Sara Correia

Metformin and male reproduction: effects on Sertoli cell metabolism.

Metformin is commonly used to treat type 2 diabetes (T2D). While new clinical applications have been ascribed to metformin, including treatment of anovulatory infertility, its effects on male reproduction have not been investigated. The Sertoli cell (SC) is crucial for germ cell development, exerting metabolic control of spermatogenesis, therefore, we investigated the effects of metformin on SC metabolism.

Regucalcin is broadly expressed in male reproductive tissues and is a new androgen-target gene in mammalian testis.

Regucalcin (RGN) is a calcium (Ca(2)(+))-binding protein which regulates intracellular Ca(2)(+) homeostasis by modulating the activity of enzymes regulating Ca(2)(+) concentration and enhancing Ca(2)(+)-pumping activity. Several studies have described the pivotal role of proper Ca(2)(+) homeostasis regulation to spermatogenesis and male fertility. Recently, RGN was identified as a sex steroid-regulated gene in prostate and breast; however, a possible role of RGN in spermatogenesis has not been examined. In this study, the expression and localization of RGN in rat and human testis, and other rat reproductive tissues was analyzed. Moreover, we studied whether RGN protein was present in seminiferous tubule fluid (STF). Finally, we examined the effect of 5α-dihydrotestosterone (DHT) on the expression of Rgn mRNA in rat seminiferous tubules (SeT) cultured ex vivo. The results presented in this study show that RGN is expressed in Leydig and Sertoli cells, as well as in all types of germ cells of both rat and human testis. RGN is also expressed in rat prostate, epididymis, and seminal vesicles. Moreover, RGN protein is present in rat STF. The results also demonstrate that Rgn expression is age dependent in rat testis, and is upregulated by the non-aromatizable androgen DHT in rat SeT cultured ex vivo. Taken together, these findings indicate that Rgn is a novel androgen-target gene in rat testis and that it may have a role in male reproductive function, particularly in the control of spermatogenesis.

Regucalcin, a calcium-binding protein with a role in male reproduction?

Regucalcin (RGN) is a calcium (Ca(2+))-binding protein which plays an important role in the regulation of Ca(2+) homeostasis and has been shown to catalyse an important step in L-ascorbic acid biosynthesis. It is encoded by an X-linked gene and differs from other Ca(2+)-binding proteins by lacking the typical EF-hand Ca(2+)-binding domain. RGN controls intracellular Ca(2+) concentration by regulating the activity of membrane Ca(2+) pumps. Moreover, RGN has been indicated to regulate the activity of numerous enzymes and to act in the regulation of cell proliferation and apoptosis. The importance of Ca(2+) homeostasis in spermatogenesis has been demonstrated by several studies, and its disruption has been shown to cause reversible male infertility. Recently, the expression of RGN in male reproductive tissues has been described and its localization in all testicular cell types was demonstrated. In addition, RGN expression is regulated by androgens, a class of steroid hormones recognized as male germ cell survival factors and of uttermost importance for spermatogenesis. Altogether, available information suggests the hypothesis that RGN might play a role in spermatogenesis, directly or as a mediator of androgen action. This review discusses this hypothesis presenting novel data about RGN expression in human testis.

Expression pattern of G protein-coupled receptor 30 in human seminiferous tubular cells.

The role of estrogens in male reproductive physiology has been intensively studied over the last few years. Yet, the involvement of their specific receptors has long been a matter of debate. The selective testicular expression of the classic nuclear estrogen receptors (ERα and ERβ) argues in favor of ER-specific functions in the spermatogenic event. Recently, the existence of a G protein-coupled estrogen receptor (GPR30) mediating non-genomic effects of estrogens has also been described. However, little is known about the specific testicular expression pattern of GPR30, as well as on its participation in the control of male reproductive function. Herein, by means of immunohistochemical and molecular biology techniques (RT-PCR and Western blot), we aimed to present the first exhaustive evaluation of GPR30 expression in non-neoplastic human testicular cells. Indeed, we were able to demonstrate that GPR30 was expressed in human testicular tissue and that the staining pattern was consistent with its cytoplasmic localization. Additionally, by using cultured human Sertoli cells (SCs) and isolated haploid and diploid germ cells fractions, we confirmed that GPR30 is expressed in SCs and diploid germ cells but not in haploid germ cells. This specific expression pattern suggests a role for GPR30 in spermatogenesis.

Estrogens down-regulate the stem cell factor (SCF)/c-KIT system in prostate cells: Evidence of antiproliferative and proapoptotic effects.

The development of prostate cancer (PCa) is intimately associated with the hormonal environment, and the sex steroids estrogens have been implicated in prostate malignancy. However, if some studies identified estrogens as causative agents of PCa, others indicated that these steroids have a protective role counteracting prostate overgrowth. The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF), have been associated with the control of cell proliferation/apoptosis and prostate carcinogenesis, and studies show that estrogens regulate their expression in different tissues, though, in the case of prostate this remains unknown. The present study aims to evaluate the role of 17β-estradiol (E2) in regulating the expression of SCF/c-KIT in human prostate cell lines and rat prostate, and to investigate the consequent effects on prostate cell proliferation and apoptosis. qPCR, Western Blot, and immuno(cito)histochemistry analysis showed that E2-treatment decreased the expression of SCF and c-KIT both in human prostate cells and rat prostate. Furthermore, the diminished expression of SCF/c-KIT was underpinned by the diminished prostate weight and reduced proliferation index. On the other hand, the results of TUNEL labelling, the increased activity of caspase-3, and the augmented expression of caspase-8 and Fas system in the prostate of E2-treated animals indicated augmented apoptosis in response to E2. The obtained results demonstrated that E2 down-regulated the expression of SCF/c-KIT system in prostate cells, which was associated with antiproliferative and proapoptotic effects. Moreover, these findings support the protective role of estrogens in PCa and open new perspectives on the application of estrogen-based therapies.

Regucalcin is an androgen-target gene in the rat prostate modulating cell-cycle and apoptotic pathways.

Regucalcin (RGN) is a calcium (Ca2+)‐binding protein underexpressed in prostate adenocarcinoma comparatively to non‐neoplastic prostate or benign prostate hyperplasia cases. Moreover, RGN expression is negatively associated with the cellular differentiation of prostate adenocarcinoma, suggesting that loss of RGN may be associated with tumor onset and progression. However, the RGN actions over the control of prostate cell growth have not been investigated.

Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility?

The c-KIT, a tyrosine kinase receptor, and its ligand the stem cell factor (SCF) play an important role in the production of male and female gametes. The interaction of SCF with c-KIT is required for germ cell survival and growth, and abnormalities in the activity of the SCF/c-KIT system have been associated with human infertility. Recently, it was demonstrated that gonadotropic and sex steroid hormones, among others, regulate the expression of SCF and c-KIT in testicular and ovarian cells. Therefore, the hormonal (de)regulation of SCF/c-KIT system in the testis and ovary may be a cause underpinning infertility. In the present review, we will discuss the effects of hormones modulating the expression levels of SCF and c-KIT in the human gonads. In addition, the implications of hormonal regulation of SCF/c-KIT system for germ cell development and fertility will be highlighted.

The SCF/c-KIT system in the male: Survival strategies in fertility and cancer

Maintaining the delicate balance between cell survival and death is of the utmost importance for the proper development of germ cells and subsequent fertility. On the other hand, the fine regulation of tissue homeostasis by mechanisms that control cell fate is a factor that can prevent carcinogenesis. c‐KIT is a type III receptor tyrosine kinase activated by its ligand, stem cell factor (SCF). c‐KIT signaling plays a crucial role in cell fate decisions, specifically controlling cell proliferation, differentiation, survival, and apoptosis. Indeed, deregulating the SCF/c‐KIT system by attenuation or overactivation of its signaling strength is linked to male infertility and cancer, and rebalancing its activity via c‐KIT inhibitors has proven beneficial in treating human tumors that contain gain‐of‐function mutations or overexpress c‐KIT. This review addresses the roles of SCF and c‐KIT in the male reproductive tract, and discusses the potential application of c‐KIT target therapies in disorders of the reproductive system. Mol. Reprod. Dev. 81: 1064–1079, 2014.

Transgenic overexpression of regucalcin leads to suppression of thapsigargin- and actinomycin D-induced apoptosis in the testis by modulation of apoptotic pathways

Recent evidence suggested the involvement of calcium‐binding protein regucalcin (RGN) in testicular apoptosis. Herein, we investigated the role of RGN controlling apoptotic pathways in the testis by using a transgenic rat model overexpressing RGN (Tg‐RGN). Seminiferous tubules (SeT) from Tg‐RGN and their wild‐type (Wt) counterparts were cultured ex vivo in presence or absence of apoptosis inducers thapsigargin (Thap, 10−7 and 10−6 m) and actinomycin D (Act D, 0.5 and 1 μg/mL). Expression levels of key regulators of apoptosis in SeT of Tg‐RGN and Wt animals were determined by quantitative real‐time PCR and Western blot analysis. Measurement of caspase‐3 enzymatic activity was included as an end point of apoptosis. Tg‐RGN SeT treated with 10−6 m of Thap or 1 μg/mL of Act D showed a diminished enzymatic activity and gene transcription of caspase‐3, along with increased mRNA and protein expression of antiapoptotic Bcl‐2. Bcl‐2/Bax (antiapoptotic/proapoptotic) protein ratio was also enhanced in these SeT. Although caspase‐9 mRNA was increased in the SeT of Tg‐RGN treated with Thap, no differences were observed at protein level, and no differences were also found on protein levels of apoptosis‐inducing factor. mRNA expression of proapoptotic p53 and p21 was strongly decreased in Tg‐RGN SeT treated with Thap (10−6 m) or Act D (1 μg/mL). These findings demonstrated that RGN suppresses Thap‐ and Act D‐induced apoptosis in SeT by modulating the expression and activity of key apoptotic and antiapoptotic factors. Moreover, results indicate that RGN overexpression protects germ cell from apoptosis induced by noxious stimuli, which could be a relevant mechanism for fertility preservation in situations of oncological treatments.

Regucalcin counteracts tert-butyl hydroperoxide and cadmium-induced oxidative stress in rat testis.

Regucalcin (RGN) is a calcium (Ca2+)‐binding protein with multiple physiological roles and has also been linked to the suppression of oxidative stress. It is widely known that oxidative stress adversely affects spermatogenesis, disrupting the development of germ cells, and interfering with sperm function. The present study aims to analyze the role of RGN modulating testicular oxidative stress. To address this issue, seminiferous tubules (SeT) from transgenic rats overexpressing RGN (Tg‐RGN) and wild‐type (WT) were cultured ex vivo for 24 h in the presence/absence of pro‐oxidant stimuli, tert‐butyl hydroperoxide (TBHP, 250 and 500 μM) and cadmium chloride (Cd, 10 and 20 μM). Noteworthy, SeT from Tg‐RGN animals displayed a significantly higher antioxidant capacity and diminished levels of thiobarbituric acid reactive substances relatively to their WT counterparts, both in control and experimental conditions. Regarding the antioxidant defense systems, a significant increase in the activity of glutathione‐S‐transferase was found in the SeT of Tg‐RGN whereas no differences were observed in superoxide dismutase activity throughout experimental conditions. The activity of apoptosis executioner caspase‐3 was significantly increased in the SeT of WT rats treated with 250 μM of TBHP or 10 μM of Cd, an effect not seen in Tg‐RGN animals. These results showed that the SeT of Tg‐RGN animals displayed lower levels of oxidative stress and increased antioxidant defenses, exhibiting protection against oxidative damage and apoptosis. Moreover, the present findings support the antioxidant role of RGN in spermatogenesis, which may be an important issue of further research in the context of male infertility. Copyright