Electrophysiological and Morphological Evidence for Synchronized GnRH Pulse Generator Activity Among Kisspeptin/Neurokinin B/Dynorphin A (KNDy) Neurons in Goats

Neurons in the arcuate nucleus (ARC) that concomitantly express kisspeptin, neurokinin B (NKB) and dynorphin A are termed KNDy neurons and are likely candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. Our hypothesis is that KNDy neurons are functionally and anatomically interconnected to generate discrete neural signals that govern pulsatile GnRH secretion. Our goal was to address this hypothesis using electrophysiological and anatomical experiments in goats. Bilateral electrodes targeting KNDy neurons were implanted into ovariectomized goats, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys), was measured. Spontaneous and pheromone- or senktide (an NKB receptor agonist)-induced MUA volleys were simultaneously recorded from both sides of the ARC. An anterograde tracer, biotinylated dextran amine (BDA), was also injected unilaterally into the ARC of castrated male goats, and the distribution of fibers containing both BDA and NKB was examined using dual-labeling histochemistry. The results showed that MUA volleys, regardless of origin (spontaneous or experimentally induced), occur simultaneously between the right and left sides of the ARC. Tract tracing indicated that axons projecting from NKB neurons in the ARC were directly apposed to other NKB neuronal cells located bilaterally in the ARC. These results demonstrate that GnRH pulse generator activity occurs synchronously between both sides of the ARC in goats and that KNDy neurons are bilaterally interconnected in the ARC via NKB-containing fibers. Taken together, the results suggest that KNDy neurons form a neuronal circuit to synchronize burst activity among KNDy neurons and thereby generate discrete neural signals that govern pulsatile GnRH secretion.     

Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.     

Effects of brief exposure of male pheromone on multiple-unit activity at close proximity to kisspeptin neurons in the goat arcuate nucleus

Exposure of females to the male pheromone induces pulsatile release of gonadotropin-releasing hormone (GnRH) in goats. Recently, kisspeptin neurons in the arcuate nucleus (ARC) have been suggested to represent the proximate source of the GnRH pulse generator. In this study, we examined the effects of the pheromone on multiple-unit activity (MUA) in female goats fitted with recording electrodes aimed at the ARC kisspeptin neurons. In all eight goats, periodic bursts in MUA (MUA volleys), which were considered to be electrophysiological manifestations of the GnRH pulse generator, were observed. The mean intervolley interval (T) during the control period was calculated in each goat that was then exposed to the male pheromone for 1 sec at timings of 1/4 T, 1/2 T or 3/4 T after one regularly occurring MUA volley. An instantaneous rise in MUA was observed immediately after the exposure regardless of timing. Exposure at a timing of 3/4 T resulted in an MUA volley within 60 sec following the instantaneous rise in all goats. In contrast, an MUA volley was induced in only 2 goats by exposure at 1/2 T, while exposure at 1/4 T failed to induce an MUA volley in any goats. These results suggest that transmission of the pheromone signal to the ARC, represented by an instantaneous rise, activates the GnRH pulse generator. Moreover, the timing-dependent pheromone action in inducing an MUA volley indicates that the GnRH pulse generator has a refractory period for the pheromone signal after the burst.     

Dynorphin-Kappa Opioid Receptor Signaling Partly Mediates Estrogen Negative Feedback Effect on LH Pulses in Female Rats

Accumulating evidence suggests that the arcuate nucleus (ARC) kisspeptin/neurokinin B (NKB)/dynorphin (KNDy) neurons play a role in estrogen negative feedback action on pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release. The present study aimed to determine if dynorphin (Dyn) is involved in estrogen negative feedback on pulsatile GnRH/LH release. The effect of the injection of nor-binaltorphimine (nor-BNI), a kappa-opioid receptor (KOR) antagonist, into the third cerebroventricle (3V) on LH pulses was determined in ovariectomized (OVX) adult female rats with/without replacement of negative feedback levels of estradiol (low E₂). The mean LH concentrations and baseline levels of LH secretion in nor-BNI-injected, low E₂-treated rats were significantly higher compared with vehicle-treated controls. On the other hand, the nor-BNI treatment failed to affect any LH pulse parameters in OVX rats without low E₂ treatment. These results suggest that Dyn is involved in the estrogen negative feedback regulation of pulsatile GnRH/LH release. The low E₂ treatment had no significant effect on the numbers of ARC Pdyn (Dyn gene)-,Kiss1- and Tac2 (NKB gene)-expressing cells. The treatment also did not affect mRNA levels of Pdyn and Oprk1 (KOR gene) in the ARC-median eminence region, but significantly increased the ARC kisspeptin immunoreactivity. These findings suggest that the negative feedback level of estrogen suppresses kisspeptin release from the ARC KNDy neurons through an unknown mechanism without affecting the Dyn and KOR expressions in the ARC. Taken together, the present result suggests that Dyn-KOR signaling is a part of estrogen negative feedback action on GnRH/LH pulses by reducing the kisspeptin release in female rats.     

Simultaneous observation of the GnRH pulse generator activity and plasma concentrations of metabolites and insulin during fasting and subsequent refeeding periods in Shiba goats

The time course of GnRH pulse generator activity and plasma concentrations of energy substrates and insulin were simultaneously observed in female goats during 4-day fasting and subsequent refeeding in the presence or absence of estrogen for a better understanding of the mechanism of energetic control of gonadotropin secretion in ruminants. The GnRH pulse generator activity was electrophysiologically assessed with the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In estradiol-treated ovariectomized (OVX+E2) goats, the MUA volley intervals increased as fasting progressed. Plasma concentrations of non-esterified fatty acid and ketone body increased, while those of acetic acid and insulin decreased during fasting. The MUA volley intervals and plasma concentrations of those metabolites and insulin were restored to pre-fasting levels after subsequent refeeding. In ovariectomized (OVX) goats, changes in plasma metabolites and insulin concentrations were similar to those in OVX+E2 goats, but the MUA volley intervals were not altered. The present results demonstrated that fasting suppressed GnRH pulse generator activity in an estrogen-dependent manner. Changes in plasma concentrations of energy substrates and insulin during fasting were associated with the GnRH pulse generator activity in the presence of estrogen, but not in the absence of the steroid in female goats.     

Chronic Peripheral Administration of Kappa-Opioid Receptor Antagonist Advances Puberty Onset Associated with Acceleration of Pulsatile Luteinizing Hormone Secretion in Female Rats

Puberty in mammals is timed by an increase in gonadotropin-releasing hormone (GnRH) secretion. Previous studies have shown involvement of the two neuropeptides, kisspeptin and neurokinin B (NKB), in controlling puberty onset. Little is known about the role of the other key neuropeptide, dynorphin, in controlling puberty onset, although these three neuropeptides colocalize in the arcuate kisspeptin neurons. The arcuate kisspeptin neuron, which is also referred to as the KNDy neuron, has recently been considered to play a role as an intrinsic source of the GnRH pulse generator. The present study aimed to determine if attenuation of inhibitory dynorphin-kappa-opioid receptor (KOR) signaling triggers the initiation of puberty in normal developing female rats. The present study also determined if stimulatory NKB-neurokinin 3 receptor (NK3R) signaling advances puberty onset. Female Wistar-Imamichi rats were weaned and intraperitoneally implanted with osmotic minipumps filled with nor-binaltorphimine (nor-BNI), a KOR antagonist, or senktide, a NK3R agonist, at 20 days of age. Fourteen days of intraperitoneal infusion of nor-BNI or senktide advanced puberty onset, manifested as vaginal opening and the first vaginal estrus in female rats. Frequent blood sampling showed that nor-BNI significantly increased luteinizing hormone (LH) pulse frequency at 29 days of age compared with vehicle-treated controls. Senktide tended to increase this frequency, but its effect was not statistically significant. The present results suggest that the inhibitory input of dynorphin-KOR signaling plays a role in the prepubertal restraint of GnRH/LH secretion in normal developing female rats and that attenuation of dynorphin-KOR signaling and increase in NKB-NK3R signaling trigger the onset of puberty in female rats.     

Neuroendocrine control of reproductive function in ruminants

Gonadotropin‐releasing hormone (GnRH) is a key molecule in the control of reproduction in mammals. It is generally thought that the secretion of GnRH into the pituitary portal vessels is governed by two distinct neural mechanisms: the pulsatile and surge mode centers. The former is called the GnRH pulse generator, and this neural substrate plays a role as the master regulator of the reproductive function. An electrophysiological technique for monitoring the neural activity of the GnRH pulse generator has been established in the Shiba goat. The central actions of several neuropeptides have been assessed using this system. Results suggest that several neuropeptides including neuropeptide Y, cholecystokinin‐octapeptide and melanocortins are involved in the regulation of the GnRH pulse generator activity in the goat. Each input of those neuropeptides likely represents a unique mechanism conveying specific information about changes in the internal and external environments such as olfactory signals, nutrition, stress, and steroidal milieu, to the GnRH pulse generator. Further elucidations of actions of neurotransmitters on the GnRH pulse generator may serve for better understanding of the neuroendocrine control of reproduction in the ruminant.     

The role of kisspeptin and gonadotropin inhibitory hormone in the seasonal regulation of reproduction in sheep

Sheep are seasonal breeders, experiencing an annual period of reproductive quiescence in response to increased photoperiod during the late-winter into spring and renaissance during the late summer. The nonbreeding (anestrous) season is characterized by a reduction in the pulsatile secretion of GnRH from the brain, in part because of an increase in negative feedback activity of estrogen. Neuronal populations in the hypothalamus that produce kisspeptin and gonadotropin-inhibitory hormone (GnIH) appear to be important for the seasonal shift in reproductive activity, and the former are also mandatory for puberty onset. Kisspeptin cells in the arcuate nucleus (ARC) and preoptic area appear to regulate GnRH neurons and transmit sex-steroid feedback signals to these neurons. Moreover, kisspeptin expression in the ARC is markedly up-regulated at the onset of the breeding season, as too are the number of kisspeptin fibers in close apposition to GnRH neurons. The lower levels of kisspeptin seen during the nonbreeding season can be "corrected" by infusion of kisspeptin, which causes ovulation in seasonally acyclic females. The role of GnIH is less clear, but mounting evidence supports a role for this neuropeptide in the inhibitory regulation of both GnRH secretion and gonadotropin release from the pituitary gland. Contrary to kisspeptin, GnIH expression is markedly reduced at the onset of the breeding season. In addition, the number of GnIH fibers in close apposition to GnRH neurons also decreases during this time. Importantly, exogenous GnIH treatment can block both the pulsatile release of LH and the preovulatory LH surge during the breeding season. In summary, it is most likely the integrated function of both these neuropeptide systems that modulate the annual shift in photoperiod to a physiological change in fertility.     

Involvement of neurokinin receptors in the control of pulsatile luteinizing hormone secretion in rats

It has recently been shown that neurokinin B, a tachykinin, is associated with GnRH pulse generation in sheep and goats. The aim of the present study was to clarify the role of tachykinin receptors in the control of LH secretion in rats. To this end, we evaluated the effect of CS-003, an antagonist for all three neurokinin receptors (NK1, NK2 and NK3 receptors), on pulsatile LH secretion in both sexes of rats with different routes of administration. Both oral and third ventricular administration of CS-003 suppressed LH secretion in both sexes of gonadectomized animals. Furthermore, intact male rats with oral administration of CS-003 showed decreased serum testosterone levels, which might be due to suppressed LH secretion. None of the three subtype-specific neurokinin receptor antagonists showed a significant effect on LH secretion in ovariectomized rats when each antagonist was singly administered. The present results suggest that neurokinins play a role in the control of pulsatile GnRH/LH secretion via multiple neurokinin receptors in both male and female rats.     

Central estrogen action sites involved in prepubertal restraint of pulsatile luteinizing hormone release in female rats

The present study aimed to determine estrogen feedback action sites to mediate prepubertal restraint of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release in female rats. Wistar-Imamichi strain rats were ovariectomized (OVX) and received a local estradiol-17β (estradiol) or cholesterol microimplant in several brain areas, such as the medial preoptic area (mPOA), paraventricular nucleus, ventromedial nucleus and arcuate nucleus (ARC), at 20 or 35 days of age. Six days after receiving the estradiol microimplant, animals were bled to detect LH pulses at 26 or 41 days of age, representing the pre- or postpubertal period, respectively. Estradiol microimplants in the mPOA or ARC, but not in other brain regions, suppressed LH pulses in prepubertal OVX rats. Apparent LH pulses were found in the postpubertal period in all animals bearing estradiol or cholesterol implants. It is unlikely that pubertal changes in responsiveness to estrogen are due to a change in estrogen receptor (ER) expression, because the number of ERα-immunoreactive cells and mRNA levels of Esr1, Esr2 and Gpr30 in the mPOA and ARC were comparable between the pre- and postpubertal periods. In addition, kisspeptin or GnRH injection overrode estradiol-dependent prepubertal LH suppression, suggesting that estrogen inhibits the kisspeptin-GnRH cascade during the prepubertal period. Thus, estrogen-responsive neurons located in the mPOA and ARC may play key roles in estrogen-dependent prepubertal restraint of GnRH/LH secretion in female rats.     

Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats

Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the anti-rat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fos-expressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E(2))-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERalpha immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E(2)-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.     

Central somatostatin-somatostatin receptor 2 signaling mediates lactational suppression of luteinizing hormone release via the inhibition of glutamatergic interneurons during late lactation in rats

Reproductive function is suppressed during lactation owing to the suckling-induced suppression of the kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and subsequent suppression of luteinizing hormone (LH) release. Our previous study revealed that somatostatin (SST) neurons mediate suckling-induced suppression of LH release via SST receptor 2 (SSTR2) in ovariectomized lactating rats during early lactation. This study examined whether central SST-SSTR2 signaling mediates the inhibition of ARC Kiss1 expression and LH release in lactating rats during late lactation and whether the inhibition of glutamatergic neurons, stimulators of LH release, is involved in the suppression of LH release mediated by central SST-SSTR2 signaling in lactating rats. A central injection of the SSTR2 antagonist CYN154806 (CYN) significantly increased ARC Kiss1 expression in lactating rats on day 16 of lactation. Dual in situ hybridization revealed that few ARC Kiss1-positive cells co-expressed Sstr2, and some of the ARC Slc17a6 (a glutamatergic neuronal marker)-positive cells co-expressed Sstr2. Furthermore, almost all ARC Kiss1-positive cells co-expressed Grin1, a subunit of N-methyl-D-aspartate (NMDA) receptors. The numbers of Slc17a6/Sstr2 double-labeled and Slc17a6 single-labeled cells were significantly lower in lactating dams than in non-lactating rats whose pups had been removed after parturition. A central injection of an NMDA antagonist reversed the CYN-induced increase in LH release in lactating rats. Overall, these results suggest that central SST-SSTR2 signaling, at least partly, mediates the suppression of ARC Kiss1 expression and LH release by inhibiting ARC glutamatergic interneurons in lactating rats.     

哺乳动物性晚熟相关基因的研究进展

人和哺乳动物性的发育和成熟源于下丘脑性腺激素释放激素（gonadotropin releasing hormone,GnRH）脉冲式释放。GnRH释放受到抑制或破坏,就会导致性腺机能减退,人在发育前和发育期就会出现发育迟缓和性发育不良,表现为无精症或闭经;动物则表现为初情期延迟、生殖能力下降等表型。编码促性腺激素及其受体基因的突变可能引起哺乳动物性晚熟。笔者简要介绍了GPR54、GnRH/GnRHR、FSH/FSHR、LH/LHR基因与哺乳动物性晚熟的关系。     

Induction of ovulation in anestrous mares with pulsatile administration of gonadotropin-releasing hormone

Four seasonally anestrous mares (Standardbred), housed under a nonstimulatory photoperiod of 8 hours light:16 hours dark, were administered gonadotropin-releasing hormone (GnRH) in a pulsatile pattern (50 or 250 micrograms of GnRH/hour) for 8 to 18 days during February and March 1985. Treatment with GnRH, irrespective of dose or month, induced an increase in serum luteinizing hormone from a mean pretreatment value typical of anestrus (0.58 +/- 0.02 ng/ml +/- SE) to 10.84 +/- 1.27 ng/ml on day 8 of GnRH treatment. Ovulation in the 4 mares occurred 8.8 +/- 0.7 days after the initiation of pulsatile GnRH administration. In each instance, ovulation was followed by a functional corpus luteum, as indicated by a luteal phase (defined as the number of days on which serum levels of progesterone were greater than 1.0 ng/ml) which lasted 14.5 +/- 0.6 days. These results indicate that infusion of GnRH in a pulsatile pattern is effective in inducing follicular development and ovulation in anestrous mares in the absence of a stimulatory photoperiod.     

Interactions of photoperiod, testosterone, and naloxone on GnRH and LH pulse parameters in the male sheep

This study tested the hypothesis that the effects of the opiate antagonist naloxone on GnRH (and LH) secretion is affected by photoperiod length and testosterone (T) concentrations. The effect of infusing naloxone on GnRH and LH pulse patterns was determined in four groups of orchidectomized sheep: long day (LD) photoperiod treated with T, LD without T (LDC), short day photoperiod (SD) with T, SDC (n = 5-7/group). Hypophyseal-portal and jugular blood samples were collected at 10 min intervals for 4 h before and 4 h during naloxone infusion (1 mg/kg/h). Neither photoperiod nor T affected either mean GnRH or LH whereas naloxone (P < 0.01) increased both. LD photoperiod (P < 0.01), T (P < 0.01) and naloxone (P < 0.01) all increased LH pulse amplitude whereas only naloxone increased GnRH pulse amplitude (P < 0.01). There was an interaction (P < 0.01) between steroid and naloxone on LH, but not GnRH, pulse amplitude. Both LD photoperiod and T increased both LH and GnRH (P < 0.01) interpulse-interval (IPI). Naloxone decreased GnRH IPI (P < 0.01). The LH/GnRH pulse amplitude ratio was (P < 0.02) increased by T--likely a secondary response to the T-induced increase in IPI. These results are interpreted as showing that in the ram the endogenous opiate peptides regulate both GnRH pulse frequency and amplitude, but that their specific role is modulated by photoperiod and T. These results do not support the concept that the opiate peptides are the primary mediators of the negative feedback effects of T.     

Role of noradrenergic receptors in the bed nucleus of the stria terminalis in regulating pulsatile luteinizing hormone secretion in female rats

The bed nucleus of the stria terminalis (BNST) is one of the brain areas densely innervated by noradrenergic neurons originating in the brain stem. The present study aims to determine the role of noradrenergic receptors in the BNST in regulating pulsatile luteinizing hormone (LH) secretion in female rats. Ovariectomized (OVX) or estrogen-primed OVX (OVX+E2) rats received three 1-h-interval injections of 0.05 micromol of noradrenaline (NA), phenylephrine (alpha1-adrenergic receptor agonist), clonidine (alpha2-agonist), or isoproterenol (beta-agonist) into the BNST. Injection of NA or alpha1-adrenergic agonist into the BNST strongly suppressed pulsatile LH secretion in OVX+E2 rats with a significant (P < 0.05) decrease in the mean LH level for 3 h and LH pulse frequency, but alpha2-and beta-agonists did not affect any of the LH pulse parameters. In OVX animals, alpha1- and alpha2-adrenergic agonists caused a significant change in LH pulse frequency and amplitude, respectively, though the effect was not as apparent as the NA- or alpha1-agonist-induced changes in OVX+E2 animals. These results indicate that NA inputs to the BNST suppress pulsatile LH secretion via alpha1-adrenergic receptors and that estrogen enhances this suppression.     

Gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) secretion from perifused equine pituitaries.

In vitro responsiveness of the horse anterior pituitary (AP) gonadotropes to single and multiple GnRH challenges was examined. The pituitaries were collected from reproductively sound mares in estrus (n = 5) and diestrus (n = 5). Uniform 0.5 mm AP slices were subdivided using a 3 mm biopsy punch and then bisected for use in the perifusion chamber. Four bisected sections per chamber were perifused at 0.5 ml/min at 37 C for 560 min in Medium 199 saturated with 95% 0(2)/5% CO2. Ten minute fractions were collected after an initial 2 hr equilibration period. Four different treatment regimes of GnRH (10(-10) M) were evaluated: (A) three consecutive 10 min GnRH pulses separated by 80 and 100 min, respectively; (B) a single 120 min GnRH infusion; (C) a 10 min GnRH pulse followed 80 min later by a 120 min GnRH infusion and (D) two 10 min GnRH pulses separated by 60 min followed 80 min later by a 120 min GnRH infusion. Estimated total pituitary LH content was higher in estrous than diestrus mares (p less than 0.05). The total amount of LH released in response to GnRH tended to be greater in estrus than diestrus (p less than 0.1), whereas the percentage of LH released in estrus and diestrus was similar. An increase in the area under the LH response curve was noted with each successive 10 min pulse of GnRH during both estrus and diestrus (p less than 0.05), demonstrating a self-priming effect of GnRH. In addition, a significant increase in the peak LH amplitude (p less than 0.05) and the slope to peak amplitude (p less than 0.05) were observed for the 120 min GnRH pulse in regime C and D indicating that prior exposure to short-term pulses of GnRH increased the acute LH secretory response. These results suggest that in the cycling mare (1) the responsiveness of the pituitary (amount of LH released as percent of total LH) is similar in both estrus and diestrus, however, the magnitude of the LH response (total microgram amount of LH released) differs with the stage of the estrous cycle, being highest in estrus, and appears to be related, in part, to pituitary LH content and (2) GnRH self-priming occurs independently of the stage of the estrous cycle. Furthermore, we have demonstrated that the pulsatile mode of GnRH can act directly on the anterior pituitary to dictate the pulsatile release pattern of LH in the cycling mare.     

KISS1 Gene Expression in the Developing Brain of Female Pigs in Pre- and Peripubertal Periods

Puberty is associated with an increase in gonadotropin secretion as a result of an increase in gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is considered to play a key role in puberty onset in many mammalian species, including rodents, ruminants and primates. The present study aimed to determine if changes in hypothalamic expression of the KISS1 gene, encoding kisspeptin, are associated with the onset of puberty in pigs. The animals (n=4 in each group) were perfused with 4% paraformaldehyde at 0, 1, 2, 3 and 4 months old, as prepubertal stages, and at 5 months old, as the peripubertal stage, following each blood sampling. KISS1 gene expressions in coronal sections of brains were visualized by in situ hybridization. Plasma luteinizing hormone (LH) was measured by radioimmunoassay. KISS1 mRNA signals were observed in the arcuate nucleus (ARC) at all ages examined without any significant difference in the number of KISS1-expressing cells, indicating that the KISS1 gene is constantly expressed in the ARC throughout pubertal development in pigs. The plasma LH concentration was the highest in 0-month-old piglets and significantly decreased in the 1- and 2 month-old groups (P<0.05), suggesting a developing negative feedback mechanism affecting gonadotropin release during the prepubertal period. Considering the potent stimulating effect of kisspeptin on gonadotropin release in prepubertal pigs, kisspeptin secretion rather than kisspeptin synthesis may be responsible for the onset of puberty in pigs.     

Stimulation of Dopamine D1 Receptors Increases Activity of Periventricular Somatostatin Neurons and Suppresses Concentrations of Growth Hormone

The selective dopamine D₁ receptor agonist, SKF38393, stimulates release of somatostatin (SS) from perifused bovine hypothalamic slices. Therefore, we hypothesized that SKF38393 activates SS neurons, which, via release of SS, would suppress concentrations of growth hormone (GH) in serum in calves. Our objectives were to determine whether SKF38393: (1) increases the percent of immunoreactive c-Fos protein and Fos-related antigens (Fos/FRA) detected in somatostatin neurons in periventricular (PeVN) and arcuate (ARC) hypothalamic nuclei; (2) reduces concentrations of GH in serum; (3) suppresses growth hormone-releasing hormone (GHRH)-induced release of GH. Meal-fed steers were used to perform these objectives because a synchronous pulse of GH occurs 1–2 hr before feeding in steers allowed access to feed for 2 hr each day. In Experiment 1, two groups of four Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW). Steers were injected i.v. with a lethal dose of sodium pentobarbital 100 min later and their brains were fixed with 4% paraformaldehyde. Dual-label immunohistochemistry was performed on 40 μm free-floating sections using antiserum to SS and to Fos/FRA on sections containing PeVN and ARC nuclei. More SS neurons were detected in the PeVN than in the ARC. The percent of SS neurons with immunoreactive Fos/FRA present was 2.9-fold higher in SKF38393-treated compared with vehicle-injected steers in the PeVN, but was unchanged in the ARC. In Experiment 2, eight Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW) 140 min before meal-feeding. In contrast to controls, concentrations of GH in serum of SKF38393-treated steers did not increase during the 140 min before meal-feeding. In Experiment 3, eight Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW), then 100 min later, each steer was injected i.v. with [Leu²⁷, Hse⁴⁵] bGHRH^1–45 lactone (0.2 μg/kg BW). Bovine GHRH stimulated release of GH into serum in both groups, but concentrations of GH were lower in SKF38393-treated steers. These results show that stimulation of D₁ receptors selectively increases activity of SS neurons in the PeVN, and this increased activity is associated with suppressed basal- and GHRH-induced release of GH in serum of meal-fed steers.