P-420 Involvement of Interluekin-6 (IL-6) in the development of different stages of spermatogenesis in vitro, using spermatogonial cells isolated from normal and busulfan-treated immature mice.
Authors: Ali N , Lunenfeld E , Huleihel M 
Published: June 2023

Abstract

Study question
Can Interluekin-6 (IL-6) induce spermatogonial cells from seminiferous tubules of normal or busulfan-treated mice to develop spermatogenesis in vitro using methylcellulose culture system (MCS)?

Summary answer
IL-6 induced the development of spermatogonial cells from seminiferous tubules of normal or busulfan-treated mice to premeiotic, meiotic and post-meiotic cells in vitro.

What is known already
Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. These microenvironmental factors are modified following pathological condition, such as chemotherapy, which may lead to subfertility or sterility. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro; however, this was not yet achieved.

Study design, size, duration
Sexually immature mice (7-day-old) were used as normal or were intraperitoneally (i.p) injected with busulfan (45 mg/kg) to isolate cells from their seminiferous tubules (STs). Cells were enzymatically isolated from the STs of the mice and were cultured in methylcellulose culture system (MCS), as a 3-dimensinal in vitro system. Fresh media without (CT) or with IL-6 were added to the cultures from the beginning, and after two weeks. The cultures were determined after 4 weeks

Participants/materials, setting, methods
Isolated cells from the STs were cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF and in the presence/absence of IL-6 (1,10 or 100 pg/ml) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/organoids were examined microscopically and quantified for the development of cells of the different stages of spermatogenesis and Sertoli cells functional markers by immunofluorescence staining (IF) and/or qPCR analyses.

Main results and the role of chance
Addition of IL-6 to MCS that contained isolated cells from STs of normal immature mice significantly increased the development of premeiotic cells (VASA), meiotic (BOULE) and meiotic/postmeiotic cells (ACROSIN) as examined by specific IF and/or qPCR analyses. Addition of IL-6 also differently affected the expression levels of Sertoli cell functionality markers (androgen receptor, androgen binding protein, transferrin, GDNF, FSHR). Furthermore, addition of IL-6 to isolated cells from STs of busulfan-treated immature mice also increased the development of VASA-, BOULE- and ACROSIN-positive cells (as examined by IF and qPCR analyses). However, the effect of IL-6 was more potent in the development of different stages of spermatogenesis in in vitro cultures that contained isolated cells from STs of normal compared busulfan-treated mice. Also, addition of IL-6 distinctly affected the expression levels of Sertoli cell functional markers in cultures of normal and busulfan-treated mice.

Limitations, reasons for caution
This in-vitro culture used animal models. The bio-activity of the developed post-meiotic needs to be confirmed

Wider implications of the findings
These findings indicate the possible involvement of IL-6 in the development of spermatogenesis in vitro. This effect could be directly on spermatogonial cells and/or through Sertoli cells. Our result could suggest IL-6 as a potential factor to be used in developing future in vitro therapeutic strategies for male fertility preservation

Trial registration number
not applicable