GnRH-induced Luteinizing Hormone Release Research Articles

Modulatory Effects of Ethinyl Estradiol Plus Drospirenone Contraceptive Pill on Spontaneous and GnRH-Induced LH Secretion

Background: Combined oral contraceptives (COCs) work mostly by preventing the pre-ovulatory gonadotropin surge, but the action of COCs on spontaneous episodic and GnRH (gonadotropin-releasing hormone)-induced LH (luteinizing hormone) release has been poorly evaluated. Oral contraceptives are known to act on the spontaneous hypothalamic–pituitary functions reducing both GnRH and gonadotropin release and blocking ovulation. Aim: To evaluate spontaneous and GnRH-induced LH release during both phases of the menstrual cycle or under the use of the contraceptive pill. Methods: A group of 12 women, subdivided into two groups, volunteered for the study. Group A (n = 6, controls) received no treatments, while Group B (n = 6) received a 21 + 7 combination of ethinyl-estradiol (EE) 30 µg + drospirenone (DRSP) 3 mg. Both groups were evaluated twice: Group A during follicular and luteal phases, Group B during pill assumption and during the suspension interval, performing a pulsatility test, GnRH stimulation test, and hormonal parameters evaluation. Spontaneous and GnRH-induced secretory pulses were evaluated, as well as the instantaneous secretory rate (ISR). Results: COC treatment lowered LH and FSH (follicle stimulating hormone) levels significantly if compared to the follicular phase of spontaneous cycles. During the suspension interval, hormone levels rapidly rose and became comparable to those of the follicular phase of the control group. The LH pulse frequency under COC administration during the suspension interval was similar to that observed during the follicular phase (2.6 ± 0.3 pulses/180 min and 2.3 ± 0.2 pulses/180 min, respectively). The GnRH-induced LH peaks were greater in amplitude and duration than those observed after ISR computation in both groups. The GnRH-induced LH release during the luteal phase of the control subjects was higher than in the follicular phase (51.2 ± 12.3 mIU/mL and 14.9 ± 1.8 mIU/mL, respectively). Conversely, subjects under COC showed a GnRH-induced LH response similar during COC and during the suspension interval. Conclusions: Our data support that the EE + DRSP preparation acts on both spontaneous pulsatile release and GnRH-induced LH release during the withdrawal period of the treatment, and that after 5–7 days from the treatment suspension, steroidal secretion from the ovary is resumed, such as that of androgens. This suggests that in hyperandrogenic patients, a suspension interval as short as 4 days might be clinically better.

Modulation of anterior pituitary cell luteinizing hormone secretory activity by neuropeptide Y in early pregnant pigs.

Numerous reports indicate that neuropeptide Y (NPY) is implicated in the regulation of anterior pituitary (AP) gonadotropin secretion in prepuberal and cyclic females. This study was performed to explain whether NPY directly affects basal and gonadotropin-releasing hormone- (GnRH-) induced luteinizing hormone (LH) secretion and intracellular accumulation by AP cells and whether NPY and its receptor (Y1 and Y2) mRNAs are expressed in the AP gland in pregnant pigs (days 14 - 16). Additionally, the expression of genes encoding β subunit of luteinizing hormone (β-LH) and GnRH receptor (GnRH-R) was determined in porcine AP gland. Anterior pituitaries were dispersed with trypsin and the obtained pituitary cells (2 x 105 cells/ml) were pre-incubated (72 h) and then experimentally treated for 3.5 h with GnRH (100 ng/ml), NPY (10-8, 10-7 and 10-6 M) alone, or in chosen combinations with GnRH. The LH contents in culture media and cellular homogenates were measured by radioimmunoassay. The expression of NPY, Y1 and Y2 NPY receptors, GnRH-R and β-LH mRNAs in the AP gland was determined by the real-time PCR method. NPY (10-8, 10-7 and 10-6 M) had no effect on basal LH secretion or basal and GnRH-induced LH accumulation in AP cells. The lowest dose of NPY (10-8 M) decreased, while higher doses of NPY only showed a tendency to inhibit GnRH-induced release of LH from AP cells. The presence of mRNAs for NPY and its receptor Y2, as well as β-LH and GnRHR was confirmed in the porcine AP gland. These results indicate that NPY and one of its receptors (Y2) are expressed in the porcine anterior pituitary and this neuropeptide might be involved in the modulation of LH secretion by AP cells in pregnant pigs.

A prepartum diet supplemented with oilseeds high in oleic or linoleic acid reduced GnRH-induced LH release in dairy cows during second week postpartum.

BackgroundThe objective was to determine the effect of prepartum diets supplemented with rolled canola seed (high in oleic acid) or sunflower seed (high in linoleic acid) on luteinizing hormone (LH) pulsatility and gonadotropin releasing hormone (GnRH)-induced LH release during early postpartum.MethodsThirty-one pregnant Holstein cows, blocked by body condition score, parity and expected calving date, were assigned to 1 of 3 prepartum diets supplemented with 8 % rolled canola or sunflower seed, or no oilseed (control) during the last 35 d of gestation. Blood samples were collected at Weeks (wk)-3, 0, +1 and +2, relative to calving, to determine non-esterified fatty acids (NEFA), Beta-hydroxy butyric acid (BHBA) and glucose. Additional blood samples were collected during wk1 (n = 5 per treatment) or wk2 (n = 5 or 6 per treatment), for 6 h, to measure LH pulsatility; thereafter, 100 mcg GnRH was administrated i.m., and blood was sampled for 4 h more, to measure GnRH-induced LH release.ResultsDietary treatment did not affect prepartum energy balance, but cows fed the control diet were in a deeper state of negative energy balance during wk2, than those fed canola (P = 0.03) or sunflower (P = 0.01). Prepartum diets did not influence the mean plasma concentration of BHBA and glucose. However, NEFA concentration during wk2 was greater in control cows than those fed sunflower (P = 0.03) or canola (P = 0.07). Prepartum diets did not affect LH pulsatility (i.e. mean, minimum, maximum concentration, pulse frequency, and amplitude during wk1 and 2). GnRH-induced LH release did not differ among dietary treatments during wk1 but the mean GnRH-induced LH release during wk2 was either greater (P = 0.02) and tended to be greater (P = 0.09) in control cows than in those fed canola and sunflower, respectively.ConclusionsPrepartum diets did not affect LH pulsatility and GnRH-induced LH release during the first week postpartum, but cows fed a diet supplemented with oilseeds high in oleic or linoleic acid released less LH than control cows, in response to an exogenous GnRH challenge during the second week postpartum.

The non-steroidal mycoestrogen zeranol suppresses luteinizing hormone secretion from the anterior pituitary of cattle via the estradiol receptor GPR30 in a rapid, non-genomic manner

Picomolar concentrations of estradiol produce rapid suppression of GnRH-induced luteinizing hormone (LH) secretion from the anterior pituitary (AP) of cattle via G-protein-coupled receptor 30 (GPR30). Zeranol is a strong estrogenic metabolite derived from zearalenone, a non-steroidal mycoestrogen produced by Fusarium that induces reproductive disorders in domestic animals. The hypothesis was tested that zeranol suppresses GnRH-induced LH release from the AP of cattle via GPR30 in a rapid, non-genomic manner. The AP cells (n=15) were cultured for 3 days in steroid-free conditions and then treated them with estradiol (0.001–10nM) or zeranol (0.001–100nM) for 5min before GnRH stimulation. Pre-treatment with 0.001–0.1nM estradiol suppressed GnRH-stimulated LH secretion. Pre-treatment with zeranol at concentrations of 0.001nM (P<0.01), 0.01nM (P<0.01), 0.1nM (P<0.05), and 1nM (P<0.05), but not at concentrations of 10 and 100nM, also inhibited GnRH-stimulated LH secretion from AP cells. Pre-treatment for 5min with a GPR30-specific antagonist, G36, inhibited estradiol or zeranol suppression of LH secretion from cultured AP cells. Cyclic AMP measurements and quantitative PCR analyses revealed that pre-treatment with small amounts of estradiol (P<0.05) or zeranol (P<0.01) decreased cAMP, but not gene expressions of the LHα, LHβ, or FSHβ subunits in the AP cells. Hence, zeranol may suppress luteinizing hormone secretion from the AP of cattle via GPR30 in a rapid, non-genomic manner.

Concentrations of luteinizing hormone and ovulatory responses in dairy cows before timed artificial insemination (2013)

The objective of this study was to determine the incidence of spontaneous and gonadotropinreleasing hormone (GnRH)-induced surges of luteinizing hormone (LH) and ovulatory responses in lactating dairy cows enrolled in a timed artificial insemination (TAI) protocol. Cows (n = 70) in a single herd were assigned to one of two presynchronization protocols: Pre- 10 or PG-3-G. Cows assigned to the Pre-10 treatment received 2 injections of prostaglandin F2α(PGF2α) 14 days apart (Presynch), with the second injection administered 10 days before the onset of a TAI protocol. Cows assigned to the PG-3-G treatment received an injection of prostaglandin F2α(PGF2α), then 3 days later an injection of GnRH (GnRH-1) 7 days before the onset of a TAI protocol. All cows received the first GnRH injection (GnRH-2) of the Ovsynch protocol and a PGF2αinjection 7 days later, then cows received the breeding injection of GnRH (GnRH-3) at either 56 or 72 hours after PGF2α, with insemination occurring 12 to 16 hours after the second GnRH injection. Blood samples were collected during the study to monitor serum changes in LH, progesterone, and estradiol to determine why ovulatory responses to GnRH-induced LH release did not approach 100% when follicle dominance and adequate follicle size was achieved. Presynchronization administration of GnRH-1 increased the incidence of LH surges and ovulation rates in cows presynchronized with PG-3-G compared with Pre-10. Incidence of ovulation and occurrence of LH surges did not differ after GnRH-2, but more LH was released in Pre-10 than PG-3-G cows. Luteolysis, LH surge incidence, and ovulation rates were similar among the 4 treatment-time combinations after GnRH-3. Pregnancy per TAI was decreased in Pre-10 at 56 hours compared with Pre-10 at 72 hours and PG-3-G at 56 and 72 hours. We concluded that administration of GnRH 56 hours before breeding decreased pregnancy per TAI compared with administration of GnRH at 72 hours when cows were presynchronized with Pre-10. Presynchronization with PG-3-G resulted in acceptable pregnancy per TAI with GnRH administration occurring at either 56 or 72 hours before TAI. The flexibility of GnRH timing with the PG-3-G presynchronization protocol may be an advantage compared with the Pre-10 protocol for dairy cattle when programmed for a TAI at first service.; Dairy Day, 2013, Kansas State University, Manhattan, KS, 2013; Dairy Research, 2013 is known as Dairy Day, 2013

Effect of suckling on basal and GnRH-induced LH release in post-partum dairy buffaloes

Suckling, a common practice in smallholder dairy-farming systems in the developing world, delays the onset of post-partum ovarian activity in dairy buffalo. The present study was designed to assess the effect of suckling on pituitary function in lactating buffaloes 25–35 days post-partum. Six suckled and nine non-suckled buffaloes were challenged intravenously with a bolus injection of GnRH (20 μg buserelin acetate; Receptal). Heparinized venous blood samples were collected at 15 min intervals for 2 h before and up to 4 h after GnRH for luteinizing hormone (LH) estimation. Pretreatment basal LH concentrations were similar in the suckled (0.6 ± 0.2 ng/ml) and the non-suckled (0.5 ± 0.1 ng/ml) buffaloes. All but one suckled buffaloes released a LH surge, starting 15–60 min post-GnRH treatment, which lasted for 180–225 min. While one suckled buffalo did not respond to GnRH, the LH response in the remaining suckled buffaloes was significantly less than in the non-suckled buffaloes in terms of peak LH concentrations (14.3 ± 2.7 ng/ml versus 26.2 ± 4.3 ng/ml) and area under the LH curve (1575.6 ± 197.4 mm 2 versus 2108.9 ± 323.9 mm 2). The LH response was least in suckled buffaloes challenged with GnRH while in the luteal phase of an oestrus cycle and with plasma progesterone concentration >1 ng/ml. In conclusion, suckling suppressed pituitary responsiveness to exogenous GnRH challenge in post-partum buffaloes.

Role of Ca 2+ in the secretory and biosynthetic response of porcine gonadotropes to substance P and gonadotropin-releasing hormone

Substance P has been previously shown to stimulate luteinizing hormone (LH) secretion and synergistically enhance gonadotropin-releasing hormone (GnRH)-evoked LH release from cultured pig pituitary cells. To investigate the mechanisms involved in these responses, the effects of substance P (100 nM; 4 h) and/or GnRH (10 nM, 4 h) on LH release, LH intracellular content, and βLH mRNA accumulation were evaluated in the absence or presence of extracellular Ca 2+. Likewise, the effects of substance P on the dynamics of cytosolic free Ca 2+ concentration ([Ca 2+] i) were examined in single cells. Extracellular Ca 2+ deprivation abolished both substance P- and GnRH-stimulated LH release, as well as their synergistic interaction. The substance P antagonist d-Arg1, d-Phe5,-Trp7,9,Leu11-substance P (100 nM) blocked the stimulatory effect of substance P on LH release and its interaction with GnRH without affecting GnRH-induced LH secretion. Whereas substance P did not modify βLH transcript levels, GnRH stimulated βLH mRNA accumulation through a mechanism dependent upon extracellular Ca 2+. Substance P directly increased [Ca 2+] i in a 30% of gonadotropes by causing two distinct types of response kinetics with single-peak (predominant, 83.3%) or sustained-plateau profiles. Reduction of external [Ca 2+] decreased by half the percent of responsive cells, which only showed single-peak profiles. Taken together, our results demonstrate that the ability of substance P to stimulate basal and GnRH-induced LH release is exerted directly upon gonadotropes, is extracellular Ca 2+-dependent and does not seem to require net increases in βLH mRNA levels. Moreover, [Ca 2+] i measurements revealed that although substance P action in pig gonadotropes is strongly dependent on extracellular Ca 2+ influx, it would also involve intracellular Ca 2+ mobilization. Finally, extracellular Ca 2+ also plays a requisite role to sustain GnRH-stimulated increases in both βLH mRNA and LH release.

Interaction between anterior pituitary prodynorphin and the secretion of luteinizing hormone (LH)

The purpose of present research was to investigate the possible involvement of prodynorphin (proDYN)-derived peptides acting locally within the anterior pituitary (AP) on the effects of estradiol-17 β (E2) and gonadotropin-releasing hormone (GnRH) on the release of luteinizing hormone (LH). Exposure of bovine AP cells in primary suspension cultures to E2 increased ( P<0.05) the spontaneous release of proDYN-derived peptides and also augmented ( P<0.05) the GnRH-induced release of LH. Both of these E2-induced responses required either high E2 dosages or prolonged exposure to produce significant changes, but there were a few cases in which the association between E2-induced changes in both parameters was absent. Therefore, it seems unlikely that proDYN-derived AP peptides mediate the effects of E2 on GnRH-induced LH release. Using another approach, cultured cells were exposed for 48 h to an antisense oligodeoxynucleotide (oligo) targeted against the translation initiation site of bovine proDYN. Compared with the two control treatments (scrambled oligo sequence or no oligo treatment), the antisense treatment decreased ( P<0.05) the quantity of LH released in response to challenge of the cells with 5 nM GnRH. There were no concurrent changes in cellular contents of proDYN-derived peptides or mRNA for LH- β, but the antisense treatment tended to decrease ( P<0.10) the relative abundance of proDYN mRNA. In summary, proDYN-derived peptides probably do not mediate direct intrapituitary effects of E2 on LH, but the antisense treatment interfered in an unknown way with GnRH-induced release of LH from cultured AP cells.

Enhancement by Prolactin of the GnRH-Induced Release of LH from Dispersed Anterior Pituitary Cells of the Bullfrog ( Rana catesbeiana)

The response of enzymatically dispersed anterior pituitary cells of the bullfrog ( Rana catesbeiana) to gonadotropin-releasing hormone (GnRH) was studied by monitoring the release of luteinizing hormone (LH) into the culture medium. The cells responded to GnRH by releasing LH according to the incubation time and to the GnRH concentration. The responsiveness to GnRH became less conspicuous as the cell density was reduced. Addition of prolactin (PRL) to the medium enhanced the responsiveness to the secretagogue, and addition of antiserum against PRL lowered the responsiveness to a certain extent. Immunohistochemical studies of sectioned pituitaries revealed that PRL cells most frequently located in contact with LH cells. The possibility that PRL acts directly on gonadotrophs to enhance their responsiveness to GnRH was suggested.

Inhibitory effect of leukotrienes on luteinizing hormone release

The aim of (his work was to study the effect of high concentrations of leukotrienes on luteinizing hormone (LH) secretion in rat anterior pituitary cells. We also investigated the effect of leukotrienes in parallel with gonadotropin-releasing hormone (GnRH) action. Experiments were on cells gained from trypsinized pituitaries of female rats. Tests were performed by superfusion of the cells attached to cytodex-1 carrier beads. The LH content in samples of perfusate was estimated by radioimmunoassay. This work reports 48% inhibition of basic LH release by action of leukotriene C4 in superfused cells when applied continuously at a concentration of WO nmol/l. Moreover, we have shown that leukotrienes suppressed GnRH-induced LH secretion in rat pituitary cells when applied in parallel to GnRH (1 nmol/l) as a 4-min pulse at a concentration of 0.1 nmol/l. GnRH-induced LH release was reduced to 66% of its value by leukotriene (LT) B4 (0.1 nmol/l) action; also to 54% by LTC4, 66% by LTD4 and 74% by LTE4 action. In contrast, arachidonic acid (50 pmol/l) and its other 5-lipoxy-genase metabolites: 5-hydroperoxyeicosatetraenoic acid (5-HPETE) (50 pmol/l), or 5-hydroxyeicosatetraenoic acid (5-HETE) (50 pmol/l), had no inhibitory effect on GnRH-induced LH release. Arachidonic acid and 5-HETE potentiated GnRH-induced LH release up to 249% and 429%, respectively, when applied in parallel with GnRH (1 nmol/l) as a 4-min pulse at a concentration of 10 pmol/l. In our earlier work we have shown that several leukotrienes are potent stimulants of LH release. The present report documents the finding that the 5-lipoxygenase pathway is also involved in the inhibitory regulation of hormone release in anterior pituitary cells.

Melatonin inhibition of GnRH-induced LH release from neonatal rat gonadotroph: involvement of Ca2+ not cAMP

Melatonin inhibits gonadotropin-releasing hormone-induced release of luteinizing hormone (LH) from the neonatal rat gonadotrophs. The second messenger involved is not known, although there are several candidates, including adenosine 3',5'-cyclic monophosphate (cAMP) and intracellular free Ca2+. The present study addresses the question of which second messenger mediates melatonin inhibition of LH release. We found that the effect of melatonin was not prevented by cAMP protagonists, including 8-bromo-cAMP, dibutyryl cAMP, 3-isobutyl-1-methylxanthine, and forskolin. However, treatments that enhanced Ca2+ influx masked the effects of melatonin, and treatments that blocked Ca2+ influx mimicked the effects of melatonin. Moreover, melatonin decreased K(+)-induced LH release, which is dependent on Ca2+ influx but did not block release of LH due to thapsigargin-induced mobilization of Ca2+ from intracellular stores. These findings indicate that melatonin inhibits gonadotropin-releasing hormone-induced LH release, primarily through an action involving inhibition of Ca2+ influx, and that cAMP does not seem to be involved in this effect of melatonin.

Involvement of protein phosphatases in gonadotropin releasing hormone regulated gonadotropin secretion

The role of persistent protein phosphorylation upon gonadotropin releasing hormone (GnRH) stimulated luteinizing hormone (LH) release was investigated by the use of the selective inhibitors of protein phosphatase type 1 (PP1) and 2A (PP2A), okadaic acid (OA) and calyculin A. Pre-incubation of cultured rat pituitary cells with OA (24 h) or calyculin A (30 min) resulted in inhibition of GnRH-stimulated LH release with significant inhibition being detected at 10 nM and 30 nM for OA and calyculin A, respectively. The inactive OA analog norokadone and the protein tyrosine phosphatase inhibitor vanadyl hydroperoxide had no significant effect on GnRH-induced LH release. The stimulatory effects of the protein kinase C (PKC) activator 12- O-tetradecanoylphorbol 13-acetate (TPA, 50 ng/ml) or the Ca 2+ ionophore, ionomycin (1 μm), upon LH release were also abolished by pretreatment with OA (10–20 nM) or calyculin A (30nM). Stimulation of LH release by high K + (28 mM) or residual LH release stimulated by GnRH in Ca 2+-free medium were also blocked by OA. These observations indicate that protein dephosphorylation is involved positively in GnRH-stimulated LH release. The site of action of the protein phosphatases PPI and PP2A is most likely downstream to Ca 2+ elevation and PKC activation by GnRH.

Cellular Mechanism of Melatonin Action in Neonatal Rat Pituitary

Melatonin inhibits gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) release from neonatal rat anterior pituitary. Melatonin has been shown to decrease the concentration of several second messengers in neonatal pituitary, but it is not known which of them transduces the melatonin effect on LH release. In order to determine the mechanism of melatonin action, we tested the effect of melatonin on GnRH-induced LH release in the presence of specific drugs affecting second messengers. The calcium channel antagonist nifedipine inhibited LH release from cultured pituitary to a similar degree as melatonin and prevented the inhibitory effect of melatonin on LH release. The calcium channel agonist Bay K potentiated the GnRH-stimulated LH release and reduced the inhibitory effect of melatonin on LH release. These data strongly suggest that melatonin inhibits LH release via inhibition of calcium influx through voltage-sensitive channels. The cyclic AMP (cAMP) derivative 8-bromo-cAMP potentiated GnRH stimulation of LH release but did not prevent the melatonin-induced inhibition of LH release. However, when used in combination with Bay K, which reduced only partially the melatonin effect by itself, 8-bromo-cAMP completely blocked the melatonin effect. This observation suggests that decreased cAMP accumulation may also be involved in transduction of the melatonin effect on LH release.

Influence of folliculo-stellate cells on biphasic luteinizing hormone secretion response to gonadotropin-releasing hormone in rat pituitary cell aggregates.

Anterior pituitary cells cultured as three-dimensional cell aggregates and incubated with gonadotropin-releasing hormone (GnRH) show a biphasic pattern of luteinizing hormone (LH) release when steroid-free bovine follicle fluid is added to the culture medium. Initially, the GnRH-induced LH release is low (lag-phase response), but LH release increases during further incubations with GnRH (primed-state response). Also, in aggregates of dispersed cells from long-term ovariectomized rats cultured for 2 days in the presence of 1% bovine follicle fluid, a low initial LH responsiveness to GnRH could be restored. Cycloheximide was found to block the induction of the primed state, indicating the protein synthesis dependency of GnRH self-priming. In aggregates from gonadotroph-enriched cell populations obtained by velocity sedimentation in a bovine serum albumin gradient, addition of 1% bovine follicle fluid to the culture medium also restored a biphasic pattern of GnRH-induced LH release. However, co-culturing the gonadotroph-enriched cell aggregates with a folliculo-stellate (FS) cell-enriched population resulted in the attenuation of the differences in LH secretion rate between early and late responses to GnRH. The present example of the attenuation by folliculo-stellate cells of pituitary hormone secretion responses demonstrates that the cells regulate the cellular processes leading to a priming of the LH response to GnRH, rather than interfering with the access of GnRH to its receptor in gonadotrophs. Finally, it was found that stimulation of the adenylate cyclase enzyme with maximal effective doses of forskolin counteracted the inhibitory effect of bovine follicle fluid on the initial LH response to GnRH, but did not completely abolish the biphasic pattern of LH release. It is concluded that coupling to the adenylate cyclase enzyme is presumably involved in the LH surge inhibiting feedback action on the pituitary cells, but also other messenger pathways and intercellular interactions between pituitary cells may play a role in establishing a biphasic LH release at the pituitary level following GnRH administration.

Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells.

The purpose of the present experiments was to examine the short- and long-term effects of estradiol-17 beta (E2), progesterone (P), and 5 alpha-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 h in steroid-free medium, pituitary cells were further cultured for 24 h in medium with or without E2 (1 nM), P (100 nM), or DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of 1 nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show that the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E2 resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E2. Conversely, P caused an acute stimulatory action (118%) on the LH released in response to GnRH and a slightly inhibitory effect (90%) after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) LH were observed in pituitary cells treated with E2 or DHT.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in luteinizing hormone concentrations after abortion, parturition, and weaning in primiparous sows

This study compared the endocrine changes associated with the capacity of gilts to return to estrus following mid-gestation abortion, removal of piglets at birth (zero weaned), during lactation, and following the removal of piglets after lactation (weaned). Serum concentrations of luteinizing hormone (LH), estradiol-17β (E 2), and progesterone (P 4) were determined in aborted gilts (Group 1; n = 5), zero-weaned gilts (Group 2; n = 6), and gilts (Group 3; n = 6) weaned after a 3 week lactation. In addition, postpartum endocrine changes were compared between Group 2 and Group 3 gilts during lactation. The day of abortion, parturition and weaning was considered Day 0. Blood samples were collected at 6 h intervals from Day —2 to Day 21 and at 15-min intervals for 8 h on Days —2, 1, 4, 7, 10 and 14. Following each 8 h sampling period, gilts were treated i.v. with gonadotropin releasing hormone (GnRH) at 0.5 μg kg −1 body weight (BW) and blood samples collected at 10-min intervals for 3 h. Estrus was detected using twice daily boar exposure. The number of gilts exhibiting estrus were four, four and one at 4.5 ± 0.3 days, 14.8 ± 0.5 days and 9 days for aborted, zero-weaned, and weaned groups, respectively. Gilts exhibited increasing LH concentrations with time following abortion, while GnRH-induced LH release was not affected by group or time. Changes in E 2 and P 4 concentrations reflected the endocrine changes associated with gestation or the return to estrus for each group. In addition, LH, E 2 and P 4 concentrations were similar in lactating and zero-weaned gilts for 7 days following parturition. It was evident that gilts aborted during mid-gestation have an increased ability to return to estrus than zero-weaned gilts, and that pituitary responsiveness to exogenous GnRH did not differ between groups nor was affected by the termination of pregnancy or lactation.

Effects of charcoal-extracted follicular fluid on reproductive function in postpartum cows

In order to determine the role of follicle-stimulating hormone (FSH) on the resumption of ovarian function in cows early postpartum (PP), bovine follicular fluid (FF) was used to selectively suppress concentrations of FSH. Calves were removed from all cows within 24 hr of birth. Follicular fluid that was treated with charcoal to remove steroids (15 ml; n=14) or serum (S) from an ovariectomized cow (15 ml, n=14) was injected i.m. twice daily from days 1 to 10 PP. Blood samples were collected before each injection and frequent samples (every 15 min for 6 hr) taken on days 5 and 10 PP. Eight cows from each group (FF and S) were slaughtered on the morning of day 11 PP and pituitaries and ovaries collected. The remaining cows (n=6) were observed for estrus. Treatment with FF delayed follicular growth (P<0.01), as evidenced by the largest follicle per cow observed at time of slaughter (3.6 ± 0.42 vs 11.5 ± 1.77 mm dia; FF vs S). The intervals from parturition to first estrus (P<0.11) and to first progesterone rise (25.3 ± 1.97 vs 18.0 ± 3.62 d; P<0.06) tended to be delayed by treatment with FF vs S. Many of the cows treated with S ovulated by day 10 PP, they were divided retrospectively into those that had ovulated by (n=9) or after (n=5) day 10 PP for analysis. Cows treated with FF had lower (P<0.05) and less variable (P<0.01) serum FSH concentrations while levels of luteinizing hormone (LH) tended (P<0.08) to be greater on days 5 and 10 PP. Follicular fluid decreased levels of FSH (P<0.001), but not LH (P>0.15), in the samples obtained twice daily compared to S-treated cows that did not ovulate by day 10 PP. Anterior pituitaries were dissociated, and cells from each cow were cultured in order to ascertain whether treatment with FF in vivo would affect gonadotropin secretion in vitro. Estradiol-17β (E) was incubated with pituitary cells to determine the effect of E on gonadotropin secretion from cells of PP cows, and to ascertain whether treatment with FF in vivo and with E in vitro would interact to affect secretion of FSH and LH in culture. After 2 d of incubation, cells were treated with 10 −9 M E or vehicle (1% ethanol). Two days later, cells were treated with gonadotropin-releasing hormone (GnRH) and after 6 hr, medium collected. Injections of FF did not affect GnRH-induced release of FSH or LH from pituitary cells in culture (P>0.20). Incubation with E decreased the release of LH in response to GnRH (P<0.005). There was a greater suppression of GnRH-induced LH release due to E pretreatment in cells from FF-treated cows than in cells from cows treated with S (P<0.03). Thus, treatment with FF decreased FSH concentrations and delayed follicular development and ovulation, even in the presence of elevated or control levels of LH. Therefore, FSH does play a role in folliculogenesis in PP cows. Based on results obtained in vitro, we conclude that E inhibited LH secretion and this phenomenon may be inherent to the PP animal. Even though FF treatment in vivo did not significantly decrease the secretion of FSH or LH in vitro, it did synergize with E resulting in a much greater suppression of LH than with either E or FF alone.

Mechanisms of secretory responses to gonadotropin-releasing hormone and phorbol esters in cultured pituitary cells. Participation of protein kinase C and extracellular calcium mobilization.

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of gonadotropin releasing hormone (GnRH) and phorbol esters in cultured pituitary cells. During incubation in normal medium, GnRH stimulated LH release with an ED50 of 0.35 nM. Incubation in Ca2+-deficient medium (Ca2+-free, 10 microM) substantially decreased but did not abolish the LH responses to GnRH. The extracellular Ca2+-dependent component of GnRH action could be mimicked by high K+ concentrations, consistent with the presence of voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs. Ca2+ channel agonist (Bay K 8644) and antagonist (nifedipine) analogs, respectively, enhanced or partially inhibited LH responses to GnRH and also to K+, the latter confirming the participation of two types of VSCC (dihydropyridine-sensitive and -insensitive) in K+-induced secretion. Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), 4 beta-phorbol-12,13-dibenzoate, and 4 beta-phorbol-12,13-diacetate, stimulated LH release with ED50s of 5, 10, and 1000 nM, respectively, and with about 70% of the efficacy of GnRH. Phorbol ester-stimulated LH secretion was decreased but not abolished by progressive reduction of [Ca2+]e in the incubation medium, and the residual LH response was identical with that elicited by GnRH in Ca2+-deficient medium. TPA increased [Ca2+]i to a peak after 20 s in normal medium but not in the absence of extracellular Ca2+, indicating that protein kinase C (Ca2+/phospholipid-dependent enzyme) promotes calcium entry but can also mediate secretory responses without changes in calcium influx and [Ca2+]i. The extracellular Ca2+-dependent action of TPA on LH release was blocked by Co2+. However, nifedipine did not alter TPA action on [Ca2+]i and LH release. These observations indicate that protein kinase C can participate in GnRH-induced LH release that is independent of Ca2+ entry, but also promotes the influx of extracellular Ca2+ through dihydropyridine-insensitive Ca2+-channels.

Role of Ca2+ and Na+ on luteinizing hormone release from the calf pituitary.

It has been proposed that gonadotropin-releasing hormone (GnRH) stimulates Ca2+ entry by activation of voltage-independent, receptor-mediated Ca2+ channels in the rat gonadotroph. Little work has been done on the role of calcium in GnRH-induced luteinizing hormone (LH) release in species other than the rat. Therefore, this study was done to compare the effects of agents that alter Ca2+ or Na+ entry on LH release from calf anterior pituitary primary cells in culture. GnRH (100 ng/ml), Ca2+ ionophore A23187 (2.5 microM), and the depolarizing agent ouabain (0.1-10 microM) all produced significant increases (P less than 0.05) in LH release; these effects were significantly reduced when the cells were preincubated with the organic Ca2+ channel blockers nifedipine (1-10 microM) and verapamil (1-10 microM) and with Co2+ (0.01-1 mM). The effect of ouabain was inhibited by tetrodotoxin (TTX; 1-10 nM) as well as by nifedipine at 0.1-10 microM. In contrast to its effect on rat pituitary LH release, TTX significantly inhibited GnRH-stimulated LH release at 1-100 nM. These results suggest that GnRH-induced LH release may employ Ca2+ as a second messenger in bovine gonadotrophs and support recent speculation that GnRH-induced Ca2+ mobilization may in part be voltage dependent.

Effect of cortisol or adrenocorticotropic hormone on luteinizing hormone secretion by pig pituitary cells in vitro

The effect of cortisol or adrenocorticotropic hormone (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) was studied in vitro using dispersed pig pituitary cells. Pig pituitary cells were dispersed with collagenase and DNAase and then grown in McCoy's 5a medium containing 10% dextran charcoal-pretreated horse serum and 2.5% fetal calf serum for 3 days. Cells were preincubated with cortisol or ACTH before GnRH was added. When pituitary cells were incubated with 400 μg cortisol/ml medium for 6 h or longer, increase basal secretion of LH was observed. However, GnRH-induced LH release was reduced by cortisol. The degree of this reduction was dependent on cortisol, and a concentration of cortisol higher than 100 μg/ml was needed. Cortisol also inhibited the 17β-estradiol-induced increase in GnRH response. ACTH-(1–24), ACTH-(1–39), or porcine ACTH had no influence on GnRH-induced LH secretion. Our results show that cortisol can act directly on pig pituitary to inhibit both normal and estradiol-sensitized LH responsiveness to GnRH.