Developing Mouse Sertoli Cells in vitro: Effects on Developing Ovaries in Co-Culture and Production of Anti-Müllerian Hormone

Abstract

Previous work in our laboratory showed that pre-Sertoli cells adopt an epithelial phenotype when cultured in the presence of reconstituted basement membrane (RBM), and so cultures were established with and without this substrate. Biological activity of isolated developing mouse Sertoli cells maintained in vitro was assessed in the current study by utilising a co-culture approach, to determine whether the cells were capable of affecting ovarian differentiation. Developing Sertoli cells isolated at embryonic day (E) 12.5 exerted a deleterious effect on E12.5 ovaries in co-culture, inducing a loss of germ cells. However, when cells were isolated a day later and co-cultured with E13.5 ovaries (after entry to meiosis has begun), germ cells survived and showed evidence of meiosis, although ovigerous cords in co-cultures were masculinised compared to those of control cultured ovaries. Thus, both stages examined showed biological effects; cultured pre-Sertoli cells explanted at E12.5 showed a negative effect on female germ cells, whereas those explanted at E13.5 masculinised ovigerous cords. The functional status of isolated developing mouse Sertoli cells in vitro was further assessed by immunocytochemistry to investigate the expression of anti-Müllerian hormone, an early product of pre-Sertoli cells. Positive immunostaining was seen in developing Sertoli cells in vitro, particularly where cells had been explanted to an RBM substrate, demonstrating that good epithelial morphology is associated with improved function. Our culture system is therefore well suited for investigating factors produced by developing Sertoli cells, their role in influencing testicular morphogenesis and their potential to perturb ovarian differentiation. We believe that this in vitro approach provides a more physiological assessment compared with the knockout mouse model, where global effects of genes with housekeeping functions can compromise overall development.

Effects of FGF9 on embryonic Sertoli cell proliferation and testicular cord formation in the mouse

“…These observations are consistent with our previous findings that ECM gel enhances cord-like aggregation and morphological maturation of the embryonic Sertoli cells in culture (Mackay et al, 1999). Functional maturity of the cultured Sertoli cells was confirmed by immunocytochemistry for the presence of AMH (Mackay et al, 2004).…”

“…sc-56). AMH was used both as a Sertoli cellspecific marker and also to assess functional status of the cells (Mackay et al, 2004), PCNA as a proliferative marker and FGFr3 as the receptor for FGF9. Smooth muscle α-actin antibody, a known smooth muscle specific marker (Skalli et al, 1986), was purchased from Sigma Aldrich (cat.no.…”

Effects of glial cell line‐derived neurotrophic factor on isolated developing mouse Sertoli cells in vitro

“…Immunocytochemical analysis was carried out using a variety of primary antibodies at various working concentrations. The following antibody dilutions were used: anti‐proliferating cell nuclear antigen (PCNA) (Santa Cruz: SC‐56) at 1 : 100; anti‐GFR‐α1 (Santa Cruz: SC‐10716) at 1 : 200; anti‐AMH (Santa Cruz: SC‐6886) at 1 : 50, as a marker for Sertoli cells (Mackay et al. 2004), and anti‐smooth muscle α‐actin antibody (Sigma cat.…”

“…1999). This has recently been applied to investigate their production of anti‐Müllerian hormone and effects on developing ovaries in co‐culture (Mackay et al. 2004) and to demonstrate that fibroblast growth factor 9 (FGF9) exerts a proliferative effect on developing Sertoli cells in vitro (Willerton et al.…”

Determination and stability of sex

“…(43) They also did not antagonize early steps of testis tubule formation in organotypic assays. (45) In fact, several expression profiling and functional studies indicate that progression of oocytes through early steps of meiosis may itself be modulated by somatic cells (e.g. Refs 43,46,47).…”