Effect Of Microgravity On Aromatase Expression In Sertoli Cells

Abstract

Cytochrome P450-aromatase catalyzes estrogen biosynthesis from C₁₉ steroids. In the testis, Sertoli cells express P450-aromatase and represent the primary source of estrogen during prepuberal age. This study focused on the effect of simulated microgravity (SM) on aromatase expression in primary mouse Sertoli cells. When cultured in Rotary Cell Culture System (RCCS), Sertoli cells, formed multicellular three dimensional spheroids (3D). Biological properties were first analyzed in terms of viability, cell cycle, expression of cytoskeletal components and growth factors in comparison to Sertoli cells cultured in spheroids at unit gravity (G). SM did not affect cell viability and proliferation, nor expression of the main cytoskeleton proteins and of growth factors like Kit Ligand (KL) and glial derived neurotrophic factor (GDNF). On the other hand, SM caused a strong increase in P450 aromatase mRNA and protein expression. Interestingly, P450-aromatase was no more inducible by 8-Br-cAMP. The presence of a functional aromatase was confirmed by enrichment of 17β-estradiol released in the medium by androgen precursors. We concluded that SM causes a significant upregulation of aromatase gene expression in Sertoli cells, leading to a consequent increase in 17β-estradiol secretion. High level of 17β-estradiol in the testis could have potentially adverse effects on male fertility and testicular cancer.

Figure 1

SM induces Sertoli cell spheroids. (A) Schematic representation of 3D Sertoli cell culture at unit gravity (G) and in Rotary Cell Culture System (SM). (B) Representative images of Sertoli cell spheroids after 48 hours of culture at G or under SM. (C) Representative sections of Sertoli cell spheroids immunostained with WT1 antiboby, a marker of Sertoli cells and stained with H&E.

Figure 2

SM does not cause significative changes in Sertoli cell viability and cell cycle. (A) Percentage of sub G1 Sertoli cells cultured at G or under SM. Sertoli were cultured for 48 h and then analyzed by FACS after propidium iodide staining. (B) Percentage of Trypan blue positive Sertoli cells detected after 48 h of culture at G or under SM. (C) Analysis of cleaved-PARP in protein extracts from Sertoli cells cultured at G or under SM showing the absence of cleaved forms in SM and G conditions. In Control lane is reported a positive control in which embryonal carcinoma cells (2102EP cell line) were treated for 24 h with 3.3 µM cisplatin. A cleaved band at around 89 kDa is shown. (D) Cell cycle analysis of Sertoli cells cultured for 48 h at G or under SM and then analyzed by FACS after propidium iodide staining. Most of the cells are in G1 phase in both culture conditions. At least three different experiments were performed. Bars represent s.d.

Figure 3

Microgravity causes a decrease in β-catenin protein expression. (A) Semiquantitative–PCR analysis of growth factors GDNF and KL in Sertoli cells cultured for 48 h at G or under SM. (B) Western blot analysis of cytoskeletal proteins in extracts from Sertoli cells cultured for 48 h at G or under SM. Densitometric analysis of β-catenin is reported on the right showing a decrease in the protein level under SM. (C) Western blot analysis of β-catenin in extracts from Sertoli cells cultured for 48 h at G or under SM, in the presence or not of GSK-3β inhibitor CHIR 99021. Treatment inhibits degradation of β-catenin caused by SM. (D) Western blot analysis of β-catenin distribution in the nuclear (N) and cytosolic (C) fractions of Sertoli cells cultured for 48 h at G or under SM. At least three different experiments were performed. Bars represent s.d. Asterisks indicate: *P < 0.05.

Figure 4

SM increases P450-aromatase expression in Sertoli cells. Sertoli cells were cultured for 48 h at G or in RCCS. (A) Real time PCR of P450-aromatase showing a strong increase of mRNA level under microgravity condition. (B) Real time-PCR for P450-aromatase in Sertoli cells treated or not with 1 mM 8-Br-cAMP. cAMP inducibility is lost under SM. (C) Semiquantitative RT-PCR of P450-aromatase and KL in Sertoli cells treated or not for 48 h with 1 mM 8-Br-cAMP. P450-aromatase expression is stimulated by 8-Br-cAMP at G but not under SM, while KL expression is stimulated by 8-Br-cAMP in both culture conditions. (D) Western blot analysis of P450-aromatase in Sertoli cells after 48 h of culture at G or under SM. Histogram on the right reports the mean of densitometric analysis of four different experiments. Values were normalized by reference to values for actin. (E) Chemiluminescence immunoassay for estrogen level in the culture medium of Sertoli cells. Sertoli cells were cultured in the presence or absence of 50 nM testosterone at G or in SM for 48 h. SM causes an increase of estrogen in the medium when cells are treated with testosterone. At least three different experiments were performed. Bars represent s.d. Asterisks indicate: *P < 0.05 and **P < 0.01.