Androgen receptor signaling regulates follicular growth and steroidogenesis in interaction with gonadotropins in the ovary during mini-puberty in mice

In females, androgens contribute to ovarian diseases such as polycystic ovarian syndrome (PCOS), but their action is also crucial for ovarian physiology, i.e., follicular growth and estradiol (E2) synthesis during reproductive life, in interaction with the gonadotropins LH and FSH. However, it is unclear whether androgens already play a role in the ovary at mini-puberty, a phase of postnatal development with active follicular growth and high E2 levels. Therefore, we analyzed the potential actions of androgens on the ovary and their possible interaction with gonadotropins during this period in mice. We used molecular-based studies and pharmacological approaches in vivo and on cultured ovaries. We found that mini-pubertal ovaries produce significant amounts of testosterone and display androgen receptor (AR) expression in growing follicles, both under the control of LH. By blocking AR signaling either in vivo or in ovarian cultures, we found that this pathway may participate in the regulation of prepubertal E2 synthesis and follicular growth, possibly by regulating the expression of a number of key intra-ovarian regulators, including FSH receptor (Fshr), the aromatase enzyme converting androgens into estrogens (Cyp19a1) and the cell cycle inhibitor p27KIP1 (Cdkn1b). We further showed that AR may stimulate FSH-mediated regulation of Cyp19a1 through its action on Fshr mRNA abundance. Overall, this work supports the idea that AR signaling is already activated in mini-pubertal ovaries to regulate E2 synthesis and follicular growth, at the interplay with LH and FSH signaling. Its early action may, thus, contribute to the implementation of early ovarian function with possible impacts on reproductive function

Deciphering the Roles & Regulation of Estradiol Signaling during Female Mini-Puberty: Insights from Mouse Models

Mini-puberty of infancy is a short developmental phase occurring in humans and other mammals after birth. In females, it corresponds to transient and robust activation of the hypothalamo-pituitary-ovarian (HPO) axis revealed by high levels of gonadotropin hormones, follicular growth, and increased estradiol production by the ovary. The roles of estradiol signaling during this intriguing developmental phase are not yet well known, but accumulating data support the idea that it aids in the implementation of reproductive function. This review aims to provide in-depth information on HPO activity during this particular developmental phase in several mammal species, including humans, and to propose emerging hypotheses on the putative effect of estradiol signaling on the development and function of organs involved in female reproduction

GnRH regulates the expression of its receptor accessory protein SET in pituitary gonadotropes.

Reproductive function is under the control of the neurohormone GnRH, which activates a G-protein-coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. GnRHR activates a complex signaling network to regulate synthesis and secretion of the two gonadotropin hormones, luteinizing hormone and follicle-stimulating hormone, both regulating gametogenesis and steroidogenesis in gonads. Recently, in an attempt to identify the mechanisms underlying GnRHR signaling plasticity, we identified the first interacting partner of GnRHR, the proto-oncogene SET. We showed that SET binds to intracellular domains of GnRHR to enhance its coupling to cAMP pathway in αT3-1 gonadotrope cells. Here, we demonstrate that SET protein is rapidly regulated by GnRH, which increases SET phosphorylation state and decreases dose-dependently SET protein level. Our results highlight a post-translational regulation of SET protein involving the proteasome pathway. We determined that SET phosphorylation upon GnRH stimulation is mediated by PKC and that PKC mediates GnRH-induced SET down-regulation. Phosphorylation on serine 9 targets SET for degradation into the proteasome. Furthermore, a non-phosphorylatable SET mutant on serine 9 is resistant to GnRH-induced down-regulation. Altogether, these data suggest that GnRH-induced SET phosphorylation on serine 9 mediates SET protein down-regulation through the proteasome pathway. Noteworthy, SET down-regulation was also observed in response to pulsatile GnRH stimulation in LβT2 gonadotrope cells as well as in vivo in prepubertal female mice supporting its physiological relevance. In conclusion, this study highlights a regulation of SET protein by the neurohormone GnRH and identifies some of the mechanisms involved

Production d’œstradiol par l’ovaire lors de la mini-puberté

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GnRHa down-regulates SET protein expression in αT3-1 gonadotrope.

<p>(a) <b>α</b>T3-1 gonadotrope cells were incubated during 2 hours with increasing GnRHa concentrations (10⁻⁹ to 10⁻⁶ M) and SET protein level was determined by Western blotting. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals and are expressed as the percentage of SET protein level in absence of GnRHa. Results are expressed as mean ± SEM from 3 to 4 independent experiments. Data were analyzed by One-way ANOVA followed by Dunnett’s test, *: p<0.01 and ***: p<0.001, compared to no GnRHa. (b) <b>α</b>T3-1 gonadotrope cells were pre-treated or not (control) with the proteasome inhibitors MG132 (MG132, 2 hours, 3 μM) or the clasto-lactacystin–Lactone (Lactacystin, 2 hours, 10 μM) before stimulation with GnRHa (100 nM) for the indicated times (0.5 and 2 hours). SET protein level was determined by Western blotting and normalized by vinculin signals. SET protein level in GnRHa stimulated cells was expressed as the percentage of respective basal SET expression levels at each time point in the presence or absence of MG132 or clasto-lactacystin–Lactone. Results are expressed as mean ± SEM from 3 to 5 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test. Distinct letters indicate significant differences between treatments (p<0.05). (c) αT3-1 gonadotrope cells were incubated with GnRHa (100 nM) for increasing periods of time and SET mRNA levels were determined by real-time RT-PCR and normalized to the mRNA levels of <i>Hprt</i>. Results are expressed as the percentage of SET mRNA level in unstimulated cells (0 h). Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by One-way ANOVA, no significant.</p

GnRH induces SET protein down-regulation in infantile mice pituitary.

<p>(a) <i>Left panel</i>—SET protein abundance was analyzed in infantile pituitaries (7–17 dpn) by Western blotting, with GAPDH used as a loading control. Bar graphs show the mean ± SEM of SET levels normalized to those of GAPDH (n = 3 to 8 pituitaries/age). A representative immunoblot of SET expression is shown. Data were analyzed by one-way ANOVA, followed by Tukey’s test with distinct letters indicating significant differences between ages (p<0.05). <i>Right panel</i>—The relative pituitary abundance of SET mRNA in infantile (7–17 dpn) females was determined by real-time RT-PCR and normalized to the mRNA levels of <i>Hprt</i> (n = 4 to 8 pituitaries/age). Bar graphs show the mean ± SEM of relative quantification. Data were analyzed by one-way ANOVA, followed by Tukey’s test with distinct letters indicating significant differences between ages (p<0.05). (b) SET protein abundance was analyzed in infantile pituitaries after treatment with the GnRH antagonist Ganirelix or saline vehicle at 12 and 13 dpn. A representative blot is shown. Bar graphs show the mean ± SEM of SET levels normalized to those of GAPDH (n = 3 to 5 pituitaries/condition). Data were analyzed by t test for unpaired groups, **: p<0.01, compared to 14 dpn saline.</p

Pulsatile native GnRH induces SET protein down-regulation in LβT2 cells.

<p>(a) LβT2 gonadotrope cells were treated with GnRHa (100 nM) for 2 hours and SET protein (<i>left panel</i>) and SET mRNA (<i>right panel</i>) levels were determined by Western blotting and by real-time RT-PCR, respectively. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals (SET protein) or by mRNA levels of <i>Hprt</i> (SET mRNA) and are expressed as percentage of the amount of SET in unstimulated cells. Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by t test for unpaired groups *: p<0.05, compared to no GnRHa. (b) LβT2 gonadotrope cells were cultured in perifusion chambers as described in “Materials and methods” and challenged or not with pulsatile GnRH (10 nM) at high and low frequencies (one pulse every 0.5 hour or one pulse every 2 hours, respectively). At the end of the incubation, proteins and mRNA were extracted and SET protein and <i>Set</i>, <i>Lhb and Fshb</i> transcripts levels were determined by Western blotting and by real-time RT-PCR, respectively. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals (SET protein) or by mRNA levels of <i>Hprt</i> (<i>Set</i>, <i>Lhb and Fshb</i> mRNA) and expressed as percentage of the amount of SET in unstimulated cells. Results are expressed as mean ± SEM from 3 independent experiments. One-way ANOVA followed by Tukey’s test, ***: p<0.001, compared to no GnRH.</p

GnRHa increases SET phosphorylation–Involvement of PKC.

<p>(a) <i>Left panel–</i>αT3-1 gonadotrope cells were plated in 6-well plates and labelled with [³²P]-orthophosphate (50 μCi/ml) as described in “Materials and methods”. Cells were stimulated (+) or not (-) with GnRHa (100 nM) for 0.5 hour followed by SET immunoprecipitation (IP SET) as described in “Materials and methods”. Immunoprecipitated SET was resolved on 10% SDS-PAGE, electrotransferred and probed with anti-SET antibody (IB SET). Phosphorylated SET (P-SET) was visualized by autoradiography using a Fuji Phosphoimager FLA7000. <i>Right panel–</i>αT3-1 gonadotrope cells were incubated (+) or not (-) with GnRHa (100 nM, 0.5 hour) and phosphoproteins were purified by chromatography as described in “Materials and methods”. Phosphorylated SET and ERK1/2 were detected by Western blotting using anti-SET and anti-total ERK1/2 antibodies, respectively. Results are representative of 4 independent experiments. (b) αT3-1 gonadotrope cells were pre-incubated or not with the PKC inhibitor GF109203X (2 μM, 1 hour) prior to GnRHa stimulation (100 nM, 0.5 hour). Phosphoproteins were purified by chromatography as described in “Materials and methods” and phosphorylated SET was detected by Western blotting using anti-SET antibody. Results are expressed as the percentage of SET protein level in absence of treatment. Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test, with distinct letters indicating significant differences between treatments (p<0.05). (c) αT3-1 gonadotrope cells were pre-incubated or not with the PKC inhibitor GF109203X (2 μM, 1 hour) prior to GnRHa stimulation (100 nM, 0.5 hour) and SET protein expression was detected by Western blotting using anti-SET antibody. Results are normalized by vinculin signals and expressed as the percentage of SET protein level in absence of treatment. Results are expressed as mean ± SEM from 3 to 4 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test, with distinct letters indicating significant differences between treatments (p<0.05).</p

Impact of serine 9 phosphorylation on SET expression level.

<p><b>(</b>a) Gonadotrope cells were transfected either with A9 or E9 His-SET mutants as described in “Materials and methods”. Thirty hours after transfection, cells were pre-incubated (+) or not (-) with MG132 (10 μM, 18 hours). The relative expression levels of both mutants were assessed by Western blotting and normalized by vinculin signals. His-SET proteins migrate on SDS-PAGE at a higher molecular weight than endogenous SET allowing their specific quantification. Results are expressed as the percentage of SET A9 protein level in absence of MG132. Results are expressed as mean ± SEM from 4 to 5 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test, with distinct letters indicating significant differences between treatments (p<0.05). (b) Gonadotrope cells were transfected either with the wild type His-SET or the His-SET A9 mutant and were treated or not with GnRHa (100 nM) for 0.5 or 2 hours. SET expression level was assessed by Western blotting and normalized by vinculin signals. Results are expressed as the percentage of SET WT or SET A9 protein levels in absence of GnRHa. Results are expressed as mean ± SEM from 4 independent experiments. Data were analyzed by One-way ANOVA followed by Dunnett’s test*: p<0.05, compared to no GnRHa.</p