(374) Physiological and Metabolomic Differences in Canine Testes with or without Spermatogenesis

Huleihel, Mahmoud^1,2,4;AbuMadeghem, Ali^1,2,4; Rubin, Eitan^1,2,4; Lunenfeld, Eitan³

 ¹The Shraga Segal Dept. Microbiology, Immunology and Genetics, ²The Center of Advanced Research and Education in Reproduction (CARER), ³Adelson School of Medicine, Ariel University, Ariel, Israel, ⁴Faculty of Health Sciences, Ben-Gurion University of the Negev. Beer-Sheva, Israel.

Abstract Text:

Spermatogenesis is the process of sperm generation that begins with the proliferation and differentiation of spermatogonial stem cells (SSCs) within the seminiferous tubules. This intricate process is influenced by various testicular autocrine and paracrine cytokines and growth factors. While SSC transplantation has been successful in restoring fertility in sterile animal models, its application in humans remains unsafe due to the lack of precise methods to isolate pure SSCs from cancer cells. Interleukin-7 (IL-7) is a cytokine primarily secreted by stromal cells in the bone marrow and thymus. It is also produced by keratinocytes, follicular dendritic cells, hepatocytes, neurons, and epithelial cells. IL-7 plays a critical role in lymphocyte maturation and differentiation through its interaction with the IL-7 receptor (IL-7R). We aimed to identify a functional membrane-associate specific SSC markers. Following identification of IL-7R in thy-1 cells in the seminiferous tubules, we aimed to examine the cellular localization and expression levels of IL-7 and its receptor and the possibility of involvement of IL-7 in the development of spermatogenesis.  

In this study, we utilized 7-day-old sexually immature mice to enzymatically isolate cells from the seminiferous tubules for in vitro culture. Spermatogonial cells expressing markers such as Thy1, α6-integrin, and c-Kit were sorted using fluorescence-activated cell sorting (FACS). RNA was extracted from these sorted cells for RNA sequencing (RNAseq) analysis. Additionally, mice aged between 1 and 12 weeks were employed to assess the localization and expression levels of IL-7 and its receptor (IL-7R) in testicular tissues. Quantitative PCR (qPCR) and immunofluorescence staining were conducted for this purpose. To evaluate the effect of IL-7 on spermatogenesis development in vitro, we employed a three-dimensional (3D) methylcellulose culture system (MCS).  Immunofluorescence and qPCR analyses were utilized to identify and quantify the various stages of spermatogenesis within the MCS. The expression levels of Sertoli and Leydig cell activity markers were evaluated by qPCR analysis.

RNA sequencing analysis (RNAseq) revealed elevated expression of IL-7R in Thy1-positive cells isolated from immature mice. Immunofluorescence staining localized IL-7R-positive cells to the periphery of the seminiferous tubules in immature mice and to meiotic and post-meiotic cells in adult mice, excluding Sertoli cells. Quantitative PCR (qPCR) analyses confirmed IL-7R expression across all age groups, peaking at 4 weeks. Interleukin-7 (IL-7) was present in all spermatogenic cells but absent in Sertoli cells, with optimal expression observed at 8 and 12 weeks. In vitro, the addition of IL-7 to cells isolated from seminiferous tubules of immature mice, cultured for 4 weeks in a three-dimensional methylcellulose system, significantly increased the percentages of meiotic and post-meiotic cells compared to controls. Furthermore, IL-7 supplementation markedly upregulated the expression of Sertoli cell activity markers, transferrin and follicle-stimulating hormone receptor (FSH-R), without affecting the Leydig cell activity marker 3β-hydroxysteroid dehydrogenase (3β-HSD).

Through RNAseq analysis of FACS-sorted spermatogonial cells, we have identified IL-7R as a novel membrane-associated and functional marker of spermatogonial stem cells (SSCs). This study is the first to demonstrate the presence and expression of both IL-7R and its ligand, IL-7, within spermatogonial cells. Our in vitro experiments further reveal that IL-7 may play a regulatory role in spermatogenesis, particularly during the meiotic and post-meiotic stages. These findings suggest that the IL-7 signaling pathway is a novel testicular system involved in spermatogenesis and could be leveraged in future strategies for male fertility preservation and restoration.