Ovine Pituitary Cells Research Articles

Use of rapid immunochromatographic tests and PCR for the detection of vaccine viruses (CPV, CDV CAV) in faeces and conjunctival sac swabs of vaccinated dogs

The pathologically changed pattern of gonadotropin secretion, responsible for ovulation disorders in fatty ewes, may result from the prolonged increase in leptin concentration as well as from diminution of leptin receptors expression in anterior pituitary cells. Leptin acting peripherally reduces the secretion of insulin – the potent inhibitor of lipolysis. Consequently, an increment in plasma fatty acids level is observed. It was also found that in ewe lambs born to obese sheep carrying twins or triplets, a high plasma level of saturated fatty acids (SFA) is in positive correlation with the delay in puberty. However, the relationship between SFA and gonadotropin secretion from the ovine pituitary cells in pubescent ewe lambs is not clear. Therefore, the aim of the study was to establish the effect of SFA on GnRH-induced secretion of FSH and LH. Pituitary glands were isolated from 7 month old ewe lambs. Pituitary cells were cultured in McCoy 5A medium without GnRH and SFA (negative control), with GnRH only (positive control), with GnRH and 10–9-10–3 M/l of the butyric (C4:0), caprylic (C8:0), lauric (C12:0), palmitic (C16:0) or stearic acid (C18:0). After 2 or 6 h of exposure to SFA followed by 2, 6, 12, 18, 24 or 30 h incubation, the media for LH and FSH analysis were collected. It was found that all used SFA reduce GnRH-induced LH and FSH secretion from pituitary cells in vitro. The most significant (P ≤ 0.05) suppressive effect was observed after 6 h exposure of cells to 10–3 M/l of caprylic acid, 10–4 M/l of palmitic acid and 10–4 M/l of stearic acid compared to the positive control.

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion

Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse array of tissues, including the bone, uterus, mammary gland, and components of the neural and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E₂) and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Incubation of cells for 12 h with E₂, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E₂- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E₂, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER.

Does a Nonclassical Signaling Mechanism Underlie an Increase of Estradiol-Mediated Gonadotropin-Releasing Hormone Receptor Binding in Ovine Pituitary Cells?1

Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

The Non-Classical Actions of Estradiol in Ovine Pituitary Cells Cannot be Mimicked in the Immortalized Mouse Gonadotroph Cell Line, AlphaT3-1 Cells.

The Non-Classical Actions of Estradiol in Ovine Pituitary Cells Cannot be Mimicked in the Immortalized Mouse Gonadotroph Cell Line, AlphaT3-1 Cells.

Effect of RF-Amide-Related Peptide-3 on Luteinizing Hormone and Follicle-Stimulating Hormone Synthesis and Secretion in Ovine Pituitary Gonadotropes

GnRH provides the primary stimulus for the reproductive axis, but original work also revealed the existence of a gonadotropin-inhibitory hormone (GnIH) in birds. In mammals, GnIH properties are displayed by a hypothalamic dodecapeptide, which is a member of the RF-amide family, namely RF-amide-related peptide (RFRP)-3. This peptide inhibits GnRH-stimulated gonadotropin secretion from ovine pituitary cells in culture, but it is not known whether there are effects on gonadotropin synthesis. The aim of the present study was to determine the effects of RFRP-3 on the expression of genes for beta-subunits of the gonadotropins in ovine pituitary cells from gonadectomized ewes and rams. Cells in primary culture were given GnRH or vehicle pulses every 8 h for 24 h with and without RFRP-3 treatment. GnRH stimulated LH and FSH secretion, which was reduced by RFRP-3. Quantitative real-time PCR revealed increased expression of LHbeta and FSHbeta subunit genes after GnRH treatment and a specific reduction in expression after RFRP-3 treatment. There was no effect on the expression of GH, proopiomelanocortin, or prolactin genes. Western blotting showed that GnRH stimulated phosphorylation of ERK (phospho-ERK-1/2), and this effect was abolished by RFRP-3. We conclude that RFRP-3 acts on the pituitary gonadotropes to inhibit synthesis of the gonadotropins, and this effect may be mediated by a reduction in the GnRH-stimulated second messenger phospho-ERK-1/2.

Low doses of estradiol partly inhibit release of GH in sheep without affecting basal levels

Estradiol increases basal growth hormone (GH) concentrations in sheep and cattle. This study sought to determine the effects of estradiol on GH-releasing hormone (GRH)-stimulated GH release in sheep. Growth hormone secretory characteristics, the GH response to GRH, and steady-state GH mRNA concentrations were determined in castrated male lambs treated with 2 different doses of estradiol 17-β for a 28-d experimental period. Although no differences between treatments in mean GH, basal GH, or GH pulse number were observed after 28 d of estradiol treatment, GH pulse amplitude was greater ( P < 0.05) in the 2.00-cm implant-treated animals than in the control and 0.75-cm implant group. The effect of estradiol treatment on GRH-stimulated GH release revealed differences between the control and estradiol-treated animals ( P < 0.05). The 15-min GH responses to 0.075 μg/kg hGRH in the control, 0.75-cm, and 2.00-cm implant groups, respectively, were 76 ± 10, 22.6 ± 2.1, and 43.6 ± 15.0 ng/mL. Growth hormone mRNA content was determined for pituitary glands from the different treatment groups, and no differences in steady-state GH mRNA levels were observed. There were no differences in the mean plasma concentrations of IGF-I, cortisol, T 3, or T 4 from weekly samples. Growth hormone release from cultured ovine pituitary cells from control sheep was not affected by estradiol after 72 h or in a subsequent 3-h incubation with estradiol combined with GRH. These data suggest that estradiol has differing actions on basal and GRH-stimulated GH concentrations in plasma, but the increase in pulse amplitude does not represent an increased pituitary sensitivity to GRH.

Estradiol-17β Inhibits Gonadotropin-Releasing Hormone-Induced Ca2+ in Gonadotropes to Regulate Negative Feedback on Luteinizing Hormone Release

In pituitary gonadotropes, estrogens have biphasic actions to cause an initial negative feedback followed by a positive feedback on LH secretion, but the mechanisms involved are not clearly understood. To investigate the feedback effects of estrogen, we used mixed ovine pituitary cell cultures (48-72 h), which were treated with 10(-9) M estradiol-17beta (E(2)) or vehicle followed by a pulse of 10(-9) M GnRH. Medium was collected for LH assay and cells extracted to determine activation of MAPK (phosphorylated ERK-1/2). E(2) treatment for 5 min reduced GnRH-induced LH release and caused phosphorylation of ERK-1/2. E(2) alone also caused phosphorylation of ERK-1/2, similar to the response evoked by GnRH alone. GnRH increased cytoplasmic intracellular free calcium concentration ([Ca(2+)](i)) and this was abolished by 2 min pretreatment with E(2) or E-bovine serum albumen conjugate. Blockade of Ca(2+) channels with nifedipine had no effect on the initial peak of GnRH-induced increase in [Ca(2+)](i) but reduced its duration by 27 +/- 6%. Depletion of intracellular Ca(2+) stores with thapsigargin prevented GnRH-induced increase in [Ca(2+)](i). Thapsigargin (10(-7) M) or nifedipine (10(-5) M) pretreatment (15 min) of cells lowered GnRH-induced LH secretion by 30 +/- 6 and 50% +/- 4%, respectively. We conclude that inhibition of the GnRH-induced increase in [Ca(2+)](i) in gonadotropes by E(2) is a likely mechanism for the negative feedback effect of E(2) on LH secretion involving a rapid nongenomic effect of E(2). Activation of the MAPK pathway by E(2) may be the mechanism for the time-delayed positive feedback effect on LH secretion at the level of the gonadotrope.

Disruption of Lipid Rafts Induces Gonadotropin Release in Ovine Pituitary and LbetaT2 Gonadotroph Cells1

In order to better understand the cellular mechanisms underlying LH and FSH secretion, we have addressed the contribution of lipid rafts to the secretion of gonadotropins. We used methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering agent, on an LbetaT2 murine gonadotroph cell line and on primary cultures of ovine pituitary cells. We found that in both systems, cholesterol depletion by MbetaCD induced a fast and substantial release of LH in the absence of natural stimulation by GnRH. In ovine pituitary cells, MbetaCD-mediated LH release was shown to be independent of protein synthesis. Twenty-four hours after MbetaCD treatment, there was no loss of cell viability and full recovery of LH secretory capabilities, as determined by GnRH or MbetaCD treatment. In addition, our data suggest the existence of a pool of LH that is not released by GnRH treatment but that is released by MbetaCD treatment. Finally, in ovine pituitary cells, MbetaCD treatment induced FSH secretion. Importantly, these in vitro data are supported by in vivo studies, because MbetaCD injected into the pituitary glands of anaesthetized sheep reproducibly induced a peak of LH release.

Kisspeptin Is Present in Ovine Hypophysial Portal Blood But Does Not Increase during the Preovulatory Luteinizing Hormone Surge: Evidence that Gonadotropes Are Not Direct Targets of Kisspeptin in Vivo

There is strong evidence that kisspeptin acts to regulate GnRH secretion, but whether there is also a component of action on the gonadotropes is not clear. Using quantitative RT-PCR, we found that G protein-coupled receptor-54 mRNA is expressed in ovine pituitary cell fractions enriched for gonadotropes as well as in somatotropes and lactotropes. To test whether kisspeptin acts directly on the pituitary gonadotropes, we first examined LH release from primary ovine pituitary cell cultures treated with kisspeptin. We found that kisspeptin treatment increased the concentration of LH in culture media by 80%, compared with control, but only in pituitary cultures from ewes during the follicular phase of the estrous cycle. After this, we determined whether kisspeptin acts on the pituitary gland in vivo. Using GnRH-replaced ovariectomized hypothalamo-pituitary-disconnected ewes, we were not able to achieve any effect of kisspeptin on LH under steady-state conditions or during the period of an estrogen-induced LH surge. Finally, we collected hypophysial portal blood samples from ovariectomized ewes and measured kisspeptin levels. Low but detectable amounts of kisspeptin were found in portal plasma, but levels were similar in ovariectomized ewes that were untreated or given estrogen to elicit an LH surge. Thus, although we observed an effect of kisspeptin on LH release in vitro in some situations, similar findings were not obtained in vivo. Moreover, the low concentrations of kisspeptin in hypophysial portal blood and the lack of any change during the period of an estrogen-induced GnRH/LH surge suggest that action on the pituitary gland is not of major consequence in terms of LH release.

Insight into the Neuroendocrine Site and Cellular Mechanism by which Cortisol Suppresses Pituitary Responsiveness to Gonadotropin-Releasing Hormone

Stress-like elevations in plasma glucocorticoids rapidly inhibit pulsatile LH secretion in ovariectomized sheep by reducing pituitary responsiveness to GnRH. This effect can be blocked by a nonspecific antagonist of the type II glucocorticoid receptor (GR) RU486. A series of experiments was conducted to strengthen the evidence for a mediatory role of the type II GR and to investigate the neuroendocrine site and cellular mechanism underlying this inhibitory effect of cortisol. First, we demonstrated that a specific agonist of the type II GR, dexamethasone, mimics the suppressive action of cortisol on pituitary responsiveness to GnRH pulses in ovariectomized ewes. This effect, which became evident within 30 min, documents mediation via the type II GR. We next determined that exposure of cultured ovine pituitary cells to cortisol reduced the LH response to pulse-like delivery of GnRH by 50% within 30 min, indicating a pituitary site of action. Finally, we tested the hypothesis that suppression of pituitary responsiveness to GnRH in ovariectomized ewes is due to reduced tissue concentrations of GnRH receptor. Although cortisol blunted the amplitude of GnRH-induced LH pulses within 1-2 h, the amount of GnRH receptor mRNA or protein was not affected over this time frame. Collectively, these observations provide evidence that cortisol acts via the type II GR within the pituitary gland to elicit a rapid decrease in responsiveness to GnRH, independent of changes in expression of the GnRH receptor.

Differential Modulation of Gonadotropin Secretion by Selective Estrogen Receptor 1 and Estrogen Receptor 2 Agonists in Ovariectomized Ewes1

The objectives of this study were to determine whether activation of estrogen receptor 1 (ESR1; also known as ERalpha), or estrogen receptor 2 (ESR2; also known as ERbeta), or both are required to: 1) acutely inhibit secretion of LH, 2) induce the preovulatory-like surge of LH, and 3) inhibit secretion of FSH in ovariectomized (OVX) ewes. OVX ewes (n = 6) were administered intramuscularly 25 micrograms estradiol (E2), 12 mg propylpyrazoletriol (PPT; a subtype-selective ESR1 agonist), 21 mg diaprylpropionitrile (DPN; a subtype-selective ESR2 agonist), or PPT + DPN. Like E2, administration of PPT, DPN, or combination of the two rapidly decreased (P < 0.05) secretion of LH. Each agonist induced a gradual, prolonged rise in secretion of LH after the initial inhibition, but neither agonist alone nor the combined agonists was able to induce a "normal" preovulatory-like surge of LH similar to that induced by E2. Compared with E2-treated ewes, the beginning of the increase in secretion of LH occurred earlier (P < 0.01) in DPN-treated ewes, later (P < 0.05) in PPT-treated ewes, and at a similar interval in ewes receiving the combined agonist treatment. Like E2, PPT decreased (P < 0.05) secretion of FSH, but the duration of suppression was much longer in PPT-treated ewes. DPN did not alter secretion of FSH in this study. Modulation of the number of GnRH receptors by PPT and DPN was examined in primary cultures of ovine pituitary cells. In our hands, both PPT and DPN increased the number of GnRH receptors, but the dose of DPN required to stimulate synthesis of GnRH receptors was 10 times higher than that of PPT. We conclude that in OVX ewes: 1) ESR1 and ESR2 mediate the negative feedback of E2 on secretion of LH at the level of the pituitary gland, 2) ESR1 and ESR2 do not synergize or antagonize the effects of each other; however, they do interact to synchronize the beginning of the stimulatory effect of E2 on secretion of LH, 3) ESR1 and ESR2 may mediate at least partially the positive feedback of E2 on LH secretion by increasing the number of GnRH receptors, and 4) only ESR1 appears to be involved in the negative feedback of E2 on secretion of FSH.

Differential Regulation of GHRH-Receptor and GHS-Receptor Expression by Long-Term In Vitro Treatment of Ovine Pituitary Cells with GHRP-2 and GHRH

GH secretion is regulated by GHRH and somatostatin via actions on their specific receptors in pituitary somatotropes. Ghrelin and synthetic analogs, GHRPs, also stimulate GH release via GHS-receptors (GHS-R). To examine the long-term effect of GHRH and/or GHRP on somatotropes, primary cultured ovine somatotropes were treated with GHRH (10(-9) and 10(-8) M) and GHRP-2 (10(-8) and 10(-7) M) for up to 2 d. After treatment, culture medium was collected for GH assay, and total RNA was extracted for RT-PCR analysis. To evaluate cell cultures used in this report, somatotrope-enriched pituitary cells were challenged by 6 h GHRH and dexamethasone (DEX) treatment. As expected, GHRH significantly decreased, whereas DEX increased, the levels of GHRHR mRNA. Combined low doses of GHRH (10(-9) M) and GHRP-2 (10(-8) M) treatment for 24 h increased accumulated GH secretion, significantly more than that induced by high doses of GHRH (10(-8) M) and GHRP-2 (10(-7) M). While levels of GHRH-R mRNA increased, GHS-R mRNA levels were decreased by low doses of GHRH and GHRP-2 for 24 h. High doses of GHRH and/or GHRP-2 for 2 d did not increase GH secretion in the second day of treatment and reduced the level of GHRH-R mRNA. High doses of GHRP-2 treatment decreased the levels of both GHRH-R and GHS-R mRNA. Low doses of GHRH and/or GHRP-2 for 2 d increased the level of GHS-R mRNA without changing GHRH-R mRNA levels. Such treatment also increased ghrelin- (10(-9) M) or ghrelin/GHRH (10(-9) M)-induced GH secretion. These results suggest that low doses of GHRP-2 and GHRH prime somatotropes for stimulation by GHRH and ghrelin.

Molecular mechanisms involved in LH release by the ovine pituitary cells

The luteinizing hormone-releasing hormone (LHRH) is a hypothalamic decapeptide and main positive regulator of luteinizing hormone (LH) secretion from pituitary cells. Insulin-like growth factor-I (IGF-1) also stimulates LH release and enhances the effect of LHRH. However, the molecular mechanisms involved in the interactions between LHRH and IGF-1 are unclear. Here, we first determined the effect of various types of LHRH [I (mammalian), II (chicken), III (lamprey), hyp9 and salmon] on both LH secretion and activation of MAPK (ERK1/2 and p38) in ovine pituitary cells. After 3 h of treatment, LH secretion was significantly higher for LHRH-I than for the other LHRH tested. Interestingly, LHRH-III had no effect at any concentration used on the LH release by ovine pituitary cells. The phosphorylation of both MAPK ERK1/2 and p38 was also significantly higher after treatment with LHRH-I than LHRH-II, salmon LHRH or hyp9. These MAPKs were not activated or only very weakly activated by LHRH-III. We then used pharmacological inhibitors to show that MAPK ERK1/2 and PKCδ participate in the LH release by ovine pituitary cells in response to LHRH-I. We identified the main substrates and signaling pathways [PI3K/Akt and MAPK (ERK1/2, p38 and JNK1/2] of IGF-1R and investigated the effect of IGF-1 on the stimulation of ovine pituitary cell LH secretion by the various LHRH. IGF-1 increases LH secretion in response to LHRH-I, LHRH-II, hyp9 and salmon LHRH but not the secretion after treatment with LHRH-III. Using specific inhibitors, we found that the MAPK ERK1/2 but not the PI3K/Akt signaling pathway is involved in the LH secretion in response to IGF-1. This is the first description of a common molecular mechanism, involving the MAPK ERK1/2, by which LHRH-R and IGF-1-R induce LH secretion in ovine pituitary cells.

A Nongenomic Action of 17β-Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone1

The objective of the present study was to determine the ability of 17beta-estradiol (E(2)) and conjugated forms of E(2) (E(2) conjugated to BSA [E(2)-BSA] and a novel conjugate, E(2) conjugated to a small peptide [E(2)-PEP]) to prevent the GnRH-induced secretion of LH and to determine the role of estradiol receptors (ERs) and ER subtypes (ERalpha, also known as ESR1, and ERbeta, also known as ESR2) in the mediation of the acute action of E(2) in primary cultures of ovine pituitary cells. Preincubation of cells for 15 min with E(2), E(2)-BSA, or E(2)-PEP prevented the GnRH-induced secretion of LH (P < 0.01). Treatment of cells with nonestrogenic steroid hormones did not affect secretion of LH when given alone, nor did these steroids impair the E(2)-induced inhibition of LH secretion (P > 0.1). Likewise, treatment of cells with the ER-antagonists tamoxifen, hydroxytamoxifen, or ICI 182 780 did not affect (P > 0.1) secretion of LH when given alone but did prevent (P < 0.01) the inhibition by E(2) and the E(2)-conjugates on GnRH-induced secretion of LH. When cells were treated with subtype-selective ER agonists, the ERalpha agonist (propylpyrazole-triol), but not the ERbeta agonist (diarylpropionitrile), decreased (P < 0.01) the GnRH-induced secretion of LH. In conclusion, the rapidity by which E(2) prevented GnRH-induced release of LH in ovine pituitary cells suggests that this inhibition is mediated via a nongenomic action of E(2). The inhibition of GnRH-induced secretion of LH proved to be steroid specific and mediated by ERs. It may occur specifically through ERalpha. The fact that E(2)-BSA or E(2)-PEP mimicked the action of E(2) suggests that this effect was mediated by an ER associated with the plasma membrane.

277. Is the high level of circulating FSH in Booroola ewes due to the Bmp R1b mutation?

Booroola ewes are characterised by having high ovulation rate and litter size caused by the action of the Booroola fecundity gene (FecB), which encodes the bone morphogenetic protein receptor (BMPR) type 1b mutation (Q249R). The product of the FecB gene influences both pituitary and ovarian activity. In both neonatal and adult Booroola ewes carrying a double copy of the FecB allele, plasma concentrations of the pituitary-derived follicle stimulating hormone (FSH) are higher than in non-carrier females. BMPR-1b is expressed in the sheep pituitary gland and recent reports have shown that in addition to GnRH, TGF-β family members such as BMP 6, 7, and 15 induce pituitary FSHβ expression. Thus BMPs may function in regulating FSH synthesis in the pituitary. We are investigating whether we can detect induction/secretion of FSH in response to BMP treatment of ovine primary pituitary cell cultures, with the aim of elucidating whether the Booroola mutation is the cause of the increased FSH secretion. Due to the high variance between cultures from different animals, and between replicates from the same animal, we are attempting to determine the best method for approaching the question. Some results from these investigations will be presented.

Activin and inhibin receptor gene expression in the ewe pituitary throughout the oestrous cycle.

In mammals, activin and inhibin are important regulators of FSH secretion. Previous studies have demonstrated that primary ovine pituitary cells express different activin receptor subtypes: activin receptor-like (ALK)2, ALK4, activin type II receptor A (ActRIIA), ActRIIB and Smad proteins in vitro. Here, we have carried out physiological studies to investigate the pattern of mRNA expression of the activin receptor subunits in the ewe pituitary throughout the oestrous cycle. The oestrous cycles of ewes were synchronized with progestagen sponges. The animals were killed 36 h (before the preovulatory surge, n=4), 48 h (during the preovulatory surge, n=4), 72 h (during the second surge of FSH, n=6) and 192 h (during the luteal phase, n=4) after sponge removal. Using Northern blots, we have shown that the levels of ALK2, ALK4 and ActRIIB mRNA were significantly higher before the preovulatory surge and during the secondary surge of FSH as compared with both during the preovulatory surge and the luteal phase, whereas the level of the ActRIIA mRNA was similar throughout the oestrous cycle. Using Western blots we have also demonstrated that the level of phospho-Smad2 did not vary during the reproductive cycle. Inhibin binding protein (InhBP/p120) and the transforming growth factor-beta type III receptor, betaglycan, have been identified as putative inhibin co-receptors. In this study, we cloned a fragment of both InhBP/p120 and betaglycan cDNAs in the ewe and showed by Northern blot that pituitary betaglycan and InhBP/p120 mRNA levels did not fluctuate across the oestrous cycle nor did they correlate with serum FSH levels.

Effect of GHRH and GHRP-2 treatment in vitro on GH secretion and levels of GH, pituitary transcription factor-1, GHRH-receptor, GH-secretagogue-receptor and somatostatin receptor mRNAs in ovine pituitary cells.

Growth hormone (GH)-releasing hormone (GHRH) and GH-releasing peptides (GHRPs) stimulate the release of GH through their specific receptors on somatotropes. Combined GHRH and GHRP administration causes a synergistic GH release in vivo by an unknown mechanism. The current study focuses on the direct action of GHRH and GHRP on several molecular targets in somatotropes. To clarify the mechanism of action, ovine somatotropes were used to measure the expression of mRNAs encoding for GH, pituitary transcription factor-1 (Pit-1), GH-secretagogue receptor (GHS-R), GHRH-R, somatostatin receptor subtypes (sst-1 and sst-2) and GH release after GHRH and GHRP-2 treatment for 0.5, 1, 1.5 and 2 h. GHRH (10 nM), GHRP-2 (100 nM) and combined GHRH-GHRP-2 increased the levels of GH mRNA and GH release from 0.5 to 2 h in a time-dependent manner. The levels of Pit-1, GHRH-R and GHS-R mRNA were increased after 0.5 h treatment of cells with GHRH and GHRP-2. The levels of sst-1 but not sst-2 mRNA were significantly increased after 0.5 and 1 h of GHRH treatment. In contrast, both sst-1 and sst-2 mRNA expression was inhibited after 0.5-2 h of GHRP treatment. These data demonstrate a direct in vitro modification of ovine somatotropes by GHRH and GHRP-2 resulting in altered GHRH-R, GHS-R, Pit-1, sst-1, sst-2 and GH gene expression; this may underlie the regulatory action of GHRH and GHRP-2 on GH secretion.

Expression and functional role of peroxisome proliferator-activated receptor-gamma in ovarian folliculogenesis in the sheep.

Peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that is activated by fatty acids and derivatives and the antidiabetic glitazones, which plays a role in the control of lipid and glucose homeostasis. In the present work, we tested the hypothesis that PPARgamma plays a role in reproductive tissues by studying its expression and function in the hypothalamo-pituitary-ovary axis in the sheep. PPARgamma 1 and PPARgamma 2 proteins and mRNAs were detected in whole ovine pituitary and ovary but not in hypothalamic extracts. In situ hybridization on ovarian section localized PPARgamma mRNA in the granulosa layer of follicles. Interestingly, PPARgamma expression was higher in small antral (1-3 mm diameter) than in preovulatory follicles (>5 mm diameter) (P < 0.001) and was not correlated with healthy status. To assess the biological activity of ovarian PPARgamma, ovine granulosa cells were transfected with a reporter construct driven by PPARgamma-responsive elements. Addition of rosiglitazone, a PPARgamma ligand, stimulated reporter gene expression, showing that endogenous PPARgamma is functional in ovine granulosa cells in vitro. Moreover, rosiglitazone inhibited granulosa cell proliferation (P < 0.05) and increased the secretion of progesterone in vitro (P < 0.05). This stimulation effect was stronger in granulosa cells from small than from large follicles. In contrast, rosiglitazone had no effect on LH, FSH, prolactin and growth hormone secretion by ovine pituitary cells in vitro. Overall, these data suggest that PPARgamma ligands might stimulate follicular differentiation in vivo likely through a direct action on granulosa cells rather than by modulating pituitary hormone secretion.

Activin signaling pathways in ovine pituitary and LbetaT2 gonadotrope cells.

In the pituitary, activin stimulates the synthesis and release of FSH. However, the activin receptor signaling pathways that mediate these effects are poorly known. We investigated these mechanisms in primary ovine pituitary cells (POP) and in the murine LbetaT2 gonadotrope cell line. POP cells and LbetaT2 cells express the different activin receptors (types IA, IB, IIA, and IIB) and the Smad proteins (Smad-2, -3, -4, and -7). In both POP and LbetaT2 cells, activin activated several signaling pathways: Smad-2, extracellular regulated kinase-1/2 (ERK1/2), p38, and phosphatidylinositol 3'-kinase (PI3K)/Akt. Phosphorylation of ERK1/2 and p38 were stimulated (3- to 6-fold) rapidly in 5 min, whereas activation of both Smad-2 and Akt (3- to 5-fold) occurred later, in 60 min. Activin also increased the association of activin receptor IIB with PI3K. Using specific inhibitors, we demonstrated that the activation of Smad-2 was partially blocked by the inhibition of PI3K but not by the inhibition of ERK1/2 or p38, suggesting a cross-talk between the Smad and PI3K/Akt pathways. In both POP and LbetaT2 cells, FSH expression and secretion in response to activin were not altered by the inhibition of PI3K/Akt, ERK1/2, or p38 pathways, whereas they were reduced by about 2-fold by expression of a dominant negative of Smad-2 or the natural inhibitory Smad-7 in LbetaT2 cells. These results indicate that activin activates several signaling pathways with different time courses in both POP and LbetaT2 cells, but only the Smad-2 pathway appears to be directly implicated in FSH expression and release in LbetaT2 cells.