GnRH-induced Luteinizing Hormone Research Articles

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.

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.

Luteal phase dynamics of follicle‐stimulating and luteinizing hormones in obese and normal weight women

Female obesity is a state of relative hypogonadotrophic hypogonadism. The aim of this study is to examine gonadotrophin secretion and response to gonadotrophin-releasing hormone (GnRH) in the luteal phase of the menstrual cycle and to investigate the pharmacodynamics and pharmacokinetics of endogenous and exogenous luteinizing hormone (LH) in obese women. Participants underwent a luteal phase frequent blood sampling study. Endogenous LH pulsatility was observed, gonadotrophin-releasing hormone (GnRH) was given in two weight-based doses, and GnRH antagonist was administered followed by recombinant LH. Regularly menstruating obese (n = 10) and normal weight (n = 10) women. Endogenous hypothalamic-pituitary function (as measured by LH pulsatility), pituitary sensitivity (GnRH-induced LH secretion), pharmacodynamics of endogenous LH and pharmacokinetics of exogenous LH were compared between the obese and normal weight groups. There were no statistically significant differences in endogenous LH pulsatility or pituitary responses to two weight-based doses of GnRH between the obese and normal weight women. There were no differences in the pharmacodynamics of endogenous LH or the pharmacokinetics of exogenous LH between the groups. FSH dynamics did not differ between the groups throughout the study. The relative hypogonadotrophic hypogonadism of obesity cannot be explained by differences in LH and FSH luteal phase dynamics or differences in endogenous LH pharmacodynamics or exogenous LH pharmacokinetics.

Myo‐inositol modulates insulin and luteinizing hormone secretion in normal weight patients with polycystic ovary syndrome

To investigate hormonal dynamics in a group of non-obese polycystic ovary syndrome (PCOS) patients under myo-inositol (MYO) administration. Hormonal profiles, insulin response to oral glucose tolerance test (OGTT) and luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) stimulation test before and after the administration of a preparation of MYO (3 g p.o. daily) mixed with lactoferrin and bromelin, in a group (n = 24) of normal weight PCOS patients. After the treatment interval, body mass index (BMI) did not change while LH, LH/follicle-stimulating hormone, 17-hydroxy-progesterone and androstenedione decreased significantly. Insulin response to OGTT was significantly reduced after the treatment interval (P < 0.05) as well as GnRH-induced LH response (P < 0.05). High-sensitivity C-reactive protein decreased significantly after the treatment interval. MYO administration positively modulates insulin sensitivity in non-obese PCOS patients without compensatory hyperinsulinemia, improving hormonal parameters. The presence of bromelin in the formulation modulated the pro-inflammatory state that characterizes PCOS, independently of BMI.

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.

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

Estriol administration modulates luteinizing hormone secretion in women with functional hypothalamic amenorrhea

To evaluate the influence of estriol administration on the hypothalamus-pituitary function and gonadotropins secretion in patients affected by functional hypothalamic amenorrhea (FHA). Controlled clinical study. Patients with FHA in a clinical research environment. Twelve hypogonadotropic patients affected by FHA. Pulsatility study of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and a gonadotropin-releasing hormone (GnRH) test (10 μg in bolus) at baseline condition and after 8 weeks of therapy with 2 mg/day of estriol. Measurements of plasma LH, FSH, estradiol (E(2)), androstenedione (A), 17α-hydroxyprogesterone (17-OHP), cortisol, androstenedione (A), testosterone (T), thyroid-stimulating hormone (TSH), free triiodothyronine (fT(3)), free thyroxine (fT(4)), and insulin, and pulse detection. After treatment, the FHA patients showed a statistically significant increase of LH plasma levels (from 0.7 ± 0.1 mIU/mL to 3.5 ± 0.3 mIU/mL) and a statistically significant increase of LH pulse amplitude with no changes in LH pulse frequency. In addition, the LH response to the GnRH bolus was a statistically significant increase. Estriol administration induced the increase of LH plasma levels in FHA and improved GnRH-induced LH secretion. These findings suggest that estriol administration modulates the neuroendocrine control of the hypothalamus-pituitary unit and induces the recovery of LH synthesis and secretion in hypogonadotropic patients with FHA.

In utero and lactational exposure to PCB 118 and PCB 153 alter ovarian follicular dynamics and GnRH‐induced luteinizing hormone secretion in female lambs

The effects of in utero and lactational exposure to two structurally different polychlorinated biphenyl (PCB) congeners on follicular dynamics and the pituitary-gonadal axis in female lambs were investigated. Pregnant ewes received corn oil, PCB 118, or PCB 153, and offspring was maintained until 60 days postpartum. Ovarian follicles were quantified using stereology. Plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured using radioimmunoassay before and after administration of a gonadotropin releasing hormone (GnRH) analog. PCB 118 exposure increased numbers of transitional, secondary, and the sum of secondary, early antral, and antral (Σsecondary-antral) follicles, PCB 153 exposure only increased the number of primary follicles. GnRH-induced LH levels were significantly elevated in the PCB 153 exposure group. We conclude that PCB 153 and PCB 118 alter follicular dynamics in lambs and modulate the responsiveness of the pituitary gland to GnRH.

Induction of mPer1 expression by GnRH in pituitary gonadotrope cells involves EGR-1

We reported earlier that gonadotropin-releasing hormone (GnRH) activates period1 ( mPer1) gene expression in immortalized gonadotropes through protein kinase C and p42/44 mitogen-activated protein kinase pathways. GnRH stimulation also leads to the upregulation of early growth response protein 1 (EGR-1), a critical transcription factor for GnRH-induced luteinizing hormone beta (LHβ) synthesis. The parallels between the GnRH–LHβ and the GnRH–mPer1 pathways led us to explore whether EGR-1 is involved in the regulation of mPer1 expression in gonadotropes. Of particular interest was the presence of an EGR-1 binding site in the proximal promoter of the mPer1 gene. Stimulation of LβT2 gonadotrope cells with a GnRH agonist caused the rapid induction of Egr-1 mRNA, which was rapidly followed by mPer1 expression. Chromatin immunoprecipitation revealed that the mPer1 promoter can bind EGR-1, while site-directed mutagenesis experiments confirmed the involvement of Egr-1 sequences in maintaining basal and allowing GnRH-stimulated mPer1 transcription. By means of RNA interference experiments, it could also be demonstrated that silencing of Egr-1 expression resulted in markedly lower mPer1 transcript levels. This silencing effect of the Egr-1 siRNA could be rescued by transfecting the cells with an EGR-1 overexpression vector. In summary, these results all point to a role for the EGR-1 protein in transactivating both the LHβ as well as the mPer1 gene in pituitary gonadotrope cells.

Neonatal Exposure to Bisphenol A Alters Reproductive Parameters and Gonadotropin Releasing Hormone Signaling in Female Rats

BackgroundBisphenol A (BPA) is a component of polycarbonate plastics, epoxy resins, and polystyrene and is found in many products. Several reports have revealed potent in vivo effects, because BPA acts as an estrogen agonist and/or antagonist and as an androgen and thyroid hormone antagonist.ObjectivesWe analyzed the effects of neonatal exposure to BPA on the reproductive axis of female Sprague-Dawley rats.MethodsFemale rats were injected subcutaneusly, daily, from postnatal day 1 (PND1) to PND10 with BPA [500 μg/50 μL (high) or 50 μg/50 μL (low)] in castor oil or with castor oil vehicle alone. We studied body weight and age at vaginal opening, estrous cycles, and pituitary hormone release in vivo and in vitro, as well as gonadotropin-releasing hormone (GnRH) pulsatility at PND13 and in adults. We also analyzed two GnRH-activated signaling pathways in the adults: inositol-triphosphate (IP3), and extracellular signal-regulated kinase1/2 (ERK1/2).ResultsExposure to BPA altered pituitary function in infantile rats, lowering basal and GnRH-induced luteinizing hormone (LH) and increasing GnRH pulsatility. BPA dose-dependently accelerated puberty onset and altered estrous cyclicity, with the high dose causing permanent estrus. In adults treated neonatally with BPA, GnRH-induced LH secretion in vivo was decreased and GnRH pulsatility remained disrupted. In vitro, pituitary cells from animals treated with BPA showed lower basal LH and dose-dependently affected GnRH-induced IP3 formation; the high dose also impaired GnRH-induced LH secretion. Both doses altered ERK1/2 activation.ConclusionsNeonatal exposure to BPA altered reproductive parameters and hypothalamic–pituitary function in female rats. To our knowledge, these results demonstrate for the first time that neonatal in vivo BPA permanently affects GnRH pulsatility and pituitary GnRH signaling.

Effect of adlay (Coix lachryma‐jobi L. var. ma‐yuen Stapf.) hull extracts on testosterone release from rat Leydig cells

Adlay has been used as a traditional Chinese medicine for the treatment of many diseases. However, few studies have reported the effects of adlay seeds on the endocrine system. In the present study, the effects of methanol extracts of adlay hull (AHM) on testosterone synthesis were studied. Rat Leydig cells were incubated with different reagents including human chorionic gonadotropin, 8-bromo-adenosine-3',5'-cyclic monophosphate, forskolin, A23187, progesterone and androstenedione in the presence or absence of AHM. The rat anterior pituitary (AP) gland was treated with gonadotropin-releasing hormone (GnRH) in vitro in the presence or absence of AHM, and the concentrations of luteinizing hormone (LH) in the media were measured. AHM decreased testosterone release via the inhibition of (1) the PKA and PKC signal transduction pathways, (2) 17beta-HSD enzyme activity in rat Leydig cells, and (3) in vitro GnRH-induced LH secretion.

The Neuroendocrine Effects of Traumatic Brain Injury

The Neuroendocrine Effects of Traumatic Brain Injury

Gonadotropin-releasing hormone-induced ovulation and luteinizing hormone release in beef heifers: Effect of day of the cycle

The COSynch protocol has been used to synchronize ovulation and facilitate fixed-time AI in beef cattle. Establishment and maintenance of pregnancy was negatively affected, in previous studies, by GnRH-induced ovulation of small dominant follicles (</=11 mm). The reason for the presence of small follicles at the second GnRH (GnRH 2) is not clear. The objectives of this study were 1) to determine the effect of ovulatory response at the first GnRH (GnRH 1) on diameter and variation in diameter of the largest follicle at GnRH 2, and 2) to determine the effect of day of the cycle (stage of a follicular wave) on GnRH-induced luteinizing hormone (LH) release, and the resulting ovulatory response after GnRH 1 and 2. Two experiments used pubertal beef heifers synchronized to be on different days of the estrous cycle (d 2, 5, 10, 15, and 18 after estrus) in which a dominant follicle would or would not respond to GnRH 1. Ovulatory response to GnRH 1 did not affect size or variation in diameter of the largest follicle at GnRH 2 in Exp. 1 or 2. In Exp. 1, ovulatory response after GnRH 1 (0/14(a), 12/13(b), 4/13(ac), 9/13(bc), and 2/10(a) in the d 2, 5, 10, 15, and 18 groups; (a-c)P < 0.05) and GnRH 2 (13/14(a), 12/13(a), 12/13(a), 2/13(b), and 2/10(b) in the d 2, 5, 10, 15, and 18 groups, respectively; (a,b)P < 0.05) was affected by day of the cycle. In Exp. 2, day of the cycle also affected the proportion of heifers ovulating after GnRH 1 (0/7(a), 8/8(b), 0/6(a) 5/8(ab), and 5/8(ab) of the d 2, 5, 10, 15, and 18 heifers, respectively; (a-c)P < 0.05) and GnRH 2 (3/7(ab), 8/8(b), 5/6(b), 1/8(a), and 2/8(a) of the d 2, 5, 10, 15, and 18 heifers, respectively; (a,b)P < 0.05). In both experiments, heifers receiving GnRH 1 on d 15 and 18 had a greater (P < 0.05) occurrence of luteolysis before PGF(2alpha) injection and expression of estrus than heifers treated on d 2, 5, and 10. The GnRH-induced LH surge was of greatest magnitude in heifers receiving GnRH 1 on d 18 of the cycle followed by d 5, 15, 10, and 2 (9,054(b), 5,774(bc), 4,672(c), 2,548(c), and 915(d) arbitrary units; respectively; (a-d)P < 0.05). In summary, ovulatory response to GnRH 1 did not affect size of the dominant follicle at GnRH 2. Day of the cycle when GnRH 1 was delivered affected dominant follicle size at GnRH 2. Treatment with GnRH 1 in the earlier part of the estrous cycle (on or before d 10) increased the proportion of dominant follicles that were large enough to respond to GnRH 2 (>/=10 mm) and increased ovulatory response after GnRH 2.

The Neuroendocrine Effects of Traumatic Brain Injury

Neuroendocrine dysfunction after traumatic brain injury (TBI) is under-diagnosed, under-treated, and may adversely affect the rate of recovery. Single or multiple pituitary-target hormone disruption occurs in up to two-thirds of persons with TBI, most commonly affecting the gonadal and growth hormone axes. The time course of decline in and recovery of pituitary function in relation to cognitive dysfunction and rehabilitation progress are not well described. This article reviews the clinical spectrum of neuroendocrine deficits after TBI and their underlying mechanisms. Future studies of the effects of hormonal replacement on recovery are recommended.

Practical Ovsynch Programs

Reproductive efficiency is a major factor contributing to efficiency, profitability and sustainability of dairy farms. Estimates of economic loss due to reproductive inefficiencies range from $1 to 5 billion per year. Given the decreasing trend of conception rates during the past 30 years, it can be anticipated that fertility of lactating dairy cows will continue to be a paramount challenge to profitable dairy herd management. The exact physiological reasons for poor fertility of lactating dairy cows are unknown. This presentation focuses on correcting limitations of Ovsynch in addition to understanding the fertility potential of an ovulatory follicle by controlling its age, size, and function. Ovsynch can improve pregnancy rates when estrus detection rates are poor and inaccurate, leading to improper timing of artificial insemination in relation to ovulation. However, inadequate synchronization rate of Ovsynch limits its effectiveness. The focus of our research program is to determine if fertility of lactating dairy cows can be increased by modifying Ovsynch to control emergence of the ovulatory follicular wave and initiate ovulation at the prime stage of development of the synchronized ovulatory follicle. Our ultimate goal is to devise an exogenous hormonal control strategy using gonadotropin-releasing hormone (GnRH) and prostaglandin F2α (PGF2α) that reduces age, yet optimizes size and function, of the ovulatory follicle of Ovsynch. Based upon strong published data, it is our contention that control of ovulation to the first GnRH of Ovsynch, appropriate timing of luteolysis and the final GnRH-induced luteinizing hormone (LH) surge are essential for the synchronous emergence, development, and synchronized ovulation of the ovulatory follicle.

Reversal of Idiopathic Hypogonadotropic Hypogonadism

Idiopathic hypogonadotropic hypogonadism, which may be associated with anosmia (the Kallmann syndrome) or with a normal sense of smell, is a treatable form of male infertility caused by a congenital defect in the secretion or action of gonadotropin-releasing hormone (GnRH). Patients have absent or incomplete sexual maturation by the age of 18. Idiopathic hypogonadotropic hypogonadism was previously thought to require lifelong therapy. We describe 15 men in whom reversal of idiopathic hypogonadotropic hypogonadism was sustained after discontinuation of hormonal therapy. We defined the sustained reversal of idiopathic hypogonadotropic hypogonadism as the presence of normal adult testosterone levels after hormonal therapy was discontinued. Ten sustained reversals were identified retrospectively. Five sustained reversals were identified prospectively among 50 men with idiopathic hypogonadotropic hypogonadism after a mean (+/-SD) duration of treatment interruption of 6+/-3 weeks. Of the 15 men who had a sustained reversal, 4 had anosmia. At initial evaluation, 6 men had absent puberty, 9 had partial puberty, and all had abnormal secretion of GnRH-induced luteinizing hormone. All 15 men had received previous hormonal therapy to induce virilization, fertility, or both. Among those whose hypogonadism was reversed, the mean serum level of endogenous testosterone increased from 55+/-29 ng per deciliter (1.9+/-1.0 nmol per liter) to 386+/-91 ng per deciliter (13.4+/-3.2 nmol per liter, P<0.001), the luteinizing hormone level increased from 2.7+/-2.0 to 8.5+/-4.6 IU per liter (P<0.001), the level of follicle-stimulating hormone increased from 2.5+/-1.7 to 9.5+/-12.2 IU per liter (P<0.01), and testicular volume increased from 8+/-5 to 16+/-7 ml (P<0.001). Pulsatile luteinizing hormone secretion and spermatogenesis were documented. Sustained reversal of normosmic idiopathic hypogonadotropic hypogonadism and the Kallmann syndrome was noted after discontinuation of treatment in about 10% of patients with either absent or partial puberty. Therefore, brief discontinuation of hormonal therapy to assess reversibility of hypogonadotropic hypogonadism is reasonable. (ClinicalTrials.gov number, NCT00392756 [ClinicalTrials.gov].).

Basal and GnRH-induced secretion of FSH and LH in anestrous versus ovariectomized bitches

The basal and gonadotropin releasing hormone (GnRH)-induced plasma concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were studied in four anestrous and four ovariectomized (OVX) bitches. Blood samples were obtained via jugular venipuncture 40 min before and 0, 10, 20, 30, 60, 90, and 120 min after the i.v. administration of synthetic GnRH in a dose of 10 μg/kg body weight. The basal plasma FSH and LH concentrations were significantly higher in the OVX bitches than in the anestrous bitches. In the anestrous bitches, the plasma FSH concentration was significantly higher than the pretreatment level at 10, 20, and 30 min, whereas the plasma LH concentration was significantly elevated at 10 and 20 min. The maximal GnRH-induced plasma FSH concentration in the anestrous bitches did not surpass the lowest plasma FSH concentration in the OVX bitches, whereas the GnRH-induced plasma LH concentrations in the anestrous bitches overlapped with the basal plasma LH concentrations in the OVX bitches. In the OVX bitches, GnRH administration did not induce a significant change in the plasma FSH concentration, whereas the plasma LH concentration increased significantly at 10 and 20 min. In conclusion, the results of the present study indicate that in anestrous bitches GnRH challenge results in increased plasma levels of both FSH and LH, whereas in the OVX bitches, in which the basal plasma FSH and LH concentrations are higher, only a rise in the plasma LH concentration is present after GnRH stimulation. The results also suggest that a test to measure plasma concentration of FSH in single samples appears to have potential in verification of neuter status in bitches.

Study of Enteral versus Parenteral Application of the Gonadotropin Releasing Hormone Agonist Gonadorelin[6-D-Phe] (D-Phe6-LHRH) on LH Secretion in Goettinger Miniature Pigs

With respect to the assessment of residue situation and as a part of preclinical trials to determine the biological activities of potential gonadotropin releasing hormone (GnRH) residues in porcine organisms the GnRH agonist Gonadorelin[6-D-Phe] (D-Phe(6)-LHRH) was administered either enterally or intramuscularly (i.m.) to female Goettinger miniature pigs in order to evaluate the GnRH-induced luteinizing hormone (LH) surge. Gilts received an (i) enteral application of 10 mg D-Phe(6)-LHRH via a probang (enteral group, n=7), (ii) i.m. injection of 0.1 mg D-Phe(6)-LHRH (parenteral group, n=5), or (iii) saline injection (control group, n=4). The GnRH and saline applications were repeated every second day with up to seven repetitions. Blood samples were collected via previously fitted jugular catheters immediately before injections, over an 8 h period in 1 h intervals beginning 2 h after injections, and at 24, 26, 28 and 30 h after applications. Enteral application of D-Phe(6)-LHRH induced an LH surge in 23 of 30 treatments. All gilts in the parenteral group exhibited LH release after each D-Phe(6)-LHRH application (P<0.05), whereas no LH surges were observed after saline injection in the control group. A significant (P<0.05) LH rise to mean maximum LH concentrations of 3.25 +/- 0.43 and 3.05 +/- 0.26 ng/ml occurred in both the enteral and parenteral groups, but there was no difference in the time interval after GnRH (2.6 +/- 0.3 vs. 2.3 +/- 0.3 h) and the mean duration of LH peak (6.5 +/- 0.4 and 6.8 +/- 0.3 h) between the treatment groups. In conclusion, (i) enteral application of 10 mg D-Phe(6)-LHRH induced LH release in a physiological range from the pituitary of female minipigs, and (ii) neither an accumulative effect nor a cumulative LH response were found after repeated GnRH application. Furthermore, (iii) in regard to consumer protection and gonadotropin secretion, D-Phe(6)-LHRH residues can be excluded from having long-term effects.

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.

Effect of the Dominant Follicle Aspiration before or after Luteinizing Hormone Surge on the Corpus Luteum Formation in the Cow

Luteinizing hormone (LH) surge and follicle rupture act as trigger to start corpus luteum (CL) formation. Thus, we aimed to investigate whether a dominant follicle that has not been exposed to an LH surge can become a functional CL. For this purpose, follicular fluid from the dominant follicles (DF) of cows was aspirated before or after a GnRH-induced LH surge, and subsequent CL formation was observed. Holstein cows were divided into four groups as follows: Luteal phase, a DF was aspirated 7 days after GnRH injection; Pre-LH surge, a DF was aspirated 42 h after PGF(2alpha) injection during the mid luteal phase; Post-LH surge, a DF was aspirated 24 h after GnRH injection following PGF(2alpha); and Intact follicle, ovulation was induced by GnRH injection after PGF(2alpha). Observation of morphological changes in the aspirated follicle using color Doppler ultrasonography and blood sampling was performed on Days 0, 3, 6, and 9 (Day 0 = follicle aspiration). CL formation following DF aspiration was observed only in the Post-LH surge group. In both the Luteal phase and Pre-LH surge groups, however, none of the cows showed local blood flow at the aspirated site or CL formation. Luteal blood flow area, CL volume, and plasma progesterone concentration in the Post-LH surge group were no different from those in the Intact follicle group. The present results clearly demonstrate that rather than follicle rupture, it is the LH surge that is essential for CL formation in cows.