Stimulated LH Release Research Articles

6594 A Rare Case of Clomiphene Induced De Quervain’s Subacute Thyroiditis

Abstract Disclosure: A. Abdelhameed: None. M. Mortagy: None. A. Elsandouby: None. A. Ghaffar: None. Introduction: Clomiphene is a selective estrogen receptor modulator that stimulates LH release by inhibiting negative feedback on the hypothalamic-pituitary axis. De Quervain’s subacute granulomatous thyroiditis is usually caused by viral infections and less often is drug-induced. We present a case of possible subacute thyroiditis induced by clomiphene. To the best of ourknowledge, this association has not been reported before. Case presentation: A 35-year-old female with a past medical history of unexplained infertility presents to the general medicine clinic with severe neck pain for the last 5 days. Her neck pain started 2 days after the completion of the clomiphene course for induction of ovulation (50 mg daily for 5 days). She denied upper respiratory symptoms. She had an extremely tender thyroid with no palpable nodules. Throat was clear. She was clinically euthyroid with no rash or itching. TSH was 3.9 mIU/L (TSH was normal 6 months prior). ESR was 40 mm/hr. Thyroid ultrasound was unremarkable. She was treated with Ibuprofen for subacute thyroiditis. In the first 2 weeks, the patient complained of increased anxiety. In the next month, she had a missed menstrual cycle. Her pain and tenderness resolved after 50 days. Repeat TSH was 2.4 mIU/L. The patient was advised not to retake clomiphene. Discussion: This is a typical presentation of subacute thyroiditis as persistent thyroid pain and tenderness with elevated ESR. The patient didn’t have the classical symptoms of preceding viral infection. However, her symptoms began immediately after completion of clomiphene course suggesting a temporal relation. The patient didn’t have any history of thyroid problems. She had increased anxiety initially followed by a missed period which suggests clinical thyroid dysfunction.Clomiphene has been associated in the literature with an increased risk of thyroid cancer. Uncontrolled thyroid dysfunction is a contraindication for clomiphene use. This might suggest an interaction between clomiphene and thyroid homeostasis. Subacute thyroiditis could be caused by drugs (e.g., lithium and amiodarone). This suggests that other drugs could lead to subacute thyroiditis such as clomiphene in our case. Conclusion: Clinicians should be aware that clomiphene could be associated with thyroid disorders such as subacute thyroiditis. It is also important to note that thyroid dysfunction is a contraindication for clomiphene use. Presentation: 6/3/2024

Oxytocin-like signal regulates Lh cells directly but not Fsh cells in the ricefield eel Monopterus albus†.

The synthesis and release of LH and FSH in the pituitary of vertebrates are differentially regulated during gonadal development and maturation. However, the underlying neuroendocrine mechanisms remain to be fully elucidated. The present study examined the possible involvement of isotocin (Ist), an oxytocin-like neuropeptide, in the regulation of Lh and Fsh in a teleost, the ricefield eel Monopterus albus. The immunoreactive isotocin receptor 2 (Istr2) was shown to be localized to Lh but not Fsh cells. In contrast, immunoreactive isotocin receptor 1 (Istr1) was not observed in either Lh or Fsh cells in the pituitary. Interestingly, Lh cells in female ricefield eels expressed Istr2 and secreted Lh in response to Ist challenge stage-dependently and in correlation with ovarian vitellogenesis. Moreover, Ist decreased Lh contents in the pituitary of female fish, indicating its stimulatory roles on Lh release in vivo. The induction of Lh release by Ist in dispersed pituitary cells was blocked by a PLC or IP3R inhibitor but not by a PKA or PKC inhibitor, indicating the involvement of the IP3/Ca2+ pathway. Collectively, the above results indicate that isotocin may bind to Istr2 to stimulate Lh release via the IP3/Ca2+ pathway, and play important roles in the ovarian maturation in ricefield eels. Furthermore, the present study suggests a novel neuroendocrine mechanism underlying the differential regulation of Lh and Fsh in vertebrates.

Lame cows have a subdued hypothalamus-pituitary axis as revealed by LH release in response to exogenous endocrine challenges

Lameness is a serious concern in cows due to its multilevel effects and increasing incidences. The ability of anterior pituitary and hypothalamus to release LH, a prime reproductive hormone, is unknown in lame cows. Our study tested this ability by exogenous endocrine challenge using Buserelin acetate (GnRH analogue) and estradiol valerate (EV). The study included moderately lame (L; n=7) either cyclic (LC; n=3) or anestrus (LA; n=4); and normal cyclic, non–lame (NL; n=5) cows. Progesterone blocks LH release, so it was lowered by injecting PG in cyclic cows. After 14 h, all cows were given a walk for 5 min and immediately thereafter injected with 0.008 mg GnRH and 4 h later with 1 mg of EV. Blood plasma progesterone, cortisol and LH were quantified, periodically. There was a significant delay in the onset (105.0 vs 75.0±7.0 vs 54.0±9.0 min) and duration (22.5±5.3 vs 80.0±10.8 vs 117.0±2.7 min) of LH surge in LA vs LC vs NL cows; the surge was absent in two LA cows. The altered LH profile was attributed to a 3 fold (in L) vs 1.6 fold (in NL) rise in cortisol. LH released (ng/ml) in response to EV was also low in LA (31.5±2.4) and LC (69.9±7.2) than NL (79.2±7.0) cows. In conclusion, lame cows subjected to walk exhibited reduced pituitary response to release LH following exogenous GnRH as also a reduced ability of hypothalamus to stimulate LH release in response to exogenous estradiol.

Characterization of the Role of NKA in the Control of Puberty Onset and Gonadotropin Release in the Female Mouse.

The tachykinin neurokinin B (NKB, Tac2) is critical for proper GnRH release in mammals, however, the role of the other tachykinins, such as substance P (SP) and neurokinin A (NKA) in reproduction, is still not well understood. In this study, we demonstrate that NKA controls the timing of puberty onset (similar to NKB and SP) and stimulates LH release in adulthood through NKB-independent (but kisspeptin-dependent) mechanisms in the presence of sex steroids. Furthermore, this is achieved, at least in part, through the autosynaptic activation of Tac1 neurons, which express NK2R (Tacr2), the receptor for NKA. Conversely, in the absence of sex steroids, as observed in ovariectomy, NKA inhibits LH through a mechanism that requires the presence of functional receptors for NKB and dynorphin (NK3R and KOR, respectively). Moreover, the ability of NKA to modulate LH secretion is absent in Kiss1KO mice, suggesting that its action occurs upstream of Kiss1 neurons. Overall, we demonstrate that NKA signaling is a critical component in the central control of reproduction, by contributing to the indirect regulation of kisspeptin release.

SAT-425 NKB Signaling in the Posterodorsal Medial Amygdala Stimulates Gonadotropin Release in a Kisspeptin-Independent Manner in Female Mice

Female reproduction is regulated by a complex signaling network, the function of which is dependent on the level of circulating estrogens. Neurokinin B (NKB) colocalizes with kisspeptin in Kiss1 neurons of the arcuate nucleus (ARC). The autosynaptic action of NKB via the neurokinin 3 receptor (NK3R) in these neurons induces kisspeptin release in the presence of sex steroids. This has led us to believe that NKB stimulates LH secretion in a kisspeptin-dependent manner despite the latter mechanism having been investigated in the persistent hypogonadal state [e.g. Kiss1r knock out (KO) mice and agonadal monkeys]. Here, we aimed to assess whether in the presence of estradiol, senktide, a NK3R specific agonist, can stimulate LH release in a kisspeptin-independent manner. Adult WT female mice were ovariectomized (OVX) or OVX and estradiol-treated (OVX+E2) and studied in parallel to Kiss1KO or Kiss1KO+E2 littermates (n=10/group). Intracerebroventricular (icv) injections of senktide (600pmol) were performed and blood samples were collected before and 25 min after icv injection. Interestingly, the presence of E2 in Kiss1KO females resulted in LH increase like that observed in WT animals. Subsequently, we aimed to locate the brain area which senktide may be acting to stimulate LH release in Kiss1KO+E2 mice. We mapped the distribution of NK3R in the mouse hypothalamus and moreover investigated the anatomical relationship of NK3R and GnRH expression with dual-label immunohistochemistry as a likely pathway of the kisspeptin-independent stimulation of LH release exerted by NKB. We observed no instances of co-expression between NK3R and GnRH cell bodies (>100 cells analyzed from a total of 16 mice) and only minimal close appositions between the two proteins in the ARC and medial septum. Thus, the kisspeptin-independent action of NKB cannot be attributed to direct stimulation of NK3R located on GnRH neurons. Interestingly, we observed that NK3R immunopositive cells in the posterodorsal medial amygdala (MePD), unlike the ARC, were upregulated in the presence of E2. Next, WT OVX+E2 and Kiss1KO+E2 females were injected with senktide (600pmol; n=5/group) in the ARC, the AVPV/PeN or the MePD; areas found to contain NK3R-expressing neurons, and blood samples were collected before and then every 15 min for 45 min. Senktide significantly stimulated LH secretion in all three areas in WT OVX+E2, but only in the MePD of Kiss1KO+E2 mice. Conversely, in the absence of E2, Kiss1KO females injected with senktide in the MePD did not show any alteration in LH, mimicking the results we obtained after icv administration. These data demonstrate that activation of NK3R in the amygdala stimulates LH release in a kisspeptin independent manner in the presence of estrogen. This novel pathway for LH release, that bypasses the kisspeptin neuron, may play an important role in linking amygdala regulated social cues with gonadotropin release.

SAT-405 Activation of Kiss1 Neurons in the Posterodorsal Medial Amygdala Stimulates Gonadotropin Release via the Release of Kisspeptin in Female Mice

Reproduction is regulated by a complex neuronal network the precise components of which are still unknown. Kisspeptins, secreted from Kiss1 neurons in the arcuate nucleus (Kiss1ARC) and anteroventral periventricular/periventricular (Kiss1AVPV/PeN) nuclei, have been directly linked to the release of GnRH from the hypothalamus and hence, LH from the anterior pituitary. However, there is a population of Kiss1 neurons in the posterodorsal medial amygdala (Kiss1MePD) whose contribution to GnRH/LH stimulation has not yet been described. Here, we aimed to determine whether the release of kisspeptin and/or other components, besides kisspeptin, within the Kiss1MePD neuron, can stimulate LH release into the peripheral circulation. In this regard, we used a chemogenetic approach to specifically activate Kiss1MePD neurons in adult Kiss1Cre/+(heterozygous state) females which we studied in parallel to Kiss1Cre/Cre(Kiss1 knock-out state) littermates (n=5/group). Females received bilateral stereotaxic injections of an adeno-associated virus (pAAV) encoding a Cre-driven Gq-coupled excitatory DREADD (pAAV5/hSyn-DIO-hm3Dq:mCherry; titer 3x1012 genome copies per ml; 1 µl per hemisphere). Following infection, mice were given 3 weeks for recovery and maximum expression of the AAV vector. On day 1 of the experiment, animals were administered an ip bolus injection of vehicle saline (0.9% NaCl; day 1) and then hM3D receptors were activated by ip injection of its agonist, clozapine N-oxide (CNO; 10 mg/kg dissolved in saline; day 2). Blood samples were collected just before saline or CNO treatment (0) and then every 15 min for 90 min. Kiss1Cre/+mice expressing hM3Dq:mCherry in the MePD and treated with CNO to activate the Kiss1MePD neurons, showed an increase in LH within 30 min after the injection (P=0.0107) compared to animals receiving saline treatment, which was sustained for a further 30 min before returning to basal levels. Furthermore, no alteration in LH was observed in Kiss1Cre/Cre(i.e., Kiss1 KO) animals treated with either saline or CNO indicating that kisspeptin is the only component within the Kiss1MePD neuron that has the ability to stimulate LH release. Analysis following the completion of pharmacological studies demonstrated that mCherry expression was evident in 89% of the targeted Kiss1MePD neurons. Interestingly, mCherry labeled projections were observed in the ARC and specifically in close contact with Kiss1ARC neurons. Thus, in the female mouse, Kiss1MePD neurons have the ability to stimulate the gonadotropic axis but only when kisspeptin is present. Furthermore, this study suggests that this action is, at least in part, via the activation of Kiss1ARC neurons which, in turn, stimulate GnRH/LH release. Overall, our data provide insight into the potential mechanism via which amygdala regulated social cues can enhance reproductive function.

OR06-1 Melanocortins Act on Kiss1 Neurons to Regulate Reproduction in Females

Reproduction, controlled by the excitatory action of the neuropeptide kisspeptin, is an energy costly function for the organism and, therefore, it is tightly regulated by metabolic factors. Among those, hypothalamic melanocortins are essential in the control of metabolism. Inactivating mutations in the proopiomelanocortin (Pomc) gene or melanocortin receptor 4 (Mc4r) cause severe obesity and subfertility in humans and mice. However, whether the reproductive impairment is a direct consequence of the lack of melanocortin signaling, or indirect resulting from the obesity phenotype, remains unknown. α-MSH (derived from Pomc) stimulates LH release and this action has been shown to be mediated by Kiss1 neurons. We evaluated the expression of Mc4r in Kiss1 neurons, and using dual fluorescence in situ hybridization (RNAscope), we showed that Kiss1 neurons of the arcuate nucleus (Kiss1ARC) and the anteroventral periventricular/periventricular nucleus (Kiss1AVPV/PeN) express Mc4r. Mc4r colocalized with 84% of Kiss1ARC and with 74% of Kiss1AVPV/PeN, suggesting that Kiss1 neurons are a direct target of melanocortins. In order to evaluate the involvement of Kiss1 neurons in the reproductive role of melanocortins, we ablated Mc4r specifically from Kiss1 neurons by generating a specific Kiss1-Mc4rKO (Kiss1-cre+/Mc4r-lox+/+) mouse line. We found that Kiss1-Mc4rKO females exhibit disrupted estrous cycles with prolonged diestrous phases and delayed puberty onset compared to their control littermates, without developing obesity during the time of the study. The reproductive phenotype of Kiss1-Mc4rKO males was not affected. When central injections of an Mc4r selective agonist were performed on Kiss1-Mc4rKO intact males and their control littermates, LH levels were significantly increased to the same extend in both groups at 15 min post-injections, suggesting that, in the male, α-MSH controls LH levels through a Kiss1 neuron independent mechanism. Taken together, these results support a direct role of melanocortins on Kiss1 neurons in the control of GnRH release in female mice. Overall, understanding the neuroendocrine mechanisms underlying the metabolic regulation of reproduction will help improve the fertility rates in patients affected by metabolic disturbances. Keywords: Kisspeptin, α-MSH, Melanocortins, Mc4r, Reproduction

OR33-4 A Single Subcutaneous Injection Of The Kisspeptin Analogue, MVT-602, Induces A More Prolonged LH Surge Compared With Kisspeptin-54 In Healthy Women

Background Kisspeptin is a hypothalamic neuropeptide that is requisite for normal reproductive health. A subcutaneous bolus of the native form of kisspeptin (kisspeptin-54; KP54) induces an LH-surge lasting 12-14hrs, to safely mature oocytes during in vitro fertilisation (IVF) treatment. The kisspeptin analogue, MVT-602, has a longer half-life (t1/2 1.5-2.2h) than native KP54 (t1/2 0.5h), which could therefore confer significant advantages as an oocyte maturation trigger in the treatment of infertility. Whilst MVT-602 has been shown to stimulate LH release in men, its effects in women have not previously been investigated. To address this, we studied the effect of MVT-602 on gonadotrophin release in healthy women for the first time. Methods A two-phase dose-finding study was undertaken during the early follicular phase (Day 1-4) of 12 healthy women aged 18-35yrs with regular menstrual cycles. During phase 1, a broad dose-range for MVT-602 was determined: three women received a single subcutaneous bolus of one of five doses of MVT-602 (0.003, 0.03, 0.1, 0.3, or 1.0nmol/kg) in successive menstrual cycles. Reproductive hormones were measured every 30min for 14hrs post-MVT-602, and again at 24hrs and 48hrs post-injection. During phase 2, an additional 9 women received either KP54 (9.6nmol/kg), or MVT-602 at doses of 0.01 or 0.03nmol/kg during the follicular phase of successive menstrual cycles, following which reproductive hormone levels were monitored every 30mins for 24hrs post-injection. Intervention groups were compared by one-way ANOVA with post hoc Tukey’s test. ResultsPhase 1: Peak serum LH levels occurred between 14-24hrs and no further increases occurred with MVT-602 at doses greater than 0.03 nmol/kg. Phase 2: Peak amplitude of serum LH was found to be similar following KP54 and MVT-602 (mean±SD of peak LH KP54 12.3 ±5.3iU/L; MVT-602 0.01nmol/kg 11.4 ±4.2iU/L; MVT-602 0.03nmol/kg 11.1 ±3.0iU/L, p>0.05). However, time to peak LH was later following MVT-602 (22hrs) when compared with KP54 (4.9hrs; P<0.0001). Thus, the area under the curve (AUC) for LH exposure was significantly increased following MVT-602 when compared with KP54 (mean±SD of AUC LH KP54 9,095 ±5,632iU.min/L; MVT-602 0.01nmol/kg 18,129 ±6,471iU.min/L, P=0.02 vs KP54; MVT-602 0.03nmol/kg 20,086 ±6,227iU.min/L, P=0.004 vs KP54). Conclusion A single subcutaneous bolus of MVT-602 in healthy women resulted in an LH surge of similar amplitude, but of longer duration than by KP54. During studies evaluating the utility of KP54 as a trigger of oocyte maturation in IVF treatment, extending the duration of LH surge with a second bolus of KP54 safely optimised oocyte maturation. Thus, the prolonged gonadotrophin surge induced by MVT-602 could be of significant utility not only to safely optimise oocyte maturation in IVF treatment, but also to treat other reproductive disorders affecting women’s health.

Do Substance P and Neurokinin A Play Important Roles in the Control of LH Secretion in Ewes?

There is now general agreement that neurokinin B (NKB) acts via neurokinin-3-receptor (NK3R) to stimulate secretion of GnRH and LH in several species, including rats, mice, sheep, and humans. However, the roles of two other tachykinins, substance P (SP) and neurokinin A, which act primarily via NK1R and NK2R, respectively, are less clear. In rodents, these signaling pathways can stimulate LH release and substitute for NKB signaling; in humans, SP is colocalized with kisspeptin and NKB in the mediobasal hypothalamus. In this study, we examined the possible role of these tachykinins in control of the reproductive axis in sheep. Immunohistochemistry was used to describe the expression of SP and NK1R in the ovine diencephalon and determine whether these proteins are colocalized in kisspeptin or GnRH neurons. SP-containing cell bodies were largely confined to the arcuate nucleus, but NK1R-immunoreactivity was more widespread. However, there was very low coexpression of SP or NK1R in kisspeptin cells and none in GnRH neurons. We next determined the minimal effective dose of these three tachykinins that would stimulate LH secretion when administered into the third ventricle of ovary-intact anestrous sheep. A much lower dose of NKB (0.2 nmol) than of neurokinin A (2 nmol) or SP (10 nmol) consistently stimulated LH secretion. Moreover, the relative potency of these three neuropeptides parallels the relative selectivity of NK3R. Based on these anatomical and pharmacological data, we conclude that NKB-NK3R signaling is the primary pathway for the control of GnRH secretion by tachykinins in ewes.

Acute regulation of plasma insulin-like peptide 3 concentrations by luteinizing hormone in male goats

Recently, it was reported that in bulls secretion of insulin-like peptide 3 (INSL3) in blood occurred in a pulsatile manner and was acutely regulated by LH. In the present study, the acute regulation of plasma INSL3 and its temporal relationships with LH and testosterone were examined in six sexually matured male goats using the following experimental design. (1) After stimulating LH release by administering a GnRH analogue, blood levels of LH, INSL3, and testosterone were monitored at 15-minute intervals for 2 hours followed by hourly intervals up to 8 hours. (2) After activation of the LH receptor by hCG blood levels of INSL3 and testosterone were determine at 15-minute intervals for 2 hours, followed by hourly intervals up to 8 hours, daily intervals up to Day 8, and finally on Day 12. (3) The release of LH, INSL3, and testosterone in normal physiology was established at 15-minute intervals for an 8-hour session. Concentrations of LH, INSL3, and testosterone in plasma were measured by enzyme-immunoassays. After GnRH treatment, mean plasma concentrations of all three hormones increased (P < 0.05) dramatically from 30 minutes and remained high until 120 minutes (LH), 75 minutes (INSL3), and 4 hours (testosterone) after treatment. After hCG treatment, mean plasma INSL3 concentrations increased (P < 0.05) from 30 minutes and remained elevated until the end of sampling on Day 12. An increase (P < 0.05) in mean plasma testosterone concentrations occurred from 15 minutes and remained high until Day 6. The mean increase (maximum per pretreatment concentration) of INSL3 concentrations after administration of GnRH and hCG was lower (P < 0.01) than that of testosterone. The secretory pattern of LH, INSL3, and testosterone in the general circulation was pulsatile with a frequency of 5.5 ± 0.6, 4.7 ± 0.5, and 2.2 ± 0.5, respectively, during the 8-hour period. Twenty out of 28 (71%) of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse. The mean increase (peak per basal concentration) of INSL3 pulses (2.1 ± 0.1 fold, n = 28) was lower (P < 0.01) than that of testosterone pulses (4.3 ± 2.2 fold, n = 13). In conclusion, secretion of INSL3 in blood occurred, like in bulls, in a pulsatile manner soon after LH pulses in male goats. The absolute concentrations of INSL3 in male goats were higher than that reported in other mammals. Insulin-like peptide 3 concentrations were acutely increased by endogenous and exogenous LH in male goats, but the rise of INSL3 was lower than that of testosterone.

Differential testosterone response to GnRH-induced LH release before and after musth in adult Asian elephant (Elephas maximus) bulls

Bull elephants exhibit marked increases in testosterone secretion during musth, and studies have shown a heightened sensitivity of the testis to GnRH-stimulated testosterone production in musth compared to nonmusth males. However, activity of the hypothalamo-pituitary-gonadal axis before or soon after musth has not been studied in detail. The aim of this study was to evaluate LH and testosterone responses to GnRH challenge in nine adult Asian elephant (Elephas maximus) bulls during three periods relative to musth: premusth, postmusth, and nonmusth. Bulls were administered 80 μg of a GnRH agonist, and blood was collected before and after injection to monitor serum hormone concentrations. The same bulls were injected with saline 2 weeks before each GnRH challenge and monitored using the same blood collection protocol. All bulls responded to GnRH, but not saline, with an increase in LH and testosterone during all three periods. The mean peak LH (1.76 ± 0.19 ng/mL; P < 0.001) and testosterone (6.71 ± 1.62 ng/mL; P = 0.019) concentrations after GnRH were higher than the respective baselines (0.57 ± 0.07 ng/mL, 3.05 ± 0.60 ng/mL). Although basal- and GnRH-induced LH secretion were similar across the stages, evaluation of the area under the curve in GnRH-treated bulls indicated that the testosterone response was greatest during premusth (2.84 ± 0.76 area units; P = 0.019) compared to postmusth (2.02 ± 0.63 area units), and nonmusth (2.01 ± 0.46 area units). This confirms earlier reports that GnRH stimulates LH release and subsequent testosterone production in bull elephants. Furthermore, although the hypothalamo-pituitary-gonadal axis is active throughout the year, the testis appears to be more responsive to LH in terms of testosterone production in the period leading up to musth, compared to the nonmusth and postmusth periods. This heightened sensitivity, perhaps as a result of LH receptor up-regulation, may prime the testis for maximal testosterone production, leading to the physiological and behavioral changes associated with musth.

In vivo pharmacological evaluation of a lactose-conjugated luteinizing hormone releasing hormone analogue

In the current study, the efficacy and pharmacokinetic profile of lactose-conjugated luteinizing hormone releasing hormone (LHRH) was examined following oral administration in male rats. A rapid and sensitive liquid chromatography/mass spectrometry technique was developed and applied for measuring the concentration of lactose[Q1][w6]LHRH (compound 1) in rat plasma in order to allow measurement of pharmacokinetic parameters. LH release was evaluated using a sandwich ELISA. Maximum serum concentration (Cmax=0.11μg/ml) was reached at 2h (Tmax) following oral administration of the compound at 10mg/kg. The half-life was determined to be 2.6h. The absolute bioavailability of the orally administered compound was found to be 14%, which was a remarkable improvement compared to zero-to-low oral bioavailability of the native peptide. Compound 1 was effective in stimulating LH release at 20mg/kg after oral administration. The method was validated at a linear range of 0.01–20.0μg/ml and a correlation coefficient of r2≥0.999. The accuracy and precision values showed the reliability and reproducibility of the method for evaluation of the pharmacokinetic parameters. These findings showed that the lactose derivative of LHRH has a therapeutic potential to be further developed as an orally active therapeutics for the treatment of hormone-dependent diseases.

Ovulation-inducing factor (OIF) induces LH secretion from pituitary cells

A substance in the seminal plasma of llamas and alpacas has been discovered that induces ovulation and growth of the corpus luteum (CL) in the female of the same species. The ovarian effects of the ovulation-inducing factor (OIF) are associated with a surge release of LH into circulation. We hypothesize that OIF stimulates LH release from gonadotroph cells in the anterior pituitary gland. Four experiments were done to determine if purified OIF isolated from llama seminal plasma stimulates LH secretion in pituitary cells using tissue from an induced ovulator (llama) and spontaneous ovulator (cattle). Anterior pituitary cells were cultured in vitro for two days, and on the third day, wells were incubated for 2h with media containing no treatment (control), GnRH or OIF. Concentrations of LH in the culture medium were measured using radioimmunoassay and compared among groups by analysis of variance. In all experiments, GnRH and OIF treatments induced more LH secretion than untreated controls (P<0.05). A dose-related effect was evident in the llama pituitary cell cultures in that mean LH concentrations were greater (P<0.05) in wells treated with a higher dose of OIF (5.41±0.28ng/mL) compared to wells treated with a lower dose (2.70±0.50ng/mL), both of which were higher (P<0.05) than in wells with no treatment (0.87±0.18ng/mL). Although OIF stimulated LH release in bovine cell cultures, a dose-related effect was not detected. We conclude that OIF stimulates LH secretion from pituitary gonadotrophs in vitro.

Mollusc gonadotropin-releasing hormone directly regulates gonadal functions: A primitive endocrine system controlling reproduction

Gonadotropin-releasing hormone (GnRH) is central to the control of vertebrate reproductive cycles and since GnRH orthologs are also present in invertebrates, it is likely that the common ancestor of bilateral animals possessed a GnRH-like peptide. In order to understand the evolutionary and comparative biology of GnRH peptides we cloned the cDNA transcripts of prepro GnRH-like peptides from two species of bivalve molluscs, the Yesso scallop Patinopecten yessoensis and the Pacific oyster Crassostrea gigas. We compared their deduced uncleaved and mature amino acid sequences with those from other invertebrates and vertebrates, and determined their sites of expression and biological activity. The two molluscan GnRH sequences increased the number of known protostome GnRHs to six different forms, indicating the current classification of protostome GnRHs requires further revision. In both molluscs, RT-PCR analysis showed that the genes were highly expressed in nervous tissue with lower levels present in peripheral tissues including the gonads, while immunocytochemistry, using anti-octopus GnRH-like peptide, demonstrated the presence of GnRH-like peptide in neural tissue. Putative scallop GnRH-like peptide stimulated spermatogonial cell division in cultured scallop testis, but the scallop GnRH-like peptide did not stimulate LH release from cultured quail pituitary cells. This is the first report of the cloning of bivalve GnRH-like peptide genes and of molluscan GnRH-like peptides that are biologically active in molluscs, but not in a vertebrate.

Identification, Expression, and Physiological Functions of Siberian Hamster Gonadotropin-Inhibitory Hormone

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. To further understand its physiological roles in mammalian reproduction, we identified its precursor cDNA and endogenous mature peptides in the Siberian hamster brain. The Siberian hamster GnIH precursor cDNA encoded two RFamide-related peptide (RFRP) sequences. SPAPANKVPHSAANLPLRF-NH(2) (Siberian hamster RFRP-1) and TLSRVPSLPQRF-NH(2) (Siberian hamster RFRP-3) were confirmed as mature endogenous peptides by mass spectrometry from brain samples purified by immunoaffinity chromatography. GnIH mRNA expression was higher in long days (LD) compared with short days (SD). GnIH mRNA was also highly expressed in SD plus pinealectomized animals, whereas expression was suppressed by melatonin, a nocturnal pineal hormone, administration. GnIH-immunoreactive (-ir) neurons were localized to the dorsomedial region of the hypothalamus, and GnIH-ir fibers projected to hypothalamic and limbic structures. The density of GnIH-ir perikarya and fibers were higher in LD and SD plus pinealectomized hamsters than in LD plus melatonin or SD animals. The percentage of GnRH neurons receiving close appositions from GnIH-ir fiber terminals was also higher in LD than SD, and GnIH receptor was expressed in GnRH-ir neurons. Finally, central administration of hamster RFRP-1 or RFRP-3 inhibited LH release 5 and 30 min after administration in LD. In sharp contrast, both peptides stimulated LH release 30 min after administration in SD. These results suggest that GnIH peptides fine tune LH levels via its receptor expressed in GnRH-ir neurons in an opposing fashion across the seasons in Siberian hamsters.

Neurokinin B and Kisspeptin: Sexual Partners or Single Agents?

Despite recent progress in our understanding of reproductive physiology, the precise mechanisms controlling puberty still remainelusive.Patientswith idiopathichypogonadotrophic hypogonadism (IHH) have pubertal failure as a result of insufficient gonadotrophin release, which is usually caused by a defect in GnRH synthesis, secretion, or activity. Inactivatingmutations inanumberofgeneshavebeen found to cause IHH, but the underlying genetic defect in many patients with IHH remains unidentified (1). Two novel neuropeptides, kisspeptin and neurokinin B, have emerged recently as “new kids on the block” and caused an explosion of research in reproductive endocrinology. Their similar anatomical and functional characteristics have prompted speculation whether a “sexual partnership” exists between them to control reproductive maturation. Kisspeptin emerged as an important new player in reproductivephysiology in2003whendeRouxetal. (2)andSeminara et al. (3) discovered that humans with inactivating mutations of the kisspeptin receptor had a phenotype of hypogonadotrophic hypogonadism. Furthermore, animals lacking kisspeptin (4, 5) or its receptor (3) have a similar phenotype. Over the past 7 yr, more than 400 subsequent publications have elucidated the effects of kisspeptin (comprehensively reviewed in Ref. 6). Notably, administration of kisspeptin potently stimulates reproductive hormone release in all species studied (6), including humans (7–9). The stimulatory action of kisspeptin on gonadotrophin release is abolished by pharmacological antagonism of GnRH, thus suggesting a hypothalamic site of action (6). Kisspeptin is expressed in specific hypothalamic nuclei, such as the anteroventral periventricular and arcuate nuclei, which each have important roles in its physiological action (6). Recently, it has emerged that neurokinin B is also vital for normal pubertal function. Topaloglu et al. (10) discovered that inactivating mutations of neurokinin B or its receptor caused hypogonadotrophic hypogonadism in humans. Recent data show that neurokinin B and kisspeptin are colocalized in the arcuate nucleus in sheep (11), mice (12), rats (13), and humans (14). However, in contrast to the robust stimulatory action of kisspeptin on gonadotrophin release demonstrated in numerous species, the effects of administering neurokinin B to animals are less clear. Neurokinin B administration has been shown stimulate LH release in sheep (15) but have no effect (16, 17) or inhibit (12, 18) LH release in rodents. It is also interesting that mice with targeted deletion of neurokinin B receptor are fertile (19), in contrast to kisspeptin or kisspeptin receptor knockout mice (3–5). It is therefore timely that, in this issue of Endocrinology, Ramaswamy et al. (20) demonstrate in elegant primate studies that a single i.v. injection of neurokinin B robustly stimulatesLHrelease.Thiswork follows fromstudiesofkisspeptin administration to primates, performed by the same researchers. As observed previously with kisspeptin administration (21), the effect of neurokinin B on LH release was abolished by GnRH receptor antagonism (20), thus suggesting a hypothalamic site of action. Ramaswamy et al. also show that neurokinin B and kisspeptin are colocalized in the arcuate nucleus of primates (20), which is consistent with the findings inrodents, sheep,andhumans(11–14).However, in contrast to previous results with kisspeptin (22), repeated hourly injection of a neurokinin B agonist did not result in sustainedLHrelease(20). Interestingly,repeatedadministrationof the neurokinin B did not affect the ability of kisspeptin to stimulate GnRH release (20). The precise animal model chosen by Ramaswamy et al. in the current study may be important; ago-

Kisspeptin Signaling Is Required for Peripheral But Not Central Stimulation of Gonadotropin-Releasing Hormone Neurons by NMDA

NMDA and kisspeptins can stimulate gonadotropin-releasing hormone (GnRH) release after peripheral or central administration in mice. To determine whether these agonists act independently or through a common pathway, we have examined their ability to stimulate GnRH/luteinizing hormone (LH) release after peripheral or central administration in Kiss1- or Gpr54 (Kiss1r)-null mutant mice. Peripheral injection of NMDA failed to stimulate GnRH/LH release in prepubertal or gonadally intact mutant male mice. Dual-labeling experiments indicated a direct activation of Kiss1-expressing neurons in the arcuate nucleus. In contrast, central injection of NMDA into the lateral ventricle increased plasma LH levels in both Kiss1 and Gpr54 mutant male mice similar to the responses in wild-type mice. Central injection of NMDA stimulated c-Fos expression throughout the hypothalamus but not in GnRH neurons, suggesting an action at the nerve terminals only. In contrast, kisspeptin-10 stimulated LH release after both central and peripheral injection but induced c-Fos expression in GnRH neurons only after central administration. Finally, central injection of NMDA induces c-Fos expression in catecholamine- and nitric oxide-producing neurons in the hypothalamus of mutant mice, indicating a possible kisspeptin-independent GnRH/LH release by NMDA through activation of these neurons. Thus, NMDA may act at both GnRH cell bodies (kisspeptin-independent) and nerve terminals (kisspeptin-dependent) in a dual way to participate in the GnRH/LH secretion in the male mouse.

Steroid effects on the in vitro LH secretion from pituitary cells of sexually immature carp (Cyprinus carpio L.) under the influence of naltrexone and morphine

Dispersed cells from sexually immature carp (footlings) pituitaries were exposed to estradiol (E&lt;sub&gt;2&lt;/sub&gt;) or testosterone (T) (both at 3 &amp;times; 10&lt;sup&gt;&amp;ndash;8&lt;/sup&gt; M) in the presence of opioid receptor antagonist naltrexone (10&lt;sup&gt;&amp;ndash;8&lt;/sup&gt; or 10&lt;sup&gt;&amp;ndash;6&lt;/sup&gt; M) and/or agonist &amp;ndash; morphine (10&lt;sup&gt;&amp;ndash;8&lt;/sup&gt; or 10&lt;sup&gt;&amp;ndash;6&lt;/sup&gt; M). Naltrexone alone at 10&lt;sup&gt;&amp;ndash;6&lt;/sup&gt; M increased the LH level as compared with the control. Morphine (10&lt;sup&gt;&amp;ndash;8&lt;/sup&gt; or 10&lt;sup&gt;&amp;ndash;6&lt;/sup&gt; M), T and E2 had no influence on LH levels. The combination of T with naltrexone (10&lt;sup&gt;&amp;ndash;8&lt;/sup&gt; M) stimulated LH release if compared with the control or with T alone. Morphine (both concentrations) with T caused significantly higher LH secretion than the control medium and T alone. Estradiol with naltrexone (10&lt;sup&gt;&amp;ndash;8&lt;/sup&gt; and 10&lt;sup&gt;&amp;ndash;6&lt;/sup&gt; M) had no influence on LH concentration. In media with E2 and morphine (10&lt;sup&gt;&amp;ndash;8&lt;/sup&gt; M) LH levels were higher than in the control and estradiol alone. The results show that in common carp sex steroids affect the response of pituitary cells to opioid agonist or antagonist giving an evidence on the role of steroids in LH release mediated by the opioid system.

The stimulatory effect of pituitary adenylate cyclase activating polypeptide (PACAP) on LH release from rat pituitary cells in vitro does not involve calcium mobilization.

The hypothalamic peptide "pituitary adenylate cyclase activating polypeptide (PACAP)" stimulates cAMP production in cultured rat pituitary cells and enhances LH release. It has been suggested that the stimulation of LH release by PACAP comprises two distinct mechanisms: a direct stimulatory action on LH secretion and a potentiation of the response of the gonadotrophes to LHRH. Thus the possibility exists that PACAP may enhance LH secretion not only by increased cAMP production but also by increasing cytosolic Ca2+ concentrations ([Ca2+]). In the present study we examined whether PACAP affects cytosolic [Ca2+] in identified rat gonadotrophes (as determined by the fura-method) and whether the suggested potentiating effect of PACAP on LHRH induced LH release is dependent on Ca2+. PACAP (1 nM) and 0.1 nM LHRH significantly increased LH concentrations in the culture medium after 5 hrs of incubation. Coincubation of cells with both peptides resulted in an additive increase of LH release. While the stimulatory effect LHRH was blunted in Ca(2+)-free medium, PACAP remained stimulatory to LH release. PACAP stimulated cAMP formation regardless whether the culture medium contained Ca2+ or not. Gonadotrophes were selected by their response to LHRH (1 microM) and were subsequently challenged with PACAP (1 microM). About 75% of gonadotrophes responded also to PACAP with an increase of cytosolic [Ca2+] which was blunted by removal of extracellular Ca2+. We suggest that in the rat pituitary the majority of the gonadotrophes are PACAP responsive as determined by an increase of cytosolic [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)

Potencies of Centrally- or Peripherally-Injected Full-Length Kisspeptin or Its C-Terminal Decapeptide on LH Release in Intact Male Rats

The aim of the present study was to compare the effects of full-length rat kisspeptin (rKp-52) with C-terminal decapeptide (Kp-10) of rat or human kisspeptin on LH release in intact male rats. Plasma LH profiles were determined by frequent blood sampling at 6-min intervals for 3 h after central or peripheral injection of kisspeptins. Intracerebroventricular (icv) injection of rKp-52 (0.1 nmol) induced a gradual increase in the plasma LH level, which remained high for the rest of the sampling period. On the other hand, icv injection of rKp-10 did not increase the plasma LH level at the same dose (0.1 nmol). A 10-times higher dose (1 nmol) of rKp-10 and hKp-10 increased the plasma LH level, but the increase was lower than that of rKp-52 icv injection. Intravenous (iv) injection of kisspeptins also stimulated LH release at 10 or 100 nmol/kg. In rKp-52 (10 nmol/kg)-treated animals, the plasma LH level reached a peak within 30 min and remained high until 60 min postinjection. The rKp-10- and hKp-10-injected animals showed a more rapid decline in plasma LH level after the peak found at around 30 min after the injections at both middle (10 nmol/kg) and high (100 nmol/kg) doses. The present study indicates that full-length kisspeptin is more effective in stimulating LH release compared with Kp-10 in male rats. The difference in LH-releasing activity may be the result of a difference in degradation of the peptides, but it is still worth determining whether an active domain other than the C-terminal decapeptide is present in full-length kisspeptin.