Follicle-Stimulating Hormone Induction of Steel Factor (SLF) mRNA in Mouse Sertoli Cells and Stimulation of DNA Synthesis in Spermatogonia by Soluble SLF

Abstract

Follicle-stimulating hormone (FSH) and its intracellular mediator, cAMP, increase the mRNA levels for the Steel factor (SLF, the c-kit ligand) in cultured primary mouse Sertoli cells. The inductive effect of cAMP is more evident in cultures from 13-day-old animals than in cultures from 18-day-old animals. Analysis through the polymerase chain reaction (PCR) indicates that (Bu)2cAMP or FSH treatment increases the levels of the mRNAs for both the potentially soluble form and the transmembrane form of SLF in cultured Sertoli cells. The ratio between mRNAs encoding the potentially soluble form and the transmembrane form of SLF increases during postnatal testis development, and it is higher in cultured Sertoli cells with respect to total testis, suggesting that, under the in vitro conditions, SLF could be produced by Sertoli cells mainly as a soluble factor. Soluble recombinant SLF stimulates, in a dose-dependent fashion, thymidine incorporation in cultures of isolated germ cell populations enriched in the mitotic stages (spermatogonia), independently of the presence of serum, whereas cAMP analogs have no effect. Autoradiographic analysis shows that SLF selectively stimulates DNA synthesis in type A spermatogonia.

Transcriptome analysis of differentiating spermatogonia stimulated with kit ligand

“…However, kit is known to play important roles also during post-natal stages of spermatogenesis (Sette et al, 2000). In the adult testis, the kit receptor is expressed in differentiating spermatogonia, but not in spermatogonial stem cells, whereas KL is expressed by Sertoli cells under FSH stimulation in both a soluble and a transmembrane form, which are generated by alternative splicing (Rossi et al, 1993). The soluble form of KL stimulates DNA synthesis in type A spermatogonia cultured in vitro, and injection of anti-kit antibodies blocks their proliferation in vivo (Rossi et al, 1993;Packer et al, 1995).…”

“…In the adult testis, the kit receptor is expressed in differentiating spermatogonia, but not in spermatogonial stem cells, whereas KL is expressed by Sertoli cells under FSH stimulation in both a soluble and a transmembrane form, which are generated by alternative splicing (Rossi et al, 1993). The soluble form of KL stimulates DNA synthesis in type A spermatogonia cultured in vitro, and injection of anti-kit antibodies blocks their proliferation in vivo (Rossi et al, 1993;Packer et al, 1995). A point mutation in the kit gene, which impairs KL-mediated activation of phosphatydilinositol 3-kinase, does not cause any significant reduction in PGCs number during embryonic development, nor in spermatogonial stem cell populations.…”

“…An interesting information coming from this set of data is that KL, which is known as a mitogenic and survival factor for differentiating spermatogonia (Rossi et al, 1993;Dolci et al, 2001) up-regulates the expression of ligands and receptors of the Tnf superfamily that might be involved in regulating the number of germ cells entering the first wave of spermatogenesis, which is characterized by an early and massive wave of apoptosis in the spermatocyte compartment (Rodriguez et al, 1997). The induction of Plzf, a known marker of spermatogonial stem cells (Buaas et al, 2004;Costoya et al, 2004), is in apparent conflict with the above described pro-differentiative pattern of gene expression set up by KL treatment of spermatogonia.…”

“…Germ cell populations highly enriched in mitotic spermatogonia were obtained as previously described from testes of 7-day-old mice (Rossi et al, 1993;Pellegrini et al, 2003;Dolci et al, 2001;Rossi et al, 2004). Briefly, germ cell suspensions were obtained by sequential collagenasehyaluronidase-trypsin digestions of freshly withdrawn testes.…”

Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro

“…In order to reliably assess the effect of exogenous growth factors on the proliferation of mouse SSCs, serum-free culture conditions were employed. It was reported that the addition of one to two growth factors could maintain cell survival in vitro (Rossi et al 1993). In our study, GDNF or LIF alone or in combinaion were supplemented to the medium and the effects on the mouse SSCs proliferation were evaluated.…”