、LIF、SCF、GDNF）或其他特定支持细胞因子[转铁蛋白、抑制素b、雄激素受体（AR）、雄激素结合蛋白（ABP）、FSH受体（FSHR）]的表达。我们的免疫荧光染色显示，与未处理的培养物（WO）相比，低渗休克（HYP）处理后的支持细胞培养物中VASA阳性生殖细胞减少/显著减少。此外，与WO（对照中的CT）相比，HYP培养物中检测的生长因子和其他因子的表达显著增加。然而，与相同处理下的WO培养物相比，添加激素显著降低了HYP培养物中生长因子的表达水平。另外，与WO相比以及与FSH和/或T处理后相比，所有其他检测的支持细胞因子的表达在HYP处理后均发生显著变化。然而，用一种或两种激素处理支持细胞培养物后，某些因子的表达水平保持正常（转铁蛋白、Fsh - r、Abp、Ar）。因此，我们的结果表明生殖细胞在支持细胞功能中起关键作用，并且FSH和T在体外通过低渗休克处理使生殖细胞耗竭后至少部分维持支持细胞正常活性方面可能发挥作用。
