Bovine Anterior Pituitary Cells Research Articles

Spike protein of SARS-CoV-2 suppresses gonadotrophin secretion from bovine anterior pituitaries.

Coronavirus disease (COVID-19), the ongoing global pandemic, is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Recent evidence shows that the virus utilizes angiotensin-converting enzyme 2 (ACE2) as a spike protein receptor for entry into target host cells. The bovine ACE2 contains key residues for binding to the spike protein receptor-binding domain. This study evaluated the hypothesis that bovine gonadotroph expresses ACE2, and spike protein suppresses luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from cultured bovine anterior pituitary (AP) cells. ACE2 mRNA expression and ACE2 protein expression were detected in the bovine AP cells using reverse transcription PCR and western blot analysis. Immunofluorescence microscopy analysis with the anti-ACE2 antibody revealed the co-localization of ACE2 and gonadotropin-releasing hormone (GnRH) receptor on the gonadotroph plasma membrane. Approximately 90% of GnRH receptor-positive cells expressed ACE2, and approximately 46% of ACE2-positive cells expressed the GnRH receptor. We cultured bovine AP cells for 3.5 days and treated them with increasing concentrations (0, 0.07, 0.7, or 7 pM) of recombinant spike protein having both S1 and S2 regions. The spike protein (0.07–7 pM) suppressed both basal and GnRH-induced LH secretion (P < 0.05). Spike protein (0.7–7 pM) suppressed GnRH-induced (P < 0.05), but not basal FSH secretion. In contrast, pre-treatment with ERK 1/2/5 inhibitor (U0126) partially restored the GnRH-induced LH and FSH secretion from the spike protein suppression. Collectively, the results indicate that gonadotrophs express ACE2, a receptor for coronavirus 2 spike protein, which in turn suppresses LH and FSH secretion from AP cells.

Influence of brain plasmalogen changes on gonadotropin secretion from the cultured bovine anterior pituitary cells

We recently discovered that the orphan G-protein–coupled receptor (GPR) 61 colocalized with GnRH receptors (GnRHRs) on the surface of most of bovine gonadotrophs. A recent study suggested that ethanolamine plasmalogen (PI) is a ligand for GPR61 in mouse neuroblastoma. Therefore, this study evaluated the hypothesis that PI alters LH and FSH secretion from cultured bovine anterior pituitary (AP) cells. We prepared bovine AP cells from postpubertal heifers (26 mo old) and cultured the cells for 3.5 d. We treated the cells with increasing concentrations (0, 5, 50, 500, 5,000, 50,000, or 500,000 pg/mL) of phosphoethanolamine PI (PEPI) extracted from the bovine brain, or l-α-lysophosphatidylethanolamine PI (LEPI) extracted from the bovine brain, for 5 min before either no treatment or GnRH stimulation. The medium samples were harvested 2 h after culture for LH and FSH assays. Phosphoethanolamine PI (50–500 pg/mL) stimulated (P < 0.05) the basal secretion of FSH but not LH. Phosphoethanolamine PI at 50 pg/mL also enhanced (P < 0.05) GnRH-induced FSH secretion. However, higher doses (500–500,000 pg/mL) of PEPI suppressed GnRH-induced FSH secretion. Moreover, 50 to 500,000 pg/mL PEPI suppressed GnRH-induced LH secretion. None of the tested concentrations of LEPI showed any effect on basal or GnRH-induced LH or FSH secretion. Pretreatment with Sma and Mad pathway inhibitors suppressed FSH secretion induced by PEPI, whereas an extracellular signal-regulated kinase pathway inhibitor blocked the PEPI-induced suppression of GnRH-stimulated LH secretion. Therefore, PEPI, but not LEPI, extracted from the bovine brain, alters FSH and LH secretion from cultured AP cells. Further studies are required to decide whether PEPI binds to GPR61 and whether PEPI plays an important role in the control of gonadotropin secretion from gonadotrophs.

Effects of methyl-beta-cyclodextrin on the release of luteiniz-ing hormone, follicle stimulating hormone, prolactin and growth hormone from cultured bovine anterior pituitary cells

The aims of the present study are to clarify the effect of methyl-beta-cyclo-dextrin (MβCD) on the secretions of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL) and growth hormone (GH) from the anterior pituitary (AP) cells of prepubertal male cattle and to compare the characteristics of secretion to those of gonadotrophic-releasing hormone (GnRH), thyrotropin- releasing hormone (TRH) and growth hormone-releasing hormone (GHRH), respectively. The AP cells prepared from six castrated Holstein calves (age: 8-11 months) were incubated for 2 h with MβCD (10-3 M, 10-2 M), GnRH (10-8 M), TRH (10-8 M) and GHRH (10-8 M) or vehicle only as a control (CTL). The amount of LH, PRL and GH released in the medium was measured by a time-resolved fluoroimmunoassay (TRFIA). The amount of FSH released in the medium was measured by the radioimmunoassay (RIA). MβCD significantly (P&lt;0.05) stimulated the secretion of LH, FSH, PRL and GH from the AP cells. Furthermore, GnRH significantly (P&lt;0.05) stimulated the secretion of LH and FSH. Also, TRH and GHRH significantly (P&lt;0.05) stimulated the secretion of PRL and GH, respectively. However, the potency of the MβCD was less compared to each respective hormone secretions in response to GnRH, TRH and GHRH (P&lt;0.05).

GPR30 mediates estrone, estriol, and estradiol to suppress gonadotropin-releasing hormone-induced luteinizing hormone secretion in the anterior pituitary of heifers.

Recent studies demonstrated that G-protein-coupled receptor 30 (GPR30) on the plasma membrane of gonadotroph cells mediates picomolar, but not nanomolar, levels of estradiol (E2) to rapidly suppress gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion in the anterior pituitary (AP). While estrone (E1) and estriol (E3) are considered “weak” estrogens that exert suppressive effects through estrogen receptors α and β, it is conceivable that they also strongly suppress GnRH-induced LH secretion via GPR30. Both E1 and E3 are likely present within the blood at picomolar or nanomolar concentrations, indicating that such concentrations are sufficient to suppress GnRH-induced LH secretion. To evaluate this possibility, bovine AP cells were cultured under steroid-free conditions and then incubated with various concentrations (0.01 pM to 10 nM) of E2, E1, or E3, prior to stimulation with GnRH. Notably, GnRH-induced LH secretion from AP cells was inhibited by 1–100 pM E2, 1–10 pM E1, and 1–100 pM E3. GnRH-induced LH secretion from AP cells was not inhibited by lower (0.01–0.1 pM) or higher (1–10 nM) concentrations of E2, E1, and E3. These suppressive effects were inhibited by pre-treatment of AP cells with the GPR30 antagonist G36, but not with the estrogen receptor alpha antagonist. Treatment with E1 or E3 also yielded decreased cytoplasmic cAMP levels in cultured AP cells pre-treated with dopamine and phosphodiesterase inhibitors. Therefore, these results suggest that GPR30 mediates the suppressive effects of E1, E3, and E2 on GnRH-induced LH secretion from bovine AP.

Method for isolating pure bovine gonadotrophs from anterior pituitary using magnetic nanoparticles and anti-gonadotropin-releasing hormone receptor antibody.

No methods are currently available for rapidly isolating gonadotrophs from the anterior pituitary (AP) in any species. We developed a method for preparing pure bovine gonadotrophs from a heterogeneous AP cell mixture by magnetic separation and our original antibody against the N terminus of bovine gonadotropin-releasing hormone receptor (GnRHR). A bovine AP cell mixture was incubated with the anti-GnRHR antibody, anti-dextran antibody-conjugated secondary antibody and dextran-coated magnetic nanoparticles for magnetic isolation. Approximately 5.2 × 106 cells were isolated per AP of Japanese Black heifers (26 months of age) and cultured, and confocal microscopy confirmed to be GnRHR- and luteinizing hormone-positive, corresponding to a purity of 100%. Approximately 44.5 µg of total protein was extracted from the pure gonadotrophs per AP.

Cytoplasmic kinases downstream of GPR30 suppress gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone secretion from bovine anterior pituitary cells.

GPR30 is known as a membrane receptor for picomolar concentrations of estradiol. The GPR30-specific agonist G1 causes a rapid, non-genomic suppression of gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion from bovine anterior pituitary (AP) cells. A few studies have recently clarified that protein kinase A (PKA) and phosphorylated extracellular signal-regulated kinase (pERK) might be involved in cytoplasmic signaling pathways of GPR30 in other cells. Therefore, we tested the hypothesis that PKA and ERK kinase (MEK) are important cytoplasmic mediators for GPR30-associated non-genomic suppression of GnRH-induced LH secretion from bovine AP cells. Bovine AP cells (n = 8) were cultured for 3 days under steroid-free conditions. The AP cells were previously treated for 30 min with one of the following: 5000 nM of PKA inhibitor (H89), 1000 nM of MEK inhibitor (U0126), or a combination of H89 and U0126. Next, the AP cells were treated with 0.01 nM estradiol for 5 min before GnRH stimulation. Estradiol treatment without inhibitor pretreatment significantly suppressed GnRH-induced LH secretion (P < 0.01). In contrast, estradiol treatment after pretreatment with H89, U0126 or their combination had no suppressive effect on GnRH-induced LH secretion. The inhibitors also inhibited the G1 suppression of GnRH-induced LH secretion. Therefore, these data supported the hypothesis that PKA and MEK (thus, also pERK) are the intracellular mediators downstream of GPR30 that induce the non-genomic suppression of GnRH-induced LH secretion from bovine AP cells by estradiol or G1.

The nonsteroidal mycoestrogen zearalenone and its five metabolites suppress LH secretion from the bovine anterior pituitary cells via the estradiol receptor GPR30 in vitro

Picomolar concentrations of estradiol produce nongenomic suppression of GnRH-induced LH secretion from the anterior pituitary (AP) of cattle via G-protein–coupled receptor 30 (GPR30). Zearalenone (ZEN) is the nonsteroidal mycoestrogen produced by Fusarium fungi and has been detected in cereal grains, animal feed, and ruminant urine worldwide. Zearalenone has a prolonged blood half-life that results from enterohepatic cycling. There are five metabolites of ZEN: α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), and zearalanone, which may persist for long periods in animals and humans after consumption of ZEN-contaminated feed. We recently reported that GPR30 bound with α-ZAL decreases cytoplasmic cAMP but not gene expression of LHα and LHβ subunits, and GPR30 bound with α-ZAL suppresses GnRH-induced LH release in bovine AP cells in vitro. We tested the hypothesis that GPR30 bound with ZEN or one of the four previously untested metabolites suppresses GnRH-induced LH release from the bovine AP cells in vitro. Anterior pituitary cells were cultured for 3 days under steroid-free conditions and were then incubated with various concentrations (0.001–10 nM) of estradiol or one of the ZEN analogs for 5 minutes before GnRH stimulation. Gonadotropin-releasing hormone–stimulated LH secretion from AP cells was inhibited by all of the test concentrations of ZEN, 0.001 to 1 nM of α-ZAL, and 0.001 to 0.1 nM of the remaining four analogs. Pretreatment for 5 minutes with a GPR30-specific antagonist, G36, inhibited estradiol- and the ZEN analog–induced suppression of LH secretion from cultured AP cells. G36 alone had no significant effect on LH secretion. The estimated order of the nongenomic inhibiting effect was ZEN, α-ZAL, zearalanone, α-ZOL, β-ZOL, and β-ZAL, which is quite different from the reported order for their genomic effects. Therefore, ZEN and all of its metabolites suppress LH secretion from the bovine AP cells via GPR30 in vitro.

Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations

The presence of gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) on gonadotrophs in the anterior pituitary (AP) is an important factor for reproduction control. However, little is known regarding GnRHR gene expression in gonadotrophs of cattle owing to the lack of an appropriate anti-GnRHR antibody. Therefore, an anti-GnRHR antibody for immunohistochemistry, flow cytometry, and immunocytochemistry assays was developed to characterize GnRHR gene expression in gonadotrophs. The anti-GnRHR antibody could suppress GnRH-induced LH secretion from cultured AP cells of cattle. The GnRHR, luteinizing hormone (LH), and follicle stimulating hormone (FSH) in the AP tissue was analyzed by fluorescence immunohistochemistry. The GnRHRs were aggregated on a limited area of the cell surface of gonadotrophs, possibly localized to lipid rafts. The LH secretion was stimulated with increasing amounts of GnRH; however, excessive concentrations (>1nM) resulted in a decrease in LH secretion. A novel method to purify gonadotrophs was developed using the anti-GnRHR antibody and fluorescence-activated cell sorting. Flow cytometric analysis using the anti-GnRHR antibody for cultured bovine AP cells, however, failed to support the hypothesis that GnRH induces GnRHR internalization and decreases GnRHR on the surface of GnRHR-positive AP cells. In contrast, immunocytochemistry using primary antibodies for cultured bovine AP cells showed that 10nM (P<0.05) and 100nM (P<0.01) GnRH, but not 0.01–1nM GnRH, increased GnRHR in the cytoplasm of LH-positive cells. In conclusion, these data suggested that GnRHRs were aggregated on the surface of gonadotrophs and GnRHR inside gonadotrophs increased with elevated concentrations of GnRH.

Expression of estradiol receptor, GPR30, in bovine anterior pituitary and effects of GPR30 agonist on GnRH-induced LH secretion

G-protein – coupled receptor 30 (GPR30) is an estradiol receptor located on the plasma membrane, and it initiates several rapid, non-genomic signaling events. GPR30 has recently been identified in rat anterior pituitary (AP); however, little is known about the role of GPR30 in controlling luteinizing hormone (LH) secretion from gonadotropes in animals. To fill this research gap, we hypothesized that GPR30 is expressed in bovine AP and mediates estradiol inhibition of gonadotropin-releasing hormone (GnRH)-induced LH release. We confirmed the expressions of GPR30 mRNA and protein by RT-PCR, western blotting, and immunohistochemistry. We cultured bovine AP cells (n=8) for 3 days in steroid-free conditions and then treated them with increasing concentrations (0.001nM, 0.01nM, 0.1nM, 1nM, and 10nM) of estradiol or a GPR30-specific agonist, G1, for 5min before GnRH stimulation. As expected, estradiol at 0.001–0.1nM inhibited the GnRH-stimulated LH secretion. However, we found also that G1 at 0.001nM was able to inhibit this secretion (P<0.05). In contrast, both estradiol and G1 at higher doses were less efficient in suppressing the GnRH-stimulated LH secretion. Neither estradiol nor G1 suppressed GnRH-stimulated follicle-stimulating hormone secretion. In separate experiments, fluorescent immunohistochemistry and immunocytochemistry revealed that approximately 50% of GPR30-positive cells express LH, and about 30% of LH-positive cells express GPR30. In conclusion, GPR30 is expressed in bovine gonadotropes and other AP cells and may partially contribute to rapid negative estradiol feedback of GnRH-induced LH secretion.

Sex steroid hormones do not enhance the direct stimulatory effect of kisspetin‐10 on the secretion of growth hormone from bovine anterior pituitary cells

The aims of the present study were to clarify the effect of kisspeptin10 (Kp10) on the secretion of growth hormone (GH) from bovine anterior pituitary (AP) cells, and evaluate the ability of sex steroid hormones to enhance the sensitivity of somatotrophic cells to Kp10. AP cells prepared from 8-11-month-old castrated calves were incubated for 12 h with estradiol (E(2), 10(-8) mol/L),progesterone (P(4), 10(-8) mol/L), testosterone (T, 10(-8) mol/L), or vehicle only (control), and then for 2 h with Kp10. The amount of GH released in the medium was measured by a time-resolved fluoroimmunoassay. Kp10 (10(-6) or 10(-5) mol/L) significantly stimulated the secretion of GH from the AP cells regardless of steroid treatments (P < 0.05), and E(2), P(4), and T had no effect on this response. The GH-releasing response to growth hormone-releasing hormone (GHRH, 10(-8) mol/L) was significantly greater than that to Kp10 (P < 0.05). The present results suggest that Kp10 directly stimulates the release of GH from somatotrophic cells and sex steroid hormones do not enhance the sensitivity of these cells to Kp10. Furthermore, they suggest that the GH-releasing effect of Kp10 is less potent than that of GHRH.

The role of sexual steroid hormones in the direct stimulation by Kisspeptin-10 of the secretion of luteinizing hormone, follicle-stimulating hormone and prolactin from bovine anterior pituitary cells

The aims of the present study were to clarify the effect of Kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) from bovine anterior pituitary (AP) cells and evaluate the ability of sex steroids to enhance the sensitivity of gonadotropic and lactotropic cells to Kp10. AP cells prepared from 7-week-old male calves were incubated for 12 h with estradiol (E 2; 10 −8 M), progesterone (P 4; 10 −8 M), testosterone (T; 10 −8 M), or vehicle only (control), and then for 2 h with Kp10 (10 −6 M). The amounts of LH, FSH and PRL released into the culture medium after the 2-h incubation period were examined. Kp10 significantly stimulated the secretion of LH from the AP cells treated with E 2 and T ( P < 0.05), but not from the P 4-treated cells. In contrast, Kp10 had no effect on the secretion of FSH regardless of the steroid treatment. Kp10 significantly stimulated the secretion of PRL ( P < 0.05), the sexual steroid hormones having no effect. The LH- or FSH-releasing response to gonadotropin-releasing hormone (GnRH; 10 −8 M) and PRL-releasing response to thyrotropin-releasing hormone (TRH; 10 −8 M) were significantly greater than those to Kp10 ( P < 0.05). The present results suggest that E 2 and T, but not P 4, enhance the sensitivity of gonadotropic cells to the secretion of LH in response to Kp10. However, Kp10 had no stimulatory effect on the secretion of FSH regardless of the effect of sex steroids. Kp10 directly stimulates the secretion of PRL from the pituitary cells, and sex steroids do not enhance the sensitivity of lactotropic cells to Kp10. Furthermore, the LH- and FSH-releasing effect and the PRL-releasing effect of Kp10 are less potent than that of GnRH and TRH, respectively.

Characteristics of prolactin-releasing response to salsolinol (SAL) and thyrotropin-releasing hormone (TRH) in ruminants

The secretion of prolactin (PRL) is stimulated by thyrotropin-releasing hormone (TRH), and inhibited by dopamine (DA). However, we have recently demonstrated that salsolinol (SAL), a DA-derived endogenous compound, is able to stimulate the release of PRL in ruminants. The aims of the present study were to compare the characteristics of the PRL-releasing response to SAL and TRH, and examine the relation between the effects that SAL and DA exert on the secretion of PRL in ruminants in vivo and in vitro. Three consecutive intravenous (i.v.) injections of SAL (5 mg/kg body weight (b.w.): 19.2 μmol/kg b.w.) or TRH (1 μg/kg b.w.: 2.8 nmol/kg b.w.) at 2-h intervals increased plasma PRL levels after each injection in goats ( P < 0.05); however, the responses to SAL were different from those to TRH. There were no significant differences in each peak value between the groups. The rate of decrease in PRL levels following the peak was attenuated in SAL-treated compare to TRH-treated animals ( P < 0.05). PRL-releasing responses to SAL were similar to those to sulpiride (a DA receptor antagonist, 0.1 mg/kg b.w.: 293.3 nmol/kg b.w.). In cultured bovine anterior pituitary (AP) cells, TRH (10 −8 M) significantly increased the release of PRL following both 15- and 30-min incubation periods ( P < 0.05), but SAL (10 −6 M) did not increase the release during the same periods. DA (10 −6 M) completely blocked the TRH-induced release of PRL for a 2-h incubation period in the AP cells ( P < 0.05). Sulpiride (10 −6 M) reversed this inhibitory effect but SAL (10 −6 M) did not have any influence on the action of DA. These results show that the mechanism(s) by which SAL releases PRL is different from the mechanism of action of TRH. Furthermore, they also show that the secretion of PRL is under the inhibitory control of DA, and SAL does not antagonize the DA receptor's action.

Anterior Pituitary Progenitor Cells Express Costimulatory Molecule 4Ig-B7-H3

Stem/Progenitor cells in the postnatal pituitary gland are embedded in a marginal cell layer around Rathke's pouch. However, the nature and behavior of anterior pituitary progenitor cells remain unclear. We established bovine anterior pituitary progenitor cell line (BAPC)-1 from the anterior pituitary gland, which expressed stem/progenitor cell-related genes and several inflammatory cytokines. To characterize and localize these pituitary progenitor cells, we produced a mAb (12B mAb) against BAPC-1. The 12B mAb recognized the 4Ig-B7-H3 molecule, which is a costimulatory molecule and negative regulator in T cell activation. WC1(+) gammadelta T cells in young bovine PBMC express the 4Ig-B7-H3 molecule, but few or no 4Ig-B7-H3-immunoreactive cells are expressed in PBMC in adult cattle. The 12B-immunoreactive cells in the bovine anterior pituitary gland were localized around Rathke's pouch and expressed IL-18 and MHC class II. However, the number of 12B-immunoreactive cells was lower in adult than in young cattle. BAPC-1 expressed IL-18 and MHC class II, and demonstrated phagocytotic activity. BAPC-1 also had the ability to promote CD25 expression in PBMC after 5 days of coculture, and blocking 4Ig-B7-H3 x 12B mAb enhanced their expression of CD25. In addition, the 12B-immunoreactive cells were observed around the pars tuberalis closely bordering the median eminence and in the blood vessels of the primary portal plexus in the anterior pituitary gland. These results suggest that an established BAPC-1 may originate from these progenitor cells, and that the progenitor cells with 4Ig-B7-H3 may play a critical role in the immunoendocrine network.

Bovine Anterior Pituitary Progenitor Cell Line Expresses Interleukin (IL)‐18 and IL‐18 Receptor

In the anterior pituitary gland, inflammatory mediators regulate cell function through an immuno-endocrine pathway. Recent studies have shown that undifferentiated stem cells act as immunomodulators. These studies prompted us to establish a progenitor cell line from the bovine anterior pituitary gland and to detail its function. First, we localised interleukin (IL)-18 by immunohistochemistry to the marginal cell layer of Rathke's pouch that is assumed to embody a stem/progenitor cell compartment of the postnatal pituitary gland. A cloned anterior pituitary-derived cell line from the bovine anterior pituitary gland was established from single cell clone by the limiting dilution method and was designated as bovine anterior pituitary-derived cell line (BAPC)-1. BAPC-1 cells constantly expressed mRNAs for IL-18 and IL-18 receptor, and grew steadily and rapidly in the medium containing epidermal growth factor and basic fibroblast growth factor. The cell line also expressed the mRNAs for the stem/progenitor cell- related factors such as Nanog, Oct-4, Ptch1, Nestin, Notch1, Hes1, Lrp and Fzd4, and the mRNAs for embryonic pituitary-related factors, such as Lhx3, PitX1 and Pit-1. The nuclei of BAPC-1 were immunostained positively for Pit-1, Hes1 and beta-catenin antibodies. Furthermore, BAPC-1 cells expressed mRNAs for cytokine such as IL-1alpha, IL-6, IL-7, IL-12 and IL-15. Stimulation of BAPC-1 cells with IL-18 increased expression of mRNAs for IL-1alpha, IL-6, IL-1beta and IL-8. At day 6 in culture, BAPC-1 cells also express growth hormone mRNA. These results strongly suggest that BAPC-1 is a stem/progenitor cell line and modulates the immuno-endocrine function of the anterior pituitary cells through its cytokine production.

Methodology for the study of the hypothalamic-pituitary hormone secretion in cattle

Studies on the neuroregulatory mechanisms on the secretion of anterior pituitary (AP) hormones in domestic animals are important because nearly all complex physiological and metabolic processes are regulated by the AP hormones. To examine them, this article considers in vivo approaches such as the techniques of intrahypothalamic injection, intracerebroventricular injection, push-pull perfusion, and microdialysis, which have been employed in our own research group for the study in cattle. Also, in vitro approaches such as bovine AP cell culture and the AP explants superfusion system are described. This article clarifies the potential of neuroendocrine study techniques in cattle.

Kisspeptin-10 stimulates the secretion of growth hormone and prolactin directly from cultured bovine anterior pituitary cells

Kisspeptins are peptide hormones encoded by the KiSS-1 gene, and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. We recently observed that kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) also has a direct stimulating effect on luteinizing hormone (LH) secretion in bovine anterior pituitary (AP) cells. In the present study, we evaluated the direct effect of kisspeptin-10 on the secretion of other pituitary hormones, growth hormone (GH) and prolactin (PRL), from bovine AP cells. The AP cells, which were prepared from 1- or 8-month-old male calves, were incubated for 2 h with the peptides. Kisspeptin-10 at 100 nM ( P < 0.05), 1000 nM ( P < 0.01) and 10,000 nM ( P < 0.01), but not at 10 nM, significantly stimulated GH secretion from the AP cells of 1-month-old calves, while in 8-month-old calves it was significantly ( P < 0.05) stimulated at 1000 nM ( P < 0.01) and 10,000 nM ( P < 0.01), but not at 10 nM and 100 nM. The response of GH to 100 nM ( P < 0.01), 1000 nM ( P < 0.05) and 10,000 nM ( P < 0.01) kisspeptin-10 in the AP cells of 1-month-old calves was significantly greater than in those of 8-month-old calves. All tested doses of kisspeptin-10 had no effect on PRL secretion from AP cells of 1-month-old calves. However, 1000 nM ( P < 0.05) and 10,000 nM ( P < 0.01), but not lower concentrations, of kisspeptin-10 significantly stimulated PRL secretion from the AP cells of 8-month-old calves. The present study is, as far as we know, the first to examine the direct actions of kisspeptin on the secretion of GH and PRL from the bovine pituitary gland. Further studies are necessary to evaluate the importance of multiple actions of kisspeptin on the pituitary of various animals in vivo.

Interaction between salsolinol (SAL) and thyrotropin-releasing hormone (TRH) or dopamine (DA) on the secretion of prolactin in ruminants

We have recently demonstrated that salsolinol (SAL), a dopamine (DA)-derived compound, is present in the posterior pituitary gland and is able to stimulate the release of prolactin (PRL) in ruminants. The aim of the present study was to clarify the effect that the interaction of SAL with thyrotropin-releasing hormone (TRH) or DA has on the secretion of PRL in ruminants. A single intravenous (i.v.) injection of SAL (5 mg/kg body weight (b.w.)), TRH (1 μg/kg b.w.), and SAL plus TRH significantly stimulated the release of PRL in goats ( P < 0.05). The cumulative response curve (area under the curve: AUC) during 120 min was 1.53 and 1.47 times greater after the injection of SAL plus TRH than either SAL or TRH alone, respectively ( P < 0.05). A single i.v. injection of sulpiride (a DA receptor antagonist, 0.1 mg/kg b.w.), sulpiride plus SAL (5 mg/kg b.w.), and sulpiride plus TRH (1 μg/kg b.w.) significantly stimulated the release of PRL in goats ( P < 0.05). The AUC of PRL during 120 min was 2.12 and 1.78 times greater after the injection of sulpiride plus TRH than either sulpiride alone or sulpiride plus SAL, respectively ( P < 0.05). In cultured bovine anterior pituitary (AP) cells, SAL (10 −6 M), TRH (10 −8 M), and SAL plus TRH significantly increased the release of PRL ( P < 0.05), but the additive effect of SAL and TRH detected in vivo was not observed in vitro. In contrast, DA (10 −6 M) inhibited the TRH-, as well as SAL-induced PRL release in vitro. All together, these results clearly show that SAL can stimulate the release of PRL in ruminants. Furthermore, they also demonstrate that the additive effect of SAL and TRH on the release of PRL detected in vivo may not be mediated at the level of the AP, but that DA can overcome their releasing activity both in vivo and in vitro, confirming the dominant role of DA in the inhibitory regulation of PRL secretion in ruminants.

Direct kisspeptin-10 stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells

Kisspeptins are peptide hormones encoded by the KiSS-1 gene and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. However, peripheral administration, as well as central administration, of kisspeptin stimulates luteinizing hormone (LH) secretion in some mammalian species. In order to evaluate the direct effects of kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) on LH secretion from bovine and porcine anterior pituitary (AP) cells, LH-releasing effects of kisspeptin-10 on AP cells were compared with GnRH in vitro. The AP cells were prepared from 1-month-old intact male calves, 8-month-old castrated male calves, or 6-month-old barrows, and then the cells were incubated for 2 h with the peptides. The 1000 nM and 10,000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from the bovine AP cells ( P < 0.05). The 100 nM and 1000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from porcine AP cells ( P < 0.05). As 10 nM of GnRH strongly stimulated LH secretion from all AP cells tested in this study, the present results suggest that kisspeptin-10 has a direct, but weak, stimulating effect on LH secretion in bovine and porcine AP cells. The present study is the first to examine the direct actions of kisspeptin on the bovine and porcine pituitary gland as far as we know. Kisspeptin might have other actions on the pituitary because the pituitary has multiple roles.

Salsolinol is present in the bovine posterior pituitary gland and stimulates the release of prolactin both in vivo and in vitro in ruminants

The aims of the present study were to determine whether salsolinol (SAL), a dopamine-related compound, is present in the bovine posterior pituitary (PP) gland, and to clarify the effect of SAL on the secretion of prolactin (PRL) in ruminants. SAL was detected in extract of bovine PP gland using high-pressure liquid chromatography with electrochemical detection (HPLC-EC). A single intravenous (i.v.) injection of SAL (5 and 10 mg/kg body weight) significantly and dose-dependently stimulated the release of PRL in goats ( P < 0.05). Plasma PRL levels reached a peak 10 min after the injection, then gradually returned to basal values in 60–80 min. The PRL-releasing pattern was similar to that in response to sulpiride (a dopamine receptor antagonist). The intracerebroventricular (i.c.v.) injection of 1 mg of SAL had no significant effect on the release of PRL in calves, however, 5 mg significantly stimulated the release ( P < 0.05) with peak values reached 30–40 min after the injection. Moreover, SAL significantly stimulated the release of PRL from cultured bovine anterior pituitary cells at doses of 10 −6 and 10 −5 M, compared to control cells ( P < 0.05). Taken together, our data clearly show that SAL is present in extract of the PP gland of ruminants, and has PRL-releasing activity both in vivo and in vitro. Therefore, this endogenous compound is a strong candidate for the factor having PRL-releasing activity that has been previously detected in extract of the bovine PP gland.

Effects of leptin on the release of luteinizing hormone, growth hormone and prolactin from cultured bovine anterior pituitary cells

ABSTRACTThe effects of leptin on the release of luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) were studied in cultured bovine anterior pituitary (AP) cells in vitro. The AP cells were obtained from fully‐fed Japanese Black steers and were incubated for 3 h with 10−13 to 10−7 mol/L of leptin after incubating in Dulbecco's modified Eagle's Medium for 3 days. Leptin significantly increased the concentration of LH in the culture medium by 45 and 44% at doses of 10−8 and 10−7 mol/L, respectively, compared with the controls (P &lt; 0.05). Leptin significantly increased the concentration of GH in the culture medium by 14 and 12% at doses of 10−8 and 10−7 mol/L, respectively (P &lt; 0.05). Leptin also significantly increased the concentration of PRL in the culture medium by 26% compared with the controls at a dose of 10−7 mol/L (P &lt; 0.05). These results show that leptin stimulates the release of LH, GH and PRL by acting directly on bovine AP cells from fully‐fed steers.