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

The 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 < 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 < 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 < 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.     

Kisspeptin‐10 stimulates the release of luteinizing hormone and testosterone in pre‐ and post‐pubertal male goats

The aims of the present study were to clarify the effect of kisspeptin‐10 (Kp10) on the secretion of luteinizing hormone (LH) and testosterone (T) in pre‐pubertal and post‐pubertal male ruminants. Four male goats (Shiba goats) were given an intravenous (i.v.) injection of Kp10 (5 µg/kg body weight (b.w.)), gonadotoropin‐releasing hormone (GnRH, 1 µg/kg b.w.), or 2 mL of saline as a control at the ages of 3 (pre‐pubertal) and 6 (post‐pubertal) months. A single i.v. injection of Kp10 significantly stimulated the release of LH and T in both groups. The area under the response curve (AUC) of LH for a 60‐min period after the i.v. injection of Kp10 was significantly greater in the pre‐pubertal goats (P < 0.05). The AUC of T for a 120 min period post‐injection did not differ between the two age groups. A single i.v. injection of GnRH also significantly stimulated the release of LH and T in both groups (P < 0.05). The secretory pattern of LH and T in response to GnRH resembled that in response to Kp10. These results show that the LH‐releasing response to Kp10 is greater in pre‐pubertal than post‐pubertal male goats. They also show that Kp10, as well as GnRH, is able to stimulate the release of T in male goats.     

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.     

Raf‐ERK1/2 signalling pathways mediate steroid hormone synthesis in bovine ovarian granulosa cells

Steroid hormones are required for normal reproductive function of female. The aim of this study was to investigate the role of Raf‐ERK1/2 on steroid hormone synthesis in bovine ovarian granulosa cells. Immunohistochemistry assay showed that both B‐Raf and C‐Raf were expressed in granulosa cells, theca cells and Sertoli cells. The protein expression of Raf or ERK1/2 was clearly decreased by Raf inhibitor GSK2118436 or ERK1/2 inhibitor SCH772984, respectively (p < 0.05). In addition, western blotting was performed for investigating the crosstalk between Raf and ERK1/2, the data showed that Raf positively regulated ERK1/2, whereas ERK1/2 had a negative feedback effect on Raf. The biosynthesis of oestradiol or testosterone was significantly decreased by treatment with GSK2118436 or SCH772984 (p < 0.05). Conversely, the progesterone biosynthesis was clearly increased by treatment with those inhibitors (p < 0.05). Furthermore, the mRNA expression of STAR, aromatase and CYP17 was blocked by Raf‐ERK1/2 signalling inhibition, which oppositely induced the mRNA expression of CYP11. Together, these findings suggested that Raf‐ERK1/2 signalling pathways mediate steroid hormone synthesis via affecting the expression of steroidogenic enzymes.     

Effect of calcitonin on adrenocorticotropic hormone secretion stimulated by corticotropin‐releasing hormone in the hen anterior pituitary

The presence of a receptor for calcitonin (CT) and the effect of chicken CT (cCT) on adrenocorticotropic hormone (ACTH) secretion stimulated by rat/human corticotropin‐releasing hormone (rhCRH) in the hen anterior pituitary were studied. The specific [¹²⁵I]cCT binding component was present in the plasma membrane of hen anterior pituitary and this binding component had properties of a receptor which has binding specificity to cCT, reversibility, saturable binding, high affinity and limited capacity. When anterior pituitary cells were incubated in vitro, cCT increased the maximal secretion of chicken ACTH stimulated by rhCRH. These results suggest that CT may act directly on the anterior pituitary via its receptor binding and enhances the ACTH secretion by CRH.     

Effects of hypothalamic dopamine on growth hormone‐releasing hormone‐induced growth hormone secretion and thyrotropin‐releasing hormone‐induced prolactin secretion in goats

The aim of the present study was to clarify the effects of hypothalamic dopamine (DA) on the secretion of growth hormone (GH) in goats. The GH‐releasing response to an intravenous (i.v.) injection of GH‐releasing hormone (GHRH, 0.25 μg/kg body weight (BW)) was examined after treatments to augment central DA using carbidopa (carbi, 1 mg/kg BW) and L‐dopa (1 mg/kg BW) in male and female goats under a 16‐h photoperiod (16 h light, 8 h dark) condition. GHRH significantly and rapidly stimulated the release of GH after its i.v. administration to goats (P < 0.05). The carbi and L‐dopa treatments completely suppressed GH‐releasing responses to GHRH in both male and female goats (P < 0.05). The prolactin (PRL)‐releasing response to an i.v. injection of thyrotropin‐releasing hormone (TRH, 1 μg/kg BW) was additionally examined in male goats in this study to confirm modifications to central DA concentrations. The treatments with carbi and L‐dopa significantly reduced TRH‐induced PRL release in goats (P < 0.05). These results demonstrated that hypothalamic DA was involved in the regulatory mechanisms of GH, as well as PRL secretion in goats.     

母马生长激素细胞和催乳激素细胞的形态计测学研究

利用免疫组织化学和形态计测学的方法 ,观察了 1 8匹成熟雌性蒙古马的脑垂体前叶生长激素细胞和催乳激素细胞的数量和面积 ,同时利用放射免疫分析方法检测了这两种激素的血浆水平。结果表明 ,每个马脑垂体前叶中 ,生长激素细胞的平均数量为 6 .42× 1 8,每个细胞的平均面积为 82 .40μm2 ;催乳激素细胞的平均数量为 6 .0 7× 1 0 8,每个细胞的平均面积为 47.31μm2 。生长激素的血浆含量平均为 2 .84ng/ m L,但个体差异较大 ,变异系数高达 78.5 % ,催乳激素的血浆含量平均为 7.2 6 ng/ m L。本研究结果揭示 :母马脑垂体生长激素细胞和催乳激素细胞的数量和面积并不是决定母马这两种激素血中浓度的唯一重要因素 ;生长激素血中浓度上的个体差异 ,可能与其搏动性分泌形式有关     

Effects of photoperiod on the secretion of growth hormone in female goats

The aim of the present study was to clarify the effect of photoperiod on the secretion of growth hormone (GH) in goats. Adult female goats were kept at 20°C with an 8‐h or 16‐h photoperiod, and secretory patterns of GH for 4 h (12.00 to 16.00 hours) were compared. In addition, the goats were kept under a 16‐h photoperiod and orally administered saline (controls) or melatonin, and the effects of melatonin on the secretion of GH were examined. GH was secreted in a pulsatile manner. There were no significant differences in pulse frequency between the 8‐ and 16‐h photoperiods; however, GH pulse amplitude tended to be greater in the group with the 16‐h photoperiod (P = 0.1), and mean GH concentrations were significantly greater in the 16‐h photoperiod (P < 0.05). The GH‐releasing response to GH‐releasing hormone (GHRH) was also significantly greater for the 16‐h photoperiod (P < 0.05). There were no significant differences in GH pulse frequency between the saline‐ and melatonin‐treated groups. However, GH pulse amplitude and mean GH concentrations were significantly greater in the saline‐treated group (P < 0.05). The present results show that a long photoperiod enhances the secretion of GH, and melatonin modifies GH secretion in female goats.     

细胞因子对牛乳腺上皮细胞体外增殖作用评价

分离纯化了乳腺上皮细胞,对细胞进行角蛋白免疫组化鉴定后,比较不同细胞因子对乳腺上皮细胞生长的影响.结果显示,生长因子EGF或HGF对牛乳腺上皮细胞的增殖具有重要作用,17β-E2不能促进牛乳腺上皮细胞的增殖,但可与EGF协同促进牛乳腺上皮细胞的增殖,表明17β3-E2对EGF诱导的乳腺上皮细胞增殖具有重要作用.     

The preliminary study on the effects of growth hormone and insulin‐like growth factor‐I on κ‐casein synthesis in bovine mammary epithelial cells in vitro

The effects of growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) on protein synthesis and gene expression of κ‐casein in bovine mammary epithelial cell in vitro were studied. The treatments were designed as follows: the growth medium without serum was set as the control group, while the treatments were medium supplemented with GH (100 ng/ml), IGF‐I (100 ng/ml), and GH (100 ng/ml) + IGF‐I (100 ng/ml). The quantity of κ‐casein protein was measured by ELISA, and the κ‐casein gene (CSN3) expression was examined by real‐time quantitative PCR (RT‐qPCR). Compared with the control group, all the experimental groups had greater (p < 0.05) expression of CSN3. The concentration of κ‐casein followed a similar response as CSN3, but the difference between the treatments and the control was not statistically significant (p > 0.05). Furthermore, no synergistic effect of GH and IGF‐I was observed for both the κ‐casein concentration and CSN3 expression. It is therefore concluded that GH or IGF‐I can independently promote the expression of CSN3 in bovine mammary epithelial cells in vitro.     

猪铜蓝蛋白的分离纯化及对垂体细胞分泌生长激素的影响

采用硫酸铵分级沉淀分离,DEAE-sepharose离子交换,羟基磷灰石吸附层析,Superdex-200凝胶过滤纯化,从猪血清中分离纯化出电泳纯的一种蛋白质.进一步研究,其相对分子质量为123 500,等电点为5.08,分子含铜原子数为6个,含糖量为7.8%,亚基数为1,经分析为猪血清铜蓝蛋白.用含不同质量浓度铜蓝蛋白(0、0.1、0.2、0.4、0.8、1.2 g/L)的无血清培养液培养已培养48 h的新生仔猪脑垂体细胞12 h,于2、4、8、12 h分别收集培养液,用放免法测定培养液中GH浓度,结果表明铜蓝蛋白质量浓度为0.2、0.4、0.8、1.2 g/L组垂体细胞生长激素的分泌显著高于0 g/L组,其中以0.4 g/L组、换液培养8 h,垂体细胞生长激素分泌最为明显.     

NMDA对培养的大鼠腺垂体细胞分泌生长激素影响的研究

应用细胞培养、结合放射免疫测定法探讨了 N-甲基 -D-天冬氨酸 (NMDA)对培养的大鼠腺垂体细胞分泌生长激素 (GH)的影响。结果表明 :在腺垂体细胞培养中 ,试验组 (分别添加1 0 - 8、1 0 - 6 、1 0 - 4M NMDA)培养液中的 GH比对照组分别提高了 67.92 % ,87.46% ,1 0 0 .67% ,差异显著 (P <0 .0 5)。试验表明 NM-DA能直接刺激离体腺垂体细胞分泌 GH,并与NMDA呈剂量依赖关系     

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

The present study was carried out to determine whether leptin or leptin (116–130) peptide amide (lep (116–130)), an active fragment of the native protein in rats, is able to stimulate the release of luteinizing hormone (LH), growth hormone (GH) or prolactin (PRL) from cultured porcine anterior pituitary (AP) cells in vitro. The AP cells were obtained from 6 month‐old pigs and were incubated for 3 h with 10^?11?10^?7 mol/L leptin or lep (116–130) after being cultured in Dulbecco's modified Eagle's medium for 3–4 days. Leptin significantly increased the concentration of LH and GH in the culture medium at concentrations of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin did not increase the concentration of PRL in the culture medium. In contrast to these results, no effects of lep (116–130) on the release of LH, GH or PRL were seen in the cultured cells. These results suggest that leptin stimulates the release of LH and GH by acting directly on porcine AP cells, and that a fragment of leptin protein comprising amino acids 116–130 is not associated with the secretion of hormones in pigs.     

The effect of lighting conditions on the rhythmicity of growth hormone secretion in Holstein steers

Growth hormone (GH) secretion regularity and the effects of lighting condition and GH‐releasing hormone (GHRH) on GH release were determined in steers. First, steers were kept under 12:12 L : D conditions (light: 06.00–18.00 hours). The animals were then subjected to a 1‐h advancement in lighting on/off conditions (05.00 and 17.00 hours, respectively). Blood was sampled for 24 h at 1‐h interval on the seventh day of each condition. Second, GHRH was injected intravenously (IV) at 12.00 and 00.00 hours under 12:12 L : D and blood was sampled at 15‐min interval for 4‐h (1 h before and 3 h after the injection). Plasma GH concentrations were measured by a radioimmunoassay. Periodicity of GH secretory profile was calculated by power spectrum analysis using the maximum entropy method. Plasma GH concentrations showed a characteristic pattern consisting of four distinct peaks. Mean periodicity of GH secretory profile was 5.7 h, and it was not altered by any change in lighting conditions. IV injection of GHRH increased GH secretion during the day and night. The increase in GH secretory volume after GHRH injection during the night was equal to that during the day. The present results suggest that GH secreted from the anterior pituitary have regularity in steers.     

Positive effect of silymarin on cell growth and differentiation in bovine and murine mammary cells

Silymarin, a naturally acknowledged hepatoprotector used in humans to treat liver diseases has been tested in murine (HC11) and bovine (BME‐UV) mammary epithelial cell lines to evaluate a possible direct effect on cell growth and differentiation in mammary gland. Silymarin enhanced cell proliferation (p < 0.05) from 10 to 1000 ng/ml in association with growth factors, (up to 20%) or alone (up to 15%) versus controls. Furthermore, silymarin (100 ng/ml) was able to increase (p < 0.05) β‐casein gene expression alone or in association with prolactin (5 μg/ml). These effects may be related with protein kinase B (AKT) activation induced by silymarin treatment (p < 0.05) and/or by a dose‐related inhibitory effect (p < 0.05) on caspase‐3 activity related to a protective role in cell apoptosis. These data suggest that silymarin should be considered a candidate to support mammary gland activity during a lactogenetic state.     

Survey on vertical infection of bovine viral diarrhea virus from fetal bovine sera in the field

Bovine viral diarrhea virus (BVDV) isolation and antibody survey were performed using 2,758 fetal bovine sera (FBS) collected from slaughterhouses in New Zealand, Australia and the Dominican Republic, and then sent to Japan to manufacture commercial serum for cell culture use. FBS in the Dominican Republic were pooled for each several individuals, and those collected in other countries were separated according to each individual and subjected to the tests. BVDV was isolated from 25 (0.91%) FBS, and the BVDV antibody was detected in 44 (1.60%) FBS. The survey on 139 sets of paired sera of a dam and her fetus revealed that neither the BVDV antibody nor BVDV was detected in all FBS from BVDV antibody-positive dams.     

Influence of a bovine respiratory disease vaccine with a temperature‐sensitive modified live or killed infectious bovine rhinotracheitis component on oestrous cycle parameters and anti‐Müllerian hormone concentration in nulliparous heifers

The objective of this study was to examine the impact of a bovine respiratory disease complex (BRDC) vaccine with a temperature‐sensitive modified live vaccine (MLV) infectious bovine rhinotracheitis (IBR) component on oestrous cycle parameters and the follicular pool. Twenty‐four Holstein heifers (12.4 ± 0.5 months) previously calfhood vaccinated with an IBR MLV component were enrolled in two replicates (Spring; n = 10 and Fall; n = 14) and were blocked by pre‐vaccination bovine viral diarrhoea (BVD) serum neutralizing (SN) titres. Upon enrolment, heifers were oestrous synchronized with sampling beginning at detected oestrus. At their second heat, heifers were vaccinated with a BRDC calfhood vaccine with a MLV (MLV; n = 12) or killed (K; n = 12) IBR component and sampled for two additional cycles. Serum samples for oestrogen (E2) and progesterone (P4) as well as ultrasound data of ovarian structures were collected every other day. Serum samples for anti‐Müllerian hormone (AMH) were collected at oestrus and mid‐cycle for each cycle, and serum for titres was collected prior to and following vaccination. Data were analysed with the PROC MIXED and GLM procedures of SAS. There was no difference in pre‐ or post‐vaccination titres between MLV and K heifers (p > .5). Vaccination had no impact on P4 concentrations, P4 area under the curve, luteal tissue area, peak E2 production or oestrous cycle length (p > .05). Cycle number did impact AMH concentration (p < .05). In MLV heifers, AMH concentration was highest in cycle 1 (p < .05) while cycles 2 and 3 did not differ (p > .05). This was also true for the K heifers in the Fall replicate (p < .05). Within cycle 2, AMH concentrations were numerically lower between vaccine types (K = 308.22 ± 33.3 pg/ml, MLV = 181.13 ± 32.9 pg/ml; p > .05). Although no differences were seen in overall cycle parameters, differences in AMH concentrations may indicate a reduction of the follicular pool following vaccination and requires further investigation.     

In vitro growth of bovine oocytes in oocyte-cumulus cell complexes and the effect of follicle stimulating hormone on the growth of oocytes

Several successful in vitro culture experiments have used oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) from early antral follicles (0.5–0.7 mm) for the growth of bovine oocytes. However, in studies related to in vitro oocyte maturation and in vitro embryo production, oocyte-cumulus cell complexes (OCCs) that have no mural granulosa cells have been widely used instead of OCGCs. The purpose of this study was to determine whether cumulus cells alone support oocyte growth. First, OCCs and OCGCs were cultured in vitro for 14 days to compare the integrity of the complexes as well as antrum formation. After 14 days, the diameter and meiotic competence of oocytes in OCCs and OCGCs were examined. Oocytes in OCCs grew fully and acquired meiotic competence similar to OCGCs, whereas antrum formation occurred later in OCCs as compared to OCGCs. Subsequently, the effects of follicle stimulating hormone (FSH) on in vitro growth of OCCs were examined for 14 days. When FSH was added to the culture medium, OCCs formed antrum-like structures one day earlier than those cultured without FSH. Oocytes cultured with 1 mIU/ml FSH grew fully and acquired meiotic competence. In contrast, when oocytes were cultured in media containing high concentrations of FSH, some of the OCCs collapsed and the number of degenerated oocytes increased. In conclusion, bovine oocytes in OCCs grow and acquire meiotic competence similar to OCGCs and, 1 mIU/ml FSH supports the development of OCCs and oocyte growth as observed in our culture system.     

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP), prostaglandin E2 (PGE2) and growth hormone releasing factor (GRF) on the release of growth hormone from cultured bovine anterior pituitary cells in vitro

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on growth hormone (GH) release was compared with that of prostaglandin E₂ (PGE₂) and growth hormone releasing factor (GRF) from cultured bovine anterior pituitary cells in vitro. Both PACAP and PGE₂ stimulated GH release at concentrations as low as 10⁻⁹ and 10⁻⁸ M, respectively, (P<0.01). However, GRF released GH at a concentration as low as 10⁻¹³ M (P<0.01). Percent increases of GH compared with controls were not significantly different among GRF, PACAP, and PGE₂ at 10⁻⁷ M; however, the increases of GH by the 10⁻⁸ M GRF, PACAP and PGE₂ were 196, 118, and 27%, respectively, (P<0.01), and 124, 65, and 1% in the 10⁻⁹ M media, respectively, (P<0.01). When GRF and somatostatin (SS) were added together, the GH releasing effect of GRF was blunted (P<0.01). Similar bluntness were observed in PACAP and PGE₂, when SS was added. The stimulatory effects of GRF and PGE₂ together were similar to that by either GRF or PGE₂ alone. When GRF and PACAP were added together, the GH released by both secretagogues was greater than that by PACAP alone (P<0.01); however, a synergistic effect was not clear when compared with GRF alone.

These findings suggest that PACAP and PGE₂ may modulate the release of GH in cattle.