Pituitary Of Male Mice Research Articles

FRI289 It Takes A Village: Musashi And Its Associated Proteins In The Adenohypophysis

Abstract Disclosure: K.L. Bronson: None. L. Hardy: None. A. Lagasse: None. A.M. MacNicol: None. Musashi is an RNA-binding protein that regulates stem and progenitor cell fates. Interestingly, we have reported expression of Musashi pituitary stem cells as expected but also in the differentiated anterior pituitary hormone-producing cell lineages as well as. The adult anterior pituitary relies on the functional cellular plasticity of its hormone-producing cell populations to maintain endocrine homeostasis when physiological demands change. Both Musashi isoforms (Musashi1 and Musashi2) are highly conserved across species and have two N-terminal RNA-recognition motifs (RRMs) and a disordered C-terminus containing two regulatory phosphorylation sites. Musashi can selectively activate or repress the translation of mRNAs depending on the transcript and cellular context. While Musashi phosphorylation is required for translational activation, Musashi does not possess inherent enzymatic activity; therefore, co-associated proteins must mediate Musashi-directed translational control. Musashi immunoprecipitation studies initially identified 50 co-associated proteins in Xenopus oocytes, but only a subset of these were directly implicated in Musashi-dependent translational activation (e.g. Elavl1 and Pabp4). The identity of Musashi-associated proteins in the anterior pituitary and their requirement for Musashi function are not known. To this end, we have immunoprecipitated Musashi from whole male mouse pituitaries for mass spectrometry analysis. 206 proteins significantly (adj p-value < 0.05) associated with Musashi1 and Musashi2, and seven of these overlapped with the Xenopus dataset: Elavl1, Pabp4, Hnrnpc, Mov10, Pura, Rps27, and Upf1. Three proteins from the mouse mass spectrometry dataset were selected for further analyses. Sam68 is a multi-functional protein that sensitizes ovaries to FSH and LH, as well as mediates the activation of leptin and insulin signaling in a variety of tissues. Ago2 is a component of the RNA-induced silencing complex that is necessary for miRNA-directed translational control and is known to associate with Musashi1. The AU-rich element binding protein Elavl1 (HuR) contributes to Musashi-dependent activation of target mRNAs in Xenopus and also stabilizes Gnrhr mRNA in LβT2 cells and sensitizes them to GnRH signaling. We will present our ongoing studies on the requirement for these protein interactions for Musashi-directed mRNA translational regulation. Presentation: Friday, June 16, 2023

Transcription factor GATA2 may potentiate follicle-stimulating hormone production in mice via induction of the BMP antagonist gremlin in gonadotrope cells

Mammalian reproduction depends on the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone, which are secreted by pituitary gonadotrope cells. The zinc-finger transcription factor GATA2 was previously implicated in FSH production in male mice; however, its mechanisms of action and role in females were not determined. To directly address GATA2 function in gonadotropes, we generated and analyzed gonadotrope-specific Gata2 KO mice using the Cre-lox system. We found that while conditional KO (cKO) males exhibited ∼50% reductions in serum FSH levels and pituitary FSHβ subunit (Fshb) expression relative to controls, FSH production was apparently normal in cKO females. In addition, RNA-seq analysis of purified gonadotropes from control and cKO males revealed a profound decrease in expression of gremlin (Grem1), a bone morphogenetic protein (BMP) antagonist. We show Grem1 was expressed in gonadotropes, but not other cell lineages, in the adult male mouse pituitary. Furthermore, Gata2, Grem1, and Fshb mRNA levels were significantly higher in the pituitaries of WT males relative to females but decreased in males treated with estradiol and increased following ovariectomy in control but not cKO females. Finally, we found that recombinant gremlin stimulated Fshb expression in pituitary cultures from WT mice. Collectively, the data suggest that GATA2 promotes Grem1 expression in gonadotropes and that the gremlin protein potentiates FSH production. The mechanisms of gremlin action have not yet been established but may involve attenuation of BMP binding to activin type II receptors in gonadotropes, facilitating induction of Fshb transcription by activins or related ligands.

Transcriptional and Genomic Regulation of Pituitary Function by Thyroid Hormone Receptor Beta

Background: The pituitary is a key target for thyroid hormone but underlying transcriptional mechanisms are poorly understood. Thyroid hormone modifies expression of hormones, including growth hormone (GH) and thyroid-stimulating hormone (TSH, thyrotropin). Wider transcriptome responses are undefined. Thyroid hormone receptor beta (TRb) encoded by THRB are expressed in the anterior pituitary and THRB mutations cause human resistance to thyroid hormone. Method: To investigate genomic genomic regulation by TRb, we derived Thrb-HAB knockin mice that express TRb protein with a tag that is biotinylated in vivo in presence of an R26-BirA allele. Specific, sensitive streptavidin pull-down facilitated Chromatin-Affinity-Purification-sequencing (ChAPseq) to identify genomic TRβ binding sites in pituitary of male mice. Hypo- and hyperthyroidism were produced using methimazole (MMI) in drinking water for 4 weeks with/without added thyroid hormone (T3) for the 4th week. Pituitaries from wild type and Thrb-KO mice were also isolated for RNA-sequencing (RNA-seq). Selected expression changes were confirmed by quantitative PCR. Epigenetic changes were determined by ChIPseq for histone acetylation and methylation and open chromatin analysis (ATAC-seq). Results: Transcriptome analysis revealed genes with statistically different expression induced by T3, including known response genes such as Tshb, Hr and Gh. Responses were impaired in Thrb-KO mice. T3 induced recruitment of TRb binding, chromatin opening and specific histone acetylation marks. Conclusion: Most T3 response genes in pituitary depend to some extent upon TRb. T3-dependent chromatin modifications indicate properties of TRb-dependent enhancer regions and a critical role for TRb in transcriptional regulation of pituitary function.

Histone deacetylase inhibitors suppress transdifferentiation of gonadotrophs to prolactin cells and proliferation of prolactin cells induced by diethylstilbestrol in male mouse pituitary.

Diethylstilbestrol (DES), an estrogen agonist, increases prolactin (PRL) cells through transdifferentiation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells to PRL cells as well as proliferation of PRL cells in adult male mouse pituitary. Since hyperacetylation of histone H3 is implicated in the regulation of activation of various genes, we examined the effect of DES on the state of histone H3 acetylation. DES significantly reduced the immunohistochemical signal for acetylated histone H3 at lysine 9 (H3K9ac) in PRL, LH and FSH cells, but not for H3K18ac or H3K23ac. DES-treated mice were injected intraperitoneally with HDAC inhibitors (HDACi), sodium phenylbutyrate (NaPB) or valproic acid (VPA), to mimic the acetylation level of histone H3. As expected, HDACi treatment restored the level of H3K9ac expression in these cells, and also inhibited DES-induced increase in PRL cells. Furthermore, NaPB and VPA also abrogated the effects of DES on the population density of both LH and FSH cells. Similarly, the numbers of proliferating and apoptotic cells in the pituitary in NaPB- or VPA-treated mice were comparable to those of the control mice. Considered together, these results indicated that the acetylation level of histone H3 plays an important role in DES-induced transdifferentiation of LH to PRL cells as well as proliferation of PRL cells.

Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland.

ABSTRACTNesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17β-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulation mechanism in the pituitary of male mouse. Therefore, we examined whether nesfatin-1/NUCB2 is expressed in the male mouse pituitary and if its expression is regulated by testosterone. As a result of PCR and western blotting, we found that a large amount of nesfatin-1/NUCB2 was expressed in the pituitary and hypothalamus. The NUCB2 mRNA expression level in the pituitary was decreased after castration, but not in the hypothalamus. In addition, its mRNA expression level in the pituitary was increased after testosterone treatment in the castrated mice, whereas, the expression level in the hypothalamus was significantly decreased after the treatment with testosterone. The in vitro experiment to elucidate the direct effect of testosterone on NUCB2 mRNA expression showed that NUCB2 mRNA expression was significantly decreased with testosterone in cultured hypothalamus tissue, but increased with testosterone in cultured pituitary gland. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the male mouse pituitary and was regulated by testosterone. This data suggests that reproductive-endocrine regulation through hypothalamus-pituitary-testis axis may contribute to NUCB2 mRNA expression in the mouse hypothalamus and pituitary gland.

Pituitary androgen receptor signalling regulates prolactin but not gonadotrophins in the male mouse.

Production of the androgen testosterone is controlled by a negative feedback loop within the hypothalamic-pituitary-gonadal (HPG) axis. Stimulation of testicular Leydig cells by pituitary luteinising hormone (LH) is under the control of hypothalamic gonadotrophin releasing hormone (GnRH), while suppression of LH secretion by the pituitary is controlled by circulating testosterone. Exactly how androgens exert their feedback control of gonadotrophin secretion (and whether this is at the level of the pituitary), as well as the role of AR in other pituitary cell types remains unclear. To investigate these questions, we exploited a transgenic mouse line (Foxg1Cre/+; ARfl/y) which lacks androgen receptor in the pituitary gland. Both circulating testosterone and gonadotrophins are unchanged in adulthood, demonstrating that AR signalling is dispensable in the male mouse pituitary for testosterone-dependent regulation of LH secretion. In contrast, Foxg1Cre/+; ARfl/y males have a significant increase in circulating prolactin, suggesting that, rather than controlling gonadotrophins, AR-signalling in the pituitary acts to suppress aberrant prolactin production in males.

Immunohistochemistry of CD38 in Different Cell Types in the Hypothalamus and Pituitary of Male Mice

Immunohistochemistry of CD38 in Different Cell Types in the Hypothalamus and Pituitary of Male Mice

Role of the Non-Canonical Notch Ligand Delta-Like Protein 1 in Hormone-Producing Cells of the Adult Male Mouse Pituitary

To better understand the role of the non-canonical Notch ligand delta-like protein 1 (DLK1), in hormone-producing cells, we studied the cell distribution and subcellular localisation of DLK1 in the pituitary of male adult 129/SvJ mice, and analysed the variations in the hormone-producing cells associated with the lack of this gene in Dlk1 knockout mice. The results obtained showed the presence of DLK1-immunoreactive (ir) cells in all hormone-producing cells of the anterior pituitary. Immunoelectron microscopy showed DLK1-ir in the rough endoplasmic reticulum and inside secretory vesicles, suggesting that DLK1 is released together with pituitary hormones. Moreover, we found that prolactin (PRL)-DLK1-ir cells are in intimate contact with follicle-stimulating hormone (FSH)-ir-DLK1-negative cells. In Dlk1 knockout mice, we detected a significantly lower number of gowth hormone (GH)-ir cells, a reduction in the FSH and PRL immunostaining intensity, and a significant decrease in FSH mRNA expression compared to wild-type mice. An increase in pituitary GH mRNA expression and serum leptin levels was also found. These findings provide evidence supporting several regulatory functions of DLK1 in the pituitary gland.

Diethylstilbestrol increases the density of prolactin cells in male mouse pituitary by inducing proliferation of prolactin cells and transdifferentiation of gonadotropic cells

Diethylstilbestrol (DES) has been implicated in mammalian abnormalities. We examined the effects of DES on follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) cells in the pituitaries of male mice treated with various doses of DES for 20 days. DES reduced the density of FSH and LH cells in a dose-dependent manner, but increased that of PRL cells. When the expression of estrogen receptor (ER) alpha and beta was assessed, an induction of ERbeta by DES was found predominantly in PRL cells. However, since these effects were abolished in ERalpha knockout mice, DES appears to act primarily through ERalpha. When the expression of Ki-67 and Pit-1 in PRL cells was examined at various time-points after DES treatment, some PRL cells became Ki-67 positive at 10-15 days, and Pit-1-positive cells were increased at 5-15 days. Furthermore, some FSH and LH cells became Pit-1 positive, and co-localized with PRL at 5-10 days. Our results indicate that DES increases PRL cells by inducing proliferation of PRL cells and transdifferentiation of FSH/LH cells to PRL cells.

Pituitary gonadotropin-releasing hormone receptor regulation in the hypogonadotrophic hypogonadal (hpg) mouse.

Recent evidence indicates that endogenous GnRH is required for maintenance of its own pituitary receptors (GnRH-R). We have measured GnRH-R in pituitaries of hypogonadotrophic hypogonadal (hpg) mice, in whom hypothalamic GnRH is deficient or absent. The GnRH-R concentration in hpg male mouse pituitaries was 10.6 +/- 1 fmol/pituitary vs. 30.9 +/- 1 fmol/pituitary in normal male littermate pituitaries. Similarly, GnRH-R in female hpg mice (15.2 +/- 1.7 fmol/pituitary) were 30% those of normal random cycling females (51.4 +/- 3.5 fmol/pituitary). There was no difference in receptor affinity (Ka = 1.5-3 C 10(9) M-1) of hpg mouse pituitaries. The pituitary LH content in hpg male and female mice was very similar (range 3.4-4.8 micrograms/pituitary) representing 5% and 19% of normal male (95 +/- 7.2 micrograms/pituitary) and female (18.1 +/- 1.5 micrograms/pituitary) values, respectively. The administration of 50 ng GnRH sc 10 times daily to male hpg mice, increased GnRH-R to 80% of normal values within 3 days. Serum FSH and pituitary FSH content rose to normal male values after 7 days of GnRH injections. However, serum LH remained undetectable and pituitary LH reached only 20% of normal male levels, even after 15 days of GnRH administration. Treatment of hpg male mice with 60 ng GnRH either once daily for 6 days, or 12 times daily for 5 days, increased GnRH-R to 50% of normal male values. Twelve daily injections of GnRH elevated serum FSH to above the normal male range, whereas daily GnRH only doubled untreated hpg levels. Pituitary FSH was stimulated to 50% of normal with 12 daily injections, whereas once daily administration elevated pituitary FSH to 30% of normal values. Both pulsatile regimes depleted pituitary LH. These data demonstrate that: 1) despite absence of bioactive GnRH, GnRH-R values are only reduced to 30% of normal in hpg mouse pituitaries, suggesting that little, if any, endogenous GnRH is required for expression of GnRH receptors. 2) Pituitary GnRH-R number rapidly increase when GnRH is administered to hpg male mice indicating that, as in the rat, GnRH positively regulates its own receptor concentration. 3) The pituitary FSH and LH responses to GnRH treatment in hpg mice depends to a different extent on the frequency and duration of GnRH administration. 4) The hpg mouse provides an ideal animal model for investigating the interaction of defined regiments of exogenous GnRH and gonadal steroids on pituitary GnRH receptor and gonadotroph function.

Disappearance of Inhibin‐like Activity in Bull Seminal Plasma Following Castration

Ejaculates from normal and castrated bulls were evaluated for the presence of inhibin‐like activity. Inhibin activity found in the seminal plasma of normal bulls became undetectable after castration. Crude protein concentrates prepared from the ejaculates of castrated bulls failed to produce inhibition of FSH secretion as measured by in vivo and in vitro tests: a) hCG‐induced uterine weight test in mice and b) LH‐RH‐induced FSH secretion in 34‐day‐old male mouse pituitary. The concentrates also lacked the ability to inhibit the binding of iodine‐125 (125I)‐labeled inhibin fraction from normal bull seminal plasma to ovine pituitary membranes. Chromatographic and electrophoretic analyses of the ejaculate from castrated bulls revealed the absence of the band corresponding to inhibin of normal bull seminal plasma.

Regional and subcellular localization of [ 3H]dihydrotestosterone in brain and pituitary of male mice

Regional and subcellular localization of [ 3H]dihydrotestosterone in brain and pituitary of male mice

An Electron Microscopical Study on Effects of Synthesized Estrogen on the Anterior Pituitary of Male Mice

An Electron Microscopical Study on Effects of Synthesized Estrogen on the Anterior Pituitary of Male Mice

Reactions of thyroid glands to juxtathyroidal implants of thyrotropic agents.

Detection and measurement of small amounts of thyrotropin, such as might be included in a single pituitary gland, is extremely difficult, if not impossible, by ordinary techniques. The following experiments, in which hypophyseal and control material were implanted in direct contact with the thyroid glands of otherwise intact test animals, were designed for this purpose. In some tests the thyrotropic materials were permitted to remain in contact with the thyroid for as long as nine months to determine the effect of prolonged localized inequality in thyroidarfunction. In other tests the treated animals weresacrificed after 100 hours of contact with the implant to conform to a commonly used interval of observation in bioassay of hypophyseal materials. In the 100-hour tests 23 200-gram guinea pigs of both sexes and 35 threemonth- old male CFWstrain mice were used. Of the guinea pigs, six were aseptically implanted with adult male mouse pituitaries, and six with mouse muscle; five were untreated.