LH Pulse Frequency Research Articles

Reproductive status affects the expression of prolactin receptor mRNA in the brain of female Damaraland mole-rats

The eusocial Damaraland mole-rat (Fukomys damarensis) represents an extreme example of reproductive skew, in that reproduction is completely blocked in female subordinate group members. It is thought that in these animals normal GnRH secretion from the hypothalamus is disrupted. Prolactin, a peptide hormone secreted from the anterior pituitary gland, has been implicated in a wide variety of functions. Well documented in rodents is its role in mediating lactational infertility. Elevated circulating prolactin levels, such as during lactation, are associated with reduced GnRH release into the portal blood and with a reduction in the frequency and amplitude of LH pulses. The present study aimed at investigating whether such a mechanism could act in reproductively suppressed female Damaraland mole-rats. By means of in situ hybridisation we studied the distribution and gene expression of the prolactin receptor (Prlr) in wild-caught female Damaraland mole-rats with different reproductive status. Substantial Prlr expression was found in several brain regions, with highest levels in the choroid plexus and moderate expression in the preoptic and tuberal hypothalamus. While in reproductive and non-reproductive females plasma prolactin levels were very low and not significantly different, quantification of the Prlr hybridisation signal revealed significant differences in relation to reproductive status. Reproductively suppressed females had increased expression of Prlr in the choroid plexus and in the arcuate nucleus (ARC) when compared to reproductive females. This suggests higher local prolactin levels in the brain of suppressed females. Together with previous findings, it could indicate that prolactin inhibits ARC kisspeptin neurons, which then would lead to reduced activation of GnRH neurons in such females.

Expression of kisspeptin protein in hypothalamus and LH profile of growing female lambs.

Kisspeptin (kp) is considered to be one of the major regulators of the induction of pubertal events via the activation of the gonadotrophin-releasing hormone-LH system. The aim of the present study was to analyse expression of immunoreactive (ir) kp in the hypothalamic neurons of female lambs from the neonatal to the peripubertal period (5 days to 32 weeks) in relation to the plasma LH pattern using immunohistochemistry and image analysis. Hypothalami were collected from female lambs (n=33) from the infantile, juvenile, prepubertal and peripubertal periods. The population of kp-ir perikarya was detected mainly in the arcuate nucleus and their number increased gradually from 5 to 16 weeks of age and was maintained at a high level up to the peripubertal stage. This was reflected by the significant (P<0.05) gradual increase in the percentage of hypothalamic area occupied by kp-ir neurons and increase in the number of kp-ir perikarya within the arcuate nucleus. The same pattern of kp immunoreactivity was observed in the median eminence. Plasma LH concentration increased from Week 5 to Weeks 12-16 and further increased at Week 32. LH pulse frequency increased from Week 5 to 32 (P<0.05). Thus, changes in kp expression reflected changes in the LH pattern during lamb growth. The data obtained provide evidence about the participation of kp in the mechanisms of ontogenic development of ovine reproductive processes.

Neurokinin 3 receptor antagonism decreases gonadotropin and testosterone secretion in healthy men.

Patients with mutations of neurokinin B (NKB) and its receptor show hypogonadotrophic hypogonadism, but there is little evidence for the importance of this pathway in reproductive function in normal men, or its functional hierarchy with kisspeptin. An open label study wherein men (n=6) were administered the NK3R antagonist MLE4901 40mg orally twice a day for 7days. Kisspeptin-10 (0.3μg/kg iv bolus) was given before and on day 7 of NK3R antagonist treatment. Subjects were healthy men. Reproductive hormones were measured before and during the NK3R antagonist administration, including frequent sampling on two occasions for analysis of pulsatile LH secretion. LH, FSH and testosterone secretion were decreased during NK3R antagonist administration. LH showed a biphasic response, being reduced after 24hours of treatment (4.5±0.6IU/L pretreatment to 1.7±0.2IU/L, P<.05), with partial recovery thereafter, but it was again decreased on day 7 (2.5±0.6IU/L, P<.05 vs pretreatment). FSH secretion was also suppressed, with a similar temporal pattern to that of LH. Testosterone secretion was decreased from 24hours (18.4±1.6 pretreatment vs 5.6±1.5nmol/L, P<.01) and remained suppressed throughout the treatment period. Analysis of LH pulsatility showed that both basal and pulsatile LH secretion were markedly suppressed but there was no detected change in LH pulse frequency. Kisspeptin-10 stimulated LH secretion, with similar responses before and during NK3R antagonist administration. These data demonstrate a central role for NKB/NK3R in the physiological regulation of reproductive function in men, and that this is functionally upstream of kisspeptin-mediated GnRH secretion.

Hypothalamic effects of progesterone on regulation of the pulsatile and surge release of luteinising hormone in female rats

Progesterone can block the oestradiol-induced GnRH/LH surge and inhibit LH pulse frequency. Recent studies reported that progesterone prevented premature LH surges during ovarian hyperstimulation in women. As the most potent stimulator of GnRH/LH release, kisspeptin is believed to mediate the positive and negative feedback effects of oestradiol in the hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nuclei, while the region-specific role of progesterone receptors in these nuclei remains unknown. This study examined the hypothesis that progesterone inhibits LH surge and pulsatile secretion via its receptor in the ARC and/or AVPV nuclei. Adult female rats received a single injection of pregnant mare serum gonadotropin followed by progesterone or vehicle. Progesterone administration resulted in a significant prolongation of the oestrous cycle and blockade of LH surge. However, microinjection of the progesterone receptor antagonist, RU486, into the AVPV reversed the prolonged cycle length and rescued the progesterone blockade LH surge, while RU486 into the ARC shortened LH pulse interval in the progesterone treated rats. These results demonstrated that progesterone’s inhibitory effect on the GnRH/LH surge and pulsatile secretion is mediated by its receptor in the kisspeptin enriched hypothalamic AVPV and ARC respectively, which are essential for progesterone regulation of oestrous cyclicity in rats.

Involvement of plasma adipokines in metabolic and reproductive parameters in Holstein dairy cows fed with diets with differing energy levels

This study aimed to investigate the association between plasma adipokine concentrations and metabolic and reproductive parameters in Holstein dairy cows fed diets with different energy levels during the peripartum period. The experiment started 1 mo before first calving and was maintained for 2 lactations. Dry matter intake and energy balance in animals fed a low-energy (LE) diet were significantly lower than that of animals fed a high-energy (HE) diet in the first lactation. Body weight, milk production, back fat thickness, and plasma concentrations of fatty acids, glucose, and insulin were not affected by diet, whereas plasma leptin and adiponectin concentrations were lower and plasma resistin concentrations higher in animals fed the LE diet. Unlike concentrations of adiponectin, plasma resistin concentrations were positively correlated with back fat thickness and plasma fatty acids concentrations and negatively correlated with dry matter intake and plasma leptin concentrations. No effect of diet was found on reproductive variables; that is, pregnancy rates at 35 or 90 d after artificial insemination (AI); numbers of small (3-5 mm), medium (>5 and ≤7 mm), and large (>7 mm) follicles; calving-to-AI and calving-to-calving intervals; and magnitude and duration of the LH surge. However, the commencement of luteal activity after first calving occurred sooner and the frequency of LH pulses was higher in the HE group than in the LE group. A significant positive correlation was found between the number of follicles (of any size) and the area under the curve of plasma resistin concentrations. The number of small follicles was also positively correlated with the nadir of plasma resistin concentrations. Taken together, these results suggest that dietary energy content in the range applied here can alter the resumption of ovarian activity and LH pulsatility without affecting fat mobilization. Plasma adipokine profiles (leptin, resistin, and adiponectin) were significantly altered by diet and negative energy balance but relationships with reproductive variables were limited to follicular growth characteristics and plasma resistin concentrations.

A neurokinin 3 receptor-selective agonist accelerates pulsatile luteinizing hormone secretion in lactating cattle.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion, which is indispensable for follicular development, is suppressed in lactating dairy and beef cattle. Neurokinin B (NKB) neurons in thearcuate nucleus of the hypothalamus are considered to play an essential role in generating the pulsatile mode of GnRH/luteinizing hormone (LH) secretion. The present study aimed to clarify the role of NKB-neurokinin 3 receptor (NK3R) signaling in the pulsatile pattern of GnRH/gonadotropin secretion in postpartum lactating cattle. We examined the effects of the administration of an NK3R-selective agonist, senktide, on gonadotropin secretion in lactating cattle. The lactating cattle, at approximately 7 days postpartum, were intravenously infused with senktide (30 or 300 nmol/min) or vehicle for 24 h. The administration of 30 or 300 nmol/min senktide significantly increased LH pulse frequency compared to in the control group during 0-4 or 20-24 h after infusion, respectively. Moreover, LH and follicle-stimulating hormone levels were gradually increased by 300 nmol/min administration of senktide during the 0-4-h sampling period. Ultrasonography of the ovaries was performed to identify the first postpartum ovulation in senktide-administered lactating cattle. The interval from calving to first postpartum ovulation was significantly shorter in the 300 nmol/min senktide-administered group than in the control group. Taken together, these findings suggest that senktide infusion elicits an increase in LH pulse frequency that may stimulate follicular development and, in turn, induce the first postpartum ovulation in lactating cattle.

Strategies to Manipulate Progesterone for Improved Ovarian and Fertility Responses in Bovines - A Review

Progesterone (P4) is a steroid hormone primarily secreted by the Corpus luteum (CL). Adequate circulating P4 concentration is essential for establishment and maintenance of pregnancy. Exposure to insufficient circulating level of P4 during the growth of the ovulatory follicle is one of the important factors that affect fertility in high producing animals. P4 has a negative feedback effect on LH secretion and it is believed that sub-luteal phase plasma P4 concentrations result in elevated LH pulse-frequency. There is a linear trend for the number of LH pulses to decrease concurrently with the increase in P4 concentrations as the estrous cycle progresses. These changes in LH pulse frequency are supposed to be associated with alterations in the process of follicular maturation and subsequent embryo survival. Beneficial effects of increased progesterone level during growth of dominant follicle or following AI can be documented from the current literature. P4 concentration during the development of dominant follicle (DF) influences the fertility since the DF of first and second wave use to grow under different P4 environment. Further a slight increase in circulating level of progesterone near the time of artificial insemination (AI) is highly detrimental for fertility. So, a control of follicle and CL development through utilization of various hormonal regimens is routinely done now days to further enhance the reproductive performance of dairy animals. Taking into account above all facts, the present review highlights the progesterone manipulation strategies to improve bovine fertility.

Prenatal Exposure of Kisspeptin Antagonist on the GonadotropinReleasing Hormone (GnRH) Expression in Rat Model of Polycystic Ovary Syndrome

Introduction: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age. Increased GnRH/luteinizing hormone (LH) pulse frequency is a characteristic of endocrine abnormalities in PCOS. Kisspeptin antagonists reduce LH pulse frequency and amplitude and are hence supposed to slow GnRH neuron activity that may adjust GnRH/LH levels in PCOS conditions.Objective: To investigate the impact of kisspeptin antagonist P271 administration during prenatal life to reduce GnRH expression in adulthood in prenatally androgenized (PNA) rats as a model of PCOS.Materials and methods: PNA rats (n=9) and controls (n=9) received P271 on day 20 of their prenatal life, and they were examined in adulthood (110-120 days). The ability of P271 to alter GnRH mRNA expression, and plasma levels of gonadotropins and steroid hormones were tested using reverse transcription q-Real-time PCR and ELISA methods, respectively.Results: In this study, based on the result of Generalized Estimating Equation (GEE) model, we found that GnRH expression in PCOS+P271 rats decreased compared to PCOS rats in the diestrous phase. In addition, P271 administration reduced gonadal steroid and gonadotropin levels in both PCOS and non-PCOS rats.Conclusion: In conclusion prenatal administration of kisspeptin antagonists can reduce GnRH expression and LH, FSH, T, P4 and E2 levels in PCOS rats in later life.

Short-term supplementation with maize increases ovulation rate in goats when dietary metabolizable energy provides requirements for both maintenance and 1.5 times maintenance

This study aimed to evaluate ovarian follicular dynamics in goats submitted to synchronization of estrus and supplemented with diets that differed in the metabolizable energy source and amount of energy. The experiment was carried out using 42 does allocated into three treatments, fed for 9 days with a ration providing 1.0 times maintenance containing maize (1 MM, n = 14) or without maize (1 M, n = 14) or a ration providing 1.5 times maintenance containing maize (1.5 MM, n = 14). Estrus was synchronized with two injections of cloprostenol given 7 days apart. Does were also treated with intravaginal progesterone inserts and eCG. The number of ovulations and size of the follicles were measured using ultrasonography on Days 10, 11, and 12 after the start of the dietary treatment. The interval to estrus and duration of estrus did not differ between treatments (P = 0.382). Does fed with 1 and 1.5 MM had a similar number of ovulations but a greater number of ovulations than goats fed with 1 M (P = 0.028). The mean number of small, medium, large, and total number of follicles on Days 10 to 12 of ultrasound evaluations did not differ (P = 0.204) between treatments, but mean numbers changed over time (P < 0.001). The mean frequency and amplitude of LH pulses and concentrations of glucose, insulin, leptin, and insulin growth factor-1 in plasma were not significantly affected (P > 0.258) by any of the treatments. In summary, the inclusion of maize in the ration can stimulate ovulation rate at maintenance level. Similar results between groups fed diets that included maize and provided metabolizable energy at 1.0 and 1.5 maintenance demonstrate that to increase the ovulation rate when synchronizing estrous cycles in does, dietary supplementation with maize can be restricted to provide a maintenance level of metabolizable energy only, which would reduce dietary costs.

Pulse and Surge Profiles of Luteinizing Hormone Secretion in the Mouse.

Using a new tail-tip bleeding procedure and a sensitive ELISA, we describe here the patterns of LH secretion throughout the mouse estrous cycle; in ovariectomized mice; in ovariectomized, estradiol-treated mice that model estrogen-negative and -positive feedback; and in transgenic GNR23 mice that exhibit allele-dependent reductions in GnRH neuron number. Pulsatile LH secretion was evident at all stages of the estrous cycle, with LH pulse frequency being approximately one pulse per hour in metestrous, diestrous, and proestrous mice but much less frequent at estrus (less than one pulse per 4 h). Ovariectomy resulted in substantial increases in basal and pulsatile LH secretion with pulses occurring approximately every 21 minutes. Chronic treatment with negative-feedback, estradiol-filled capsules returned LH pulse frequency to intact follicular phase levels, although pulse amplitude remained elevated. On the afternoon of proestrus, the LH surge was found to begin in a highly variable manner over a 4-hour range, lasting for more than 3 hours. In contrast, ovariectomized, estradiol-treated, positive-feedback mice exhibited a relatively uniform surge onset at approximately 0.5 hour prior to lights out. Gonadectomized wild-type and heterozygous GNR23 (∼200 GnRH neurons) male mice exhibited an LH pulse every 60 minutes. Homozygous GNR23 mice (∼80 GnRH neurons) had very low basal LH concentrations but continued to exhibit small amplitude LH pulses every 90 minutes. These studies provide the first characterization in mice of pulse and surge modes of LH secretion across the estrous cycle and demonstrate that very few GnRH neurons are required for pulsatile LH secretion.

Salsolinol—a potential inhibitor of the gonadotropic axis in sheep during lactation

This study tested the hypothesis that salsolinol, a derivative of dopamine, affects GnRH and LH secretion in lactating sheep. In the in vivo experiment, the structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1-MeDIQ), was infused into the infundibular nucleus-median eminence of sheep at the fifth wk of lactation to antagonize salsolinol's action. Simultaneously, cerebrospinal fluid from the third brain ventricle, to determine GnRH concentration, and plasma samples, to measure LH concentration, were collected. In the in vitro experiment, the anterior pituitary (AP) explants from weaned sheep were incubated in culture medium containing 2 doses of salsolinol, 20 and 100 μg/mL (S20 and S100, respectively). The concentration of LH in the collected media and relative expression of LHβ subunit messenger RNA in the AP explants were determined. No significant difference was found in mean GnRH concentration in response to 1-MeDIQ infusion, but both mean plasma LH concentration and LH pulse frequency increased significantly (P < 0.001 and P < 0.05, respectively) compared with those in controls. Significantly higher LH concentrations occurred during the first (P < 0.001), second (P < 0.001), and fourth (P < 0.05) h of 1-MeDIQ infusion. In the in vitro study, both the S20 and S100 doses of salsolinol caused a significant decrease in the mean medium LH concentration compared with that in the control (P < 0.01 and P < 0.001, respectively). Salsolinol had no effect on the relative LHβ subunit messenger RNA expression in the incubated tissue. In conclusion, salsolinol is a potential inhibitor of the secretory activity of the gonadotropic axis in lactating sheep, at least at the AP level. Although no significant changes in GnRH release were directly confirmed, an increase in the frequency of LH pulses does not allow to exclude the central action of salsolinol.

Effects of long-acting GnRH antagonist, degarelix acetate, on plasma insulin-like peptide 3, testosterone and luteinizing hormone concentrations, and scrotal circumference in male goats

We recently reported that plasma insulin-like peptide 3 (INSL3) concentrations increased soon after endogenous and exogenous stimulations of LH in male goats and bulls. However, the effects of LH suppression on INSL3 secretion are unknown in domestic animals. Here, we examined the effects of a long-acting GnRH antagonist (degarelix acetate; 4 mg/kg) on the secretions of plasma INSL3 and testosterone in two phases, an immediate and a long-term phase in male goats (n = 6; aged, 13–16 months). During the immediate phase, blood was taken at 15-minute intervals for 8 hours on Days −5, 0, and 3. The GnRH antagonist was administered after 2-hour sampling of Day 0. Moreover, a daily blood sample was taken from Day 0 to Day 7, followed by twice a week until 9 weeks and finally at week 10. The scrotal circumference was recorded before treatment and continued biweekly until week 10. Concentrations of LH, INSL3, and testosterone in plasma were determined by EIA and the pulsatile nature of secretion analyzed using pulse XP software. The mean concentrations, pulse frequency (per hour), and pulse amplitude (peak-nadir) of plasma LH and testosterone reduced from pretreatment to posttreatment Day 0 and Day 3 (P < 0.05). A decline in mean concentrations, pulse frequency, and pulse amplitude of INSL3 was exhibited on posttreatment Day 3 compared with pretreatment (P < 0.01). During long-term sampling, a decline (P < 0.01) in plasma testosterone and INSL3 concentrations was observed 1 day after treatment and remained lower until 8.5 weeks after treatment, and thereafter returned to pretreatment levels. A reduction in scrotal circumference was recorded 4 weeks after treatment and remained lower until 10 weeks after treatment (P < 0.05). In conclusion, the acute regulation of INSL3 by LH was confirmed by reduction of plasma INSL3 levels within 3 days after GnRH antagonist treatment in male goats. Although the onset of suppression of testosterone was more rapid than that of INSL3, the low levels persisted for 8.5 weeks for both hormones, and subsequently the concentrations returned to pretreatment levels. A significant reduction in testicular size was also observed. The quick, long-lasting, and transient suppression of testosterone and INSL3 after a single injection implies a potential application of this antagonist in reversible long-term chemical castration in male goats.

Interactions Between Neurokinin B and Kisspeptin in Mediating Estrogen Feedback in Healthy Women.

Context:Kisspeptin and neurokinin B (NKB) are obligate for normal gonadotropin secretion, but their hierarchy is unexplored in normal women.Objective:To investigate the interaction between kisspeptin and NKB on estrogen-regulated LH secretion.Design:Women were treated with neurokinin-3 receptor (NK3R) antagonist followed by transdermal estradiol to induce LH secretion 48 hours later, with kisspeptin-10 or vehicle infusion during estrogen administration in a 2-way crossover study.Setting:Clinical research facility.Patients or other participants:Healthy females with regular menses.Intervention(s):NK3R antagonist AZD4901 40 mg twice daily orally was taken from cycle day 4–6 for 6 days (n = 10, with 10 no treatment controls). Transdermal estradiol patches (200 μg/d) were applied after 5 days of NK3R antagonist treatment. At 24-hour estradiol treatment, women were randomized to 7-hour kisspeptin-10 (4 μg/kg/h) or vehicle iv infusion, with the alternate infusion in a subsequent cycle.Main outcome measure(s):Plasma gonadotropin and estradiol secretion.Results:After an initial suppression, LH secretion was increased 48 hours after estradiol treatment. Kisspeptin-10 increased LH secretion during the inhibitory phase, and LH remained elevated beyond the discontinuation of kisspeptin-10 infusion. NK3R antagonist decreased LH pulse frequency (0.5 ± 0.2 vs 0.7 ± 0.2 pulses/h, P < .05) and stimulated FSH response to kisspeptin-10 infusion (10.7 ± 11.0 vs 5.0 ± 3.6 IU/L, P < .05) with a nonsignificant rise in LH. The duration of LH response was blunted, with LH being lower at 48 hours (7.5 ± 4.8 vs 15.0 ± 11.4 IU/L, P < .05).Conclusions:These data demonstrate that NKB signaling regulates GnRH/LH secretion in normal women, and is predominantly proximal to kisspeptin in mediating estrogenic positive and negative feedback on LH secretion.

Neurokinin B Receptor Antagonism in Women With Polycystic Ovary Syndrome: A Randomized, Placebo-Controlled Trial

Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women, is characterized by high secretion levels of LH and T. Currently, there is no treatment licensed specifically for PCOS. The objective of this study was to investigate whether a targeted therapy would decrease LH pulse frequency in women with PCOS, subsequently reducing serum LH and T concentrations and thereby presenting a novel therapeutic approach to the management of PCOS. This study is a double-blind, double-dummy, placebo-controlled, phase 2 trial. University hospitals and private clinical research centers were included. Women with PCOS aged 18-45 years participated. Intervention included AZD4901 (a specific neurokinin-3 [NK3] receptor antagonist) at a dose of 20, 40, or 80 mg/day or matching placebo for 28 days. Change from baseline in the area under the LH serum concentration-time curve over 8 hours (area under the curve) on day 7 relative to placebo was measured. Of a total 67 randomized patients, 65 were evaluable. On day 7, the following baseline-adjusted changes relative to placebo were observed in patients receiving AZD4901 80 mg/day: 1) a reduction of 52.0% (95% confidence interval [CI], 29.6-67.3%) in LH area under the curve; 2) a reduction of 28.7% (95% CI, 13.9-40.9%) in total T concentration; and 3) a reduction of 3.55 LH pulses/8 hours (95% CI, 2.0-5.1) (all nominal P < .05). The NK3 receptor antagonist AZD4901 specifically reduced LH pulse frequency and subsequently serum LH and T concentrations, thus presenting NK3 receptor antagonism as a potential approach to treating the central neuroendocrine pathophysiology of PCOS.

The "Ram Effect": A "Non-Classical" Mechanism for Inducing LH Surges in Sheep.

During spring sheep do not normally ovulate but exposure to a ram can induce ovulation. In some ewes an LH surge is induced immediately after exposure to a ram thus raising questions about the control of this precocious LH surge. Our first aim was to determine the plasma concentrations of oestradiol (E2) E2 in anoestrous ewes before and after the “ram effect” in ewes that had a “precocious” LH surge (starting within 6 hours), a “normal” surge (between 6 and 28h) and “late» surge (not detected by 56h). In another experiment we tested if a small increase in circulating E2 could induce an LH surge in anoestrus ewes. The concentration of E2 significantly was not different at the time of ram introduction among ewes with the three types of LH surge. “Precocious” LH surges were not preceded by a large increase in E2 unlike “normal” surges and small elevations of circulating E2 alone were unable to induce LH surges. These results show that the “precocious” LH surge was not the result of E2 positive feedback. Our second aim was to test if noradrenaline (NA) is involved in the LH response to the “ram effect”. Using double labelling for Fos and tyrosine hydroxylase (TH) we showed that exposure of anoestrous ewes to a ram induced a higher density of cells positive for both in the A1 nucleus and the Locus Coeruleus complex compared to unstimulated controls. Finally, the administration by retrodialysis into the preoptic area, of NA increased the proportion of ewes with an LH response to ram odor whereas treatment with the α1 antagonist Prazosin decreased the LH pulse frequency and amplitude induced by a sexually active ram. Collectively these results suggest that in anoestrous ewes NA is involved in ram-induced LH secretion as observed in other induced ovulators.

Changes in ovarian function associated with circulating concentrations of estradiol before a GnRH-induced ovulation in beef cows

These studies were conducted to evaluate causes for differences in circulating concentrations of estradiol before a GnRH-induced ovulation. Beef cows were synchronized by an injection of GnRH on day −7 and an injection of prostaglandin F2α (PGF2α) on day 0. In experiment 1, blood samples were collected every 3 h from PGF2α on day 0 to hour 33 after PGF2α and at slaughter (hour 36 to 42; n = 10). Cows were assigned to treatment group based on circulating concentrations of estradiol (E2): HighE2 vs LowE2. At slaughter, follicular fluid (FF) and granulosa cells were collected from the dominant follicle. In experiment 2, blood samples (n = 30) were collected every 8 h from PGF2α until the dominant follicle was aspirated via ultrasound-guided follicular aspiration to collect FF and granulosa cells (hour 38 to 46). In experiment 1, HighE2 had increased abundance of 3β-hydroxysteroid dehydrogenase, cytochrome P450 aromatase, and LHR (P ≤ 0.02), and greater concentrations of estradiol and androstenedione (P ≤ 0.02) in the FF. In experiment 2, HighE2 had increased abundance of CYP11A1, 3β-hydroxysteroid dehydrogenase, cytochrome P450 aromatase, and LHR (P ≤ 0.03) vs either LowE2 or GnRHLowE2. There was a tendency (P = 0.07) for LH pulse frequency to be increased in both the GnRHLowE2 and HighE2 compared with LowE2. HighE2 cows experienced increas in circulating concentrations of estradiol compared with LowE2. In conclusion, animals with greater concentrations of circulating estradiol before fixed-time AI experienced an upregulation of the steroidogenic pathway during the preovulatory period.

The role of kisspeptin signalling in the hypothalamic-pituitary-gonadal axis--current perspective.

The discovery of kisspeptins in the recent past remoulded current understanding of the neuroendocrine axis relating to the regulation of human puberty and reproduction. Kisspeptins have been recognised to act upstream of GnRH and have been shown to play a vital role in the control of the hypothalamic-pituitary-gonadal axis via regulation of gonadotrophin secretion, onset of puberty, and control of fertility. KNDy (kisspeptin/neurokinin-B/dynorphin) neurons have been suggested to modulate GnRH pulsatile secretion, which is required to support reproductive function in both sexes. They have also been involved in mediating both positive and negative sex steroid feedback signals to GnRH neurons and serve as a vital connection between reproduction and metabolic status of the body. When kisspeptin is administered to healthy humans, and in patients with reproductive disorders, it strongly and directly stimulates GnRH and subsequent LH secretion and enhances LH pulse frequency. These observations suggest that kisspeptins are a potential novel therapeutic approach for treating disorders with either pathologically reduced or augmented gonadotrophins pulsatile secretion and is currently a focus of translational research. Kisspeptins have also been identified in several peripheral reproductive organs, indicating their role in modulation of ovarian function, embryo implantation, and placentation, but a great deal of work remains to be done to explore further in this regard, and the evidence is only available from studies done on animal models. In this review we will mainly focus on current available evidence related to the role of kisspeptins in controlling GnRH pulse frequency, specifically their role in puberty, fertility, and reproduction. We will also be appraising other factors that regulate the kiSS1/Kisspeptin/GPR-54 system.

Plasma insulin-like peptide 3 concentrations are acutely regulated by luteinizing hormone in pubertal Japanese Black beef bulls

Insulin-like peptide 3 (INSL3) is a major secretory product of testicular Leydig cells. The mechanism of acute regulation of INSL3 secretion is still unknown. The present study was undertaken in pubertal beef bulls to (1) determine the temporal relationship of pulsatile secretion among LH, INSL3, and testosterone and (2) monitor acute regulation of INSL3 secretion by LH using GnRH analogue and hCG. Blood samples were collected from Japanese Black beef bulls (N = 6) at 15-minute intervals for 8 hours. Moreover, blood samples were collected at −0.5, 0, 1, 2, 3, 4, 5, and 6 hours after GnRH treatment and −0.5, 0, 2, 4, and 8 hours on the day of treatment (Day 0), and Days 1, 2, 4, 8, and 12 after hCG treatment. Concentrations of LH, INSL3, and testosterone determined by EIAs indicated that secretion in the general circulation was pulsatile. The frequency of LH, INSL3, and testosterone pulses was 4.7 ± 0.9, 3.8 ± 0.2, and 1.0 ± 0.0, respectively, during the 8-hour period. Seventy percent of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse had occurred. The mean increase (peak per basal concentration) of testosterone pulses was higher (P < 0.001) than that of INSL3 pulses. After GnRH treatment, LH concentrations increased (P < 0.01) dramatically 1 hour after treatment and remained high (P < 0.05) until the end of sampling, whereas an elevated (P < 0.05) INSL3 concentration occurred at 1, 2, 5, and 6 hours after treatment. Testosterone concentrations increased (P < 0.01) 1 hour after the treatment and remained high until the end of sampling. After hCG treatment, an increase of INSL3 concentration occurred at 2 and 4 hours, and Days 2, 4, and 8 after treatment (P < 0.05), whereas in case of testosterone, concentrations remained high (P < 0.01) until Day 8 after treatment. The increase (maximum per pretreatment concentration) of INSL3 concentrations after injecting GnRH or hCG was much lower (P < 0.001) than that of testosterone. In conclusion, secretion of INSL3 in blood of bulls occurred in a pulsatile manner. We inferred an acute regulation of INSL3 by LH in bulls because INSL3 concentrations increased immediately after endogenous and exogenous LH stimulation. The increase of INSL3 concentrations by LH was much lower than that of testosterone in bulls.

Induction of ovulation in anestrus ewes using a dopamine receptor antagonist

Estradiol decreases the pulse frequency of LH during the nonbreeding season through dopaminergic neurons that results in anestrus in the ewe. Long-term administration of sulpiride, a dopamine antagonist, induced ovulation in seasonally anestrus mares. Accordingly, we tested whether sulpiride would induce ovulatory estrus in seasonally anestrus Malpura ewes. A total of 12 Malpura ewes were divided into sulpiride (at 0.6 mg/kg b.i.d.) or control groups. Anestrus was defined on the basis of the absence of heat signs for 2 months through twice-a-day heat detection during the nonbreeding season (October–November) and progesterone level of less than 1 ng/mL. Rates of estrus induction, ovulation, multiple ovulations, and lambing in the sulpiride-treated ewes were 83.3%, 100%, 16.6%, and 66.7%, respectively. The mean interval from treatment to estrus was 5.25 ± 1.49 days. Progesterone levels were elevated after ovulation significantly on Days 5 and 7 after estrus as compared to Day 0 after sulpiride treatment (P < 0.05). In contrast, none of the control group ewes showed either estrus or ovulation. There was a significant association between sulpiride treatment and estrus induction rate as well as ovulation rate (P < 0.05). It is concluded that the result provides proof of concept that the dopamine antagonist can induce ovulation in seasonally anestrus ewes.

Stress-induced inhibition of LH pulses in female rats: role of GABA in arcuate nucleus.

Stress exerts profound inhibitory effects on reproductive function by suppression of the pulsatile release of GnRH and therefore LH. Besides the corticotrophin-releasing factor (CRF), this effect also might be mediated via GABAergic signaling within the arcuate nucleus (ARC) since its inhibitory effects on LH pulses and increased activity during stress. In the present study, we investigated the role of endogenous GABAergic signaling within the ARC in stress-induced suppression of LH pulses. Ovariectomised oestradiol-replaced rats were implanted with bilateral and unilateral cannulae targeting toward the ARC and lateral cerebral ventricle respectively. Blood samples (25 μl) were taken via chronically implanted cardiac catheters every 5 min for 6 h for measurement of LH pulses. Intra-ARC infusion of GABAA receptor antagonist, bicuculline (0.2 pmol in 200 nl artificial cerebrospinal fluid (aCSF) each side, three times at 20-min intervals) markedly attenuated the inhibitory effect of lipopolysaccharide (LPS; 25 μg/kg i.v.) but not restraint (1 h) stress on pulsatile LH secretion. In contrast, restraint but not LPS stress-induced suppression of LH pulse frequency was reversed by intra-ARC administration of GABABR antagonist, CGP-35348 (1.5 nmol in 200 nl aCSF each side, three times at 20-min intervals). Moreover, intra-ARC application of either bicuculline or CGP-35348 attenuated the inhibitory effect of CRF (1 nmol in 4 μl aCSF, i.c.v.) on the LH pulses. These data indicate a pivotal and differential role of endogenous GABAA and GABAB signaling mechanisms in the ARC with respect to mediating immunological and psychological stress-induced suppression of the GnRH pulse generator respectively.