Enhancing the Maturation Potential of Male Germ Cells by a Sertoli Cell Co-Culture System

Abstract

There is a lack of an effective treatment for patients in whom no sperm can be retrieved. Maintaining immature germ cells in an in vitro environment endowed with growth promoting factors may lead to resumption of spermatogenesis and this could constitute a therapeutic modality for infertile men. The Sertoli cell is the only somatic cell that interacts directly with germ cells and promotes germ cell maturation and differentiation. The objective of our study was to compare the in vitro growth potential of a population of mixed germ cells versus a homogenous immature cell population using Sertoli cell co-culture system. Prospective-controlled study Germ cells were extracted from testicular tissue of C57BL mice (n = 10) using enzymatic digestion and divided into 2 aliquots: the first was kept as control, while the second aliquot was incubated for 1 hour with 1: 200 dilution of anti-CD117 antibody (clone 2B8) that recognizes the c-kit receptor. This aliquot was further subjected to magnetic cell sorting using CD117-conjugated microbeads to isolate the predominantly immature cells (spermatogonia/spermatocytes). A fraction of the immature germ cells was cultured with 15P-1 Sertoli cell line (1: 1). Culture conditions were: human tubal fluid (HTF) media + 10% fetal bovine serum (FBS) enriched with 500 IU/L recombinant follicle stimulating hormone (rFSH), 10 μM testosterone, 500 ng/mL insulin, and 100 ng/mL insulin growth factor-I (IGF-I). Another fraction of immature germ cells and the mixed germ cell control were cultured in only HTF media + 10% FBS. The maturation progress i.e. incidence of mature haploid cells (spermatid/sperm) was assessed after day 5 of culture using flow cytometry (DNA CycleTEST™). Following germ cell isolation, the percentage (%) of haploid cells in the immature fraction separated by MACS was 12.56 ± 7.67 vs. 45.06 ± 15.86 in the mixed germ cell control. Results after 5 days-culture are shown in the table below. The % of haploid cells in the immature fraction separated by MACS and cultured with Sertoli cells was higher compared to the immature fraction cultured with only HTF+10% FBS (p<0.0001) and compared to the mixed germ cell control (borderline significance, p=.056). Following adjustment for the initial values before culture, the immature germ cells displayed significantly higher increase in % haploid cells when cultured with Sertoli cells (41.03 ± 23.95) compared to those cultured in HTF media+10% FBS (1.25 ± 8.62, p<0.0001) and to the mixed germ cell controls (-2.24 ± 26.88, p<0.0001). Routine culture media is not capable of maintaining spermatogenesis in vitro. The homogeneously separated immature germ cells by MACS display higher growth potential than mixed germ cell populations. Sertoli cell co-cultures supplemented with growth factors and hormones may enhance male germ cell maturation in vitro. Tabled 1