Effect Of Microgravity On Aromatase Expression In Sertoli Cells

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.


Sertoli cells form spheroids in RCCS.
To investigate the effects of microgravity on Sertoli cells, we used the Rotary Cell Culture System (RCCS), a microgravity based bioreactor 19,20 . Under this condition, mouse Sertoli cells obtained from 17-days-old mice showed a round-shaped phenotype and aggregated into three dimensional (3D) multicellular spheroids (Fig. 1A). Sertoli cell cultured in RCCS were compared with 3D Sertoli cell aggregates cultured at unit gravity (G). They were obtained by plating the cells on plastic dishes pretreated with soft agar that, not allowing cell adhesion, forced the cells to grow in suspension and to form cell aggregates, similar in shape and size to those grown in RCCS (Fig. 1A,B). The cell-spheroids formed in RCCS or at G were composed almost exclusively of Sertoli cells, as revealed by immunohistochemistry with anti-WT1 antibody, a Sertoli cell marker, indicating a very high purity of cell population (Fig. 1C).
The effect of microgravity on cell survival was investigated after 48 hours of culture in RCCS by FACS (Fluorescence Activated Cell Sorter) analysis of cell cycle and by Trypan blue staining. As reported in Fig. 2A the percentage of cells in sub G1, which represented the apoptotic cells, was very low and did not change between RCCS and G conditions ( Fig. 2A). Similarly, no significant modifications in the percentage of Trypan blue positive cells could be detected (Fig. 2B), indicating that microgravity did not affect cell survival. We also analyzed the expression of PARP-1 protein as marker of apoptosis, whose cleavage in fragments of 89 and 24 kDa represents a useful hallmark of this type of cell death. We showed that cleaved PARP-1 fragments were not detectable in 3D cultures neither at G or in RCCS (Fig. 2C) confirming that Sertoli cells viability is not affected by the culture conditions used. A band corresponding to 89kDa-cleaved PARP-1 was detected in embryonal carcinoma cell line treated with cisplatin used as positive control.
During perinatal period, Sertoli cells proliferate quickly and, at around the onset of puberty, they switch from an immature, proliferative state to a mature, non-proliferative state. In mouse, immature Sertoli cells are proliferative until 10-13dpp (days post partum), at which point they permanently exit the cell cycle and are considered a stable and terminally differentiated population. However, since evidence from the literature is now challenging this dogma and it has been reported that Sertoli cells can resume proliferation 21 , we set out to investigate Sertoli cell proliferation in our cultures by FACS analysis. Sertoli cells cultured in spheroids in RCCS were mostly found in G1 phase (79.8% ± 6.4%) and no significant difference was detected between them and the cells at G (74.5% ± 7.5%) (Fig. 2D). This result suggests that Sertoli cells from 17dpp mice were in a quiescent state and SM did not influence their cell cycle progression. A similar conclusion was reached by cell cycle analysis after incorporation of BrdU, which did not give any staining of cells (data not shown). Our results indicate that SM did not induce significant alterations in viability and cell cycle progression in Sertoli cells.

Sertoli cell biological properties in RCCS.
To understand the biological response of Sertoli cells to the different environmental stimuli induced by SM, we first analyzed, by semiquantitative RT-PCR, the expression of two trophic factors such as Glial-cell-line-derived neurotrophic factor (GDNF) and Kit ligand (KL), secreted by the Sertoli cells 1 . As shown in Fig. 3A they resulted unaffected by microgravity. Next we evaluated by western blot analysis the expression of the main cytoskeletal components. We show in Fig. 3B that no alterations were observed in the expression of tubulin, actin and βIII tubulin in Sertoli cells in RCCS with respect to G condition. However, we found that microgravity caused a significant decrease in β-catenin global expression (1.13 ± 0.18 at SM respect to 1.63 ± 0.21 at G) (Fig. 3B). In normal epithelial cells β-catenin is found associated at the cellular membrane at adherent junctions 22 , while any free cytoplasmic β-catenin is phosphorylated and targeted for ubiquination-dependent degradation by a protein complex formed by APC, GSK-3, CKIα, and Axin 23 . We observed that, by interfering with this process with the use of CHIR 99021, an inhibitor of GSK-3 β, the level of β-catenin in Sertoli cells cultured in RCCS could be restored up to that of cells cultured at G (Fig. 3C), indicating that microgravity induced a significant increase in β-catenin degradation. We next investigated the distribution of β-catenin between nuclear and cytoplasmic fraction. We show that β-catenin is present in the cytoplasm and is particularly concentrated in the nuclei and that microgravity reduced the cytoplasmic form but had no effect on the nuclear form (Fig. 3D). Our results indicate that SM did not induce significative alterations in the expression of two growth factors, KL and GDNF as well as in the expression of the main cytoskeleton components such as actin and tubulin, but caused a downregulation of cytoplasmic β-catenin protein level.

SM affects aromatase expression in Sertoli cells.
In the testis of immature male rats, Sertoli cells represent primary source of estrogen, while in mature male, estrogen production in Sertoli cells strongly decreases 24 . We first aimed to evaluate, by real time PCR, the effect of microgravity on the expression of P450-aromatase in mouse prepuberal Sertoli cells. As shown in Fig. 4A, a highly significant increase of aromatase mRNA was detected in Sertoli cells cultured in RCCS, which resulted higher (0.0017 ± 0.001) with respect to culture at G (0.0004 ± 0.001). We also investigated the effects of SM on basal and 8-Br-cAMP stimulated aromatase mRNA levels by Real time-PCR. 8-Br-cAMP stimulated P450-aromatase expression in Sertoli cells cultured at G, but not in cells in RCCS, being the basal level of aromatase mRNA already high and therefore not substantially influenced by 8-Br-cAMP (Fig. 4B). On the other hand, semiquantitative PCR analysis of KL, whose expression is upregulated by 8-Br-cAMP at G, maintained this regulation also in SM (Fig. 4C). We next investigated the expression of aromatase at protein level by western blot analysis. The presence of an aromatase immunoreactive protein band of about 55 kDa was particularly faint in Sertoli cells at G but it was more readily detected under SM, indicating that microgravity caused an increase of aromatase expression (0.47 ± 0.11 at SM versus 0.173 ± 0.05 at G) also at protein level in primary mouse Sertoli cells (Fig. 4D).
Since aromatase abundance controls the levels of local estrogens, we investigated the presence of a functional P450-aromatase in Sertoli cells by measuring the amount of 17β-estradiol converted by androgen precursors (i.e. testosterone). Sertoli cells were cultured in the presence or absence of testosterone for 48 hours in RCCS or at G. At the end of treatment, the presence of 17β-estradiol was evaluated in the medium by chemiluminescence immunoassay. As shown in Fig. 4E, in absence of testosterone, the concentration of 17β-estradiol detected in the medium did not change between G and SM condition (178 pg/10 6 cells ± 34 at G versus 175 pg/10 6

Discussion
Knowledge about the effects of microgravity on male reproduction is still in progress. Here we report the effects of SM on the expression of P450-aromatase in isolated mouse Sertoli cells. Studies on the effect of lack of gravity at the cellular level are limited. In mouse, microgravity has a direct effect on male germ cells; indeed it has been demonstrated that isolated mitotic spermatogonia cultured under SM in RCCS enter into meiosis in the absence of any added exogenous factor or contact with somatic cells 25 , while mouse pachytene spermatocytes undergo a spontaneous meiotic progression 26 . Here we established a 3D culture system of primary mouse Sertoli cells in RCCS and we compared some biological properties of these cells to those cultured in 3D at unit gravity. This approach allows to study the cell autonomous testicular cell responses to weightless not dependent on the influence of hormones. In both culture conditions, Sertoli cells formed cell aggregates that were similar in size. SM did not affect cell viability and apoptosis, as revealed by FACS analysis of sub G1 population, by Trypan blue staining and by western blot analysis of PARP-1 protein. Moreover, SM did not induce alterations in cell cycle and most of the cells were in G1 phase as those cultured at unit gravity. This result is in line with previous evidence indicating that most of Sertoli cells from prepuberal 17days old mice have already arrested their proliferative state 27,28 . The expression of two important growth factors produced by Sertoli cells, GDNF and KL, was not altered under SM as well as the expression of the main cytoskeletal components. However, we showed that SM reduced the level of cytoplasmic β-catenin protein by promoting its degradation, since treatment of the cells with a specific GSK-3 β inhibitor, rescued the expression levels of the protein. Although the molecular mechanisms by which SM influences β-catenin expression remains unknown, we might hypothesize that the effect is linked to fluid shear stress to which cells are exposed in RCCS culture. In fact previous studies report that shear flow negatively regulated the level of β -catenin in colon cancer cells 29 . In these cells, shear stress is sensed and transduced by a pathway that involves laminin-5, α6β4 integrin, PI 3-kinase and Rac1 to selectively regulate the level of β-catenin, and future studies will be done to investigate this pathway also in Sertoli cells. The most relevant finding of this study is that microgravity induced a very high upregulation of P450-aromatase expression at mRNA and protein level in Sertoli cells. We showed that in mouse, similarly to rat 11 , expression of P450-aromatase is very low in non-proliferating Sertoli cells, when they are cultured at unit gravity, but it was strongly upregulated under microgravity, with consequent enrichment in estradiol production. The high level of expression of aromatase under SM caused a loss of its cAMP inducibility. Indeed basal level of aromatase mRNA was high under SM and therefore not substantially influenced by 8-Br-cAMP. This effect is specific of this gene and not of other cAMP-regulated genes in Sertoli cells such as KL, suggesting that microgravity may modify the transcription of aromatase gene acting on transcription factors involved in its cAMP regulation. All together these results indicate that increased aromatase expression could affect the hormonal environment of testicular tissue under SM conditions, thus possibly affecting also circulating hormonal levels. Indeed, it has been already reported that male transgenic mice overexpressing aromatase in the testis showed estradiol levels higher than control mice, both locally and in the serum 30 .
Many studies established the importance of estrogens in male fertility [31][32][33] , as demonstrated by experimental models of mice lacking estrogen receptors or aromatase. Mice lacking ERα are sterile and sperms recovered from the cauda epididymis show low motility 33 , while aromatase deficient mice (ArKO) develop abnormal spermatogenesis with a blockage of germ cell maturation at the spermatid stage 34 . However, it should be underlined that estrogens may have an inhibitory influence of on Leydig cell steroidogenesis 35,36 and that high levels of this hormone have adverse effects on male reproduction 37,38 . In accordance, increased estrogen levels have been observed in infertile men with low sperm production and quality 39 . The overexpression of aromatase in Sertoli cells under SM could also be associated with lower level of testosterone detected in rat and men after space flights and could be linked to increased aromatization of testosterone to estrogen 5,6 . Moreover it may be speculated that microgravity could modify the expression of aromatase also in other tissues expressing the enzyme such ovarian granulosa cells, the placental syncytiotrophoblast, adipose and skin fibroblasts, bone, and brain. One consequence in men should be a change in T/E2 (Testosterone/17β-estradiol) balance, a parameter that is among the etiologic factors in idiopathic infertile males 40 . Beside the alterations in male fertility, higher levels of estrogen could have a role in promoting testicular tumors. It has been demonstrated that in mice, overexpression of aromatase results in increased estrogen production that leads to the induction of Leydig cell tumors 30 . The prolonged exposure to an excess of estrogens might have a more general role in inducing male reproductive tract malignancies such as testicular and prostatic tumors 41 . Interestingly, among somatic testis tumors, large cell calcifying Sertoli cell tumors 42 and Sertoli cell tumors of Peutz-Jeghers syndrome have shown an enhanced expression of P450-aromatase 43,44 .
In conclusion, the results from this study highlight the relationship between microgravity, P450-aromatase expression and estrogen production in Sertoli cells and suggest that long term weightless exposure of male astronauts may affect fertility and promote testicular cancers.

Methods
Cell culture. Primary Sertoli cell enriched-cultures from 17-days-old Swiss-CD1 mice were prepared as previously described 45 . Seminiferous tubules were prepared by sequential trypsin and collagenase digestion of dealbuginized testes. Tissue explanted was cultured at 32 °C in serum free minimum essential medium (MEM) supplemented with glutamine, nonessential amino acids, penicillin and streptomycin for 3 days and then they were treated with hypotonic solution (20 mM Tris-HCl, pH 7.5) to remove remaining germ cells. Cells were cultured at unit gravity in conventional tissue culture dishes with PBS 0.7% agarose, or at microgravity in a rotary cell culture system (RCCS) (Synthecon Inc., El Rio, Houston, TX, USA). In the RCCS condition 4 × 10 6 Sertoli cells were seeded in disposable vessels with 10 ml MEM, using a rotation rate of 14 revolutions/min (RPM) 25 . Where indicated, Sertoli cells were cultured for 48 hours with 3 µM GSK3β inhibitor, CHIR 99021 (Axon Medchem) 46 or with 1 mM 8-Br-cAMP (8-bromoadenosine 3′,5′-cyclic monophosphate, Sigma-Aldrich). All tested conditions were analyzed in triplicates. Animals were maintained and killed in accordance with European Community guidelines. Experimental protocols were performed in accordance with guidelines established by the European Legislation (Directive 2010/63/EU) and approved by University of Rome Tor Vergata IACUC and by Ministry of Health (legal authorization N. 140072016-PR).

RT-PCR Analysis and quantitative Real Time PCR. Total RNA was extracted from Sertoli cells using
Trizol reagent (Invitrogen) according to the manufacturer's instructions and 1 μg was used for retrotranscription (RT) using M-MLV reverse transcriptase (Invitrogen). cDNA produced by the RT reaction was used as template for semiquantitative PCR analysis (GoTaq, Promega) or quantitative Real Time PCR performed using SSOADV Universal SYBR Green (BioRad) in a PRISM 7300 Sequence Detection System (Applied Biosystems). All the primers used are listed in Table 1.