LH Pulse Amplitude Research Articles

Longitudinal Evaluation of Reproductive Endocrine Function in Men with ACTH-Dependent Cushing Syndrome.

Hypogonadism may be caused by Cushing syndrome (CS) and may intensify its adverse consequences. To determine the frequency of male hypogonadism before and after curative surgery for CS, and its cause. Post-hoc analyses of prospective cohort studies. Clinical research center. Men with ACTH-dependent CS. Cohort 1 (C1) (n=8, age 32.5±12 y; studied 1985-1989); Cohort 2 (C2) (n=44, 42.7 ± 15.1 y; studied 1989-2021). C1: Every 20-minute blood sampling for 24h before and 1-40 months after surgical cure. Three subjects underwent GnRH stimulation tests pre- and post-surgery. C2: Hormone measurements at baseline and 6 and 12 months (M) post-cure. C1: LH, FSH, LH pulse frequency and LH response to GnRH. C2: LH, FSH, testosterone (T), free T, fT4, T3, TSH and UFC levels and frequency of hypogonadism pre- and post-surgery. C1: mean LH and LH pulse frequency increased after surgery (p < 0.05) without changes in LH pulse amplitude, mean FSH, or peak gonadotropin response to GnRH. C2: 82% had baseline hypogonadism (total T 205 ± 28 ng/dL). Thyroid hormone levels varied inversely with UFC and cortisol. LH, total and free T, and SHBG increased at 6M and 12M post surgery, but hypogonadism persisted in 51% at 6M and in 26% at 12M. Hypogonadism in men with CS is widely prevalent but reversible in ∼75% of patients one year after surgical cure and appears to be mediated through suppression of hypothalamic GnRH secretion, and modulated by thyroid hormones.

Metabolic hormonal alterations in functional hypothalamic amenorrhea and anovulation associated with Polycystic ovary syndrome

Background: Polycystic ovary syndrome (PCOS) is a multifaceted endocrine disorder affecting many women during their reproductive years. It is characterized by oligo/amenorrhea, anovulatory cycles, polycystic ovaries, and insulin resistance. This review explores the hormonal and metabolic alterations associated with PCOS, comparing them to functional hypothalamic amenorrhea (FHA). Key aspects include the abnormally high LH pulse frequency in PCOS, indicating hyperactive gonadotropin-releasing hormone (GnRH), and the role of hyperandrogenemia in exacerbating the condition by increasing LH pulse secretion from the pituitary gland. Additionally, the review examines the neuroendocrine basis for PCOS. Methods: The methodology involved analyzing neuroendocrine pathways and physical manifestations through PubMed, ScienceDirect, and Scopus databases. Findings indicate that PCOS is primarily characterized by androgen excess, ovulatory dysfunction, and disruption of the hypothalamic-pituitary-ovarian (HPO) axis. Hormonal dysregulation includes disturbances in GnRH, insulin, LH/FSH ratio, and androgens. GnRH stimulates LH and FSH release from the pituitary, regulating ovarian function, while Anti-Müllerian hormone (AMH) inhibits follicular development in PCOS. Conclusion: The review concludes by highlighting the hormonal alterations, including decreased frequency and amplitude of LH pulses, disruptions in GnRH, LH, and FSH. Genetic predispositions and disturbances in the LH/FSH ratio can lead to impaired follicle growth and polycystic ovaries. This comprehensive exploration underscores the importance of understanding the hormonal and neuroendocrine mechanisms underlying PCOS, contributing to better diagnosis and treatment strategies

Reproductive neuroendocrine defects programmed by prenatal testosterone treatment between gestational days 60-90 are amplified by postnatal obesity in sheep.

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women of reproductive age, and obesity can increase the severity and development of the PCOS phenotype. Prenatal testosterone (T) treatment between gestational days 30-90 advanced puberty and disrupted the reproductive and metabolic phenotype in female sheep, recapitulating attributes of women with PCOS, with postnatal obesity amplifying its severity. On the other hand, prenatal T treatment from gestational days 60-90 led to a much milder phenotype. We hypothesized that reproductive neuroendocrine defects programmed by prenatal T treatment between gestational days 60-90 are amplified by postnatal obesity in sheep. Suffolk ewes received T propionate (T; 100mg) or corn oil (C; vehicle) twice weekly from gestational days 60-90. At 5months of age, T lambs were assigned to either a maintenance (100% of NRC requirements) or overfed (130% NRC) diet and C lambs were fed the maintenance diet. We compared the timing of puberty (n = 15/group) determined by twice weekly measurement of progesterone concentrations, estradiol positive feedback responsiveness (n = 8/group) determined by assessing LH secretion in response to exogenous estradiol, periovulatory LH dynamics during the second breeding season (n = 8/group) following synchronization with two injections of PGF2α, and progesterone negative feedback (n = 8/group) determined by characterizing LH pulses during the mid-luteal phase between C, T-maintenance and T-overfed groups. Our findings indicate that postnatal obesity: 1) exacerbated reproductive defects and further deteriorated reproductive cyclicity during the second breeding season (adulthood); 2) did not amplify the impairment in estradiol positive feedback in delaying the timing and amplitude of the LH surge, although it reduced the total amount of LH secreted during the preovulatory LH surge; 3) amplified the reduced responsiveness to progesterone negative feedback manifested as an increase in LH pulse amplitude and peak. These observations, in addition to supporting our previous findings that prenatal T treatment results in reproductive neuroendocrine dysfunction and periovulatory disruptions, provide evidence that these neuroendocrine defects programmed between gestational days 60-90 are amplified by postnatal obesity in female sheep.

SAT608 Gonadotrope-specific Deletion Of Icer Results In Increased Lh Pulsatility But Reduced Fsh Levels In Mice

Abstract Disclosure: N.P. Boris: None. S.A. Pereira: None. G.A. Stamatiades: None. K.N. Hausken: None. H. Kim: None. R.S. Carroll: None. U.B. Kaiser: None. Background: The hypothalamic-pituitary-gonadal (HPG) axis is regulated by pulsatile secretion of hypothalamic GnRH, which acts on anterior pituitary gonadotropes to control synthesis and secretion of LH and FSH. High and low GnRH pulse frequencies preferentially stimulate Lhb and Fshb expression, respectively, through mechanisms that are not completely understood. The mouse Fshb gene promoter contains a half-AP-1/half-cAMP response element (CRE) motif; we have shown previously that this element is bound by CRE-binding protein (Creb) in vitro to increase expression in response to low GnRH pulse frequencies, but is antagonized by inducible cAMP early repressor (Icer) at higher GnRH pulse frequencies, resulting in a lower level of Fshb transcription. In this study, we aimed to identify the role of Icer in gonadotropin regulation in vivo in mice. We hypothesized that gonadotrope-specific deletion of Icer may influence pubertal timing and fertility due to an increase in Fshb expression. Methods and Results: Gonadotrope-specific deletion of Icer (GnrhrGRIC/+/Icerfl/fl; Icer KO) and control (Icerfl/fl) mice were generated, and pubertal and reproductive phenotypes were evaluated. There was no difference in body weight or in pubertal timing, evaluated by age of preputial separation in males and by vaginal opening and first estrus in females. In post-pubertal male mice, neither basal LH and FSH levels nor the LH or FSH response to acute GnRH stimulation were significantly different in Icer KO mice, compared to littermate Icerfl/fl controls. Male Icer KO mice 14 days post-gonadectomy (GDX) had significantly more LH pulses within a three-hour period than controls (Icer KO 5.83 ± 0.4 pulses vs Icerfl/fl 4.66 ± 0.3 pulses; p=0.049), but no differences in basal LH or LH pulse amplitude. Interestingly, serum FSH levels were significantly lower in Icer KO mice compared to Icerfl/fl controls 2 days post-GDX (Icer KO 20.0 ± 1.0 vs Icerfl/fl 26.0 ± 0.8; p=0.003) and 7 days post-GDX (Icer KO 18.0 ± 0.4 vs Icerfl/fl 22.5 ± 0.8, p=0.002). There were no significant differences in pituitary Fshb, Lhb, or Gnrhr mRNA levels between Icer KO and Icerfl/fl control mice 14 days post-GDX. Conclusions: Deletion of Icer from pituitary gonadotropes in mice did not affect body weight or timing of pubertal onset. However, in GDX male mice, LH pulse frequency was increased, whereas the post-GDX increase in serum FSH levels was blunted, in Icer KO mice compared to controls. These findings support a role for Icer in both LH and FSH regulation in vivo and highlight the importance of extending in vitro observations to in vivo models. Presentation: Saturday, June 17, 2023

Lesions of KNDy and Kiss1R Neurons in the Arcuate Nucleus Produce Different Effects on LH Pulse Patterns in Female Sheep.

The current model for the synchronization of GnRH neural activity driving GnRH and LH pulses proposes that a set of arcuate (ARC) neurons that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) is the GnRH pulse generator. This study tested the functional role of ovine KNDy neurons in pulse generation and explored the roles of nearby Kiss1 receptor (Kiss1R)-containing cells using lesions produced with saporin (SAP) conjugates. Injection of NK3-SAP ablated over 90% of the KNDy cells, while Kiss-SAP (saporin conjugated to kisspeptin-54) lesioned about two-thirds of the Kiss1R population without affecting KNDy or GnRH cell number. Both lesions produced a dramatic decrease in LH pulse amplitude but had different effects on LH pulse patterns. NK3-SAP increased interpulse interval, but Kiss-SAP did not. In contrast, Kiss-SAP disrupted the regular hourly occurrence of LH pulses, but NK3-SAP did not. Because Kiss1R is not expressed in KNDy cells, HiPlex RNAScope was used to assess the colocalization of 8 neurotransmitters and 3 receptors in ARC Kiss1R-containing cells. Kiss1R cells primarily contained transcript markers for GABA (68%), glutamate (28%), ESR1 (estrogen receptor-α) mRNA, and OPRK1 (kappa opioid receptor) mRNA. These data support the conclusion that KNDy neurons are essential for GnRH pulses in ewes, whereas ARC Kiss1R cells are not but do maintain the amplitude and regularity of GnRH pulses. We thus propose that in sheep, ARC Kiss1R neurons form part of a positive feedback circuit that reinforces the activity of the KNDy neural network, with GABA or glutamate likely being involved.

Temporality of ovarian steroids and LH/FSH pulse profiles encompassing selection of the dominant follicle in heifers†.

The tested hypotheses were (1) LH/FSH pulses and F2 diameter are diminished by P4 and, (2) E2 increases during the transition to deviation and alters LH/FSH pulses. On Day 5 (Day 0 = ovulation), heifers were randomized into an untreated group (HiP4, n = 11), and a prostaglandin analog treated group (NoP4, n = 10). On Day 6, a follicular wave was induced by follicle ablation. Ultrasound and blood collections were performed every 12h from Days 7 to 11. Blood was collected every 15min for 10h on Day 9 (largest follicle expected to be ~7.5mm). Estradiol was ~75% greater (0.36 ± 0.14 vs 0.63 ± 0.19pg/mL) in heifers with F1 ≥ 7.2mm than in heifers with F1 < 7.2mm. The HiP4 had smaller second largest follicle (F2) diameter, lower estradiol (P = 0.06), LH pulse baseline and peak concentrations (P < 0.007), in addition to half the frequency of LH/FSH pulses (4.1 ± 0.3 vs 9.6 ± 0.7 in 10h) than the NoP4. Within HiP4, heifers with F1 ≥ 7.2mm had ~25% fewer (P = 0.03) LH pulses compared to heifers with F1 < 7.2mm. In contrast, within the NoP4, heifers with F1 ≥ 7.2mm had ~75% greater LH (P = 0.05) and FSH (P = 0.08) pulse amplitude. We propose that greater F2 diameter at deviation in low P4 is related to greater LH baseline and peak concentrations, and greater frequency of LH/FSH pulses. A greater increase in E2 after F1 reaches ~7.2mm results in further stimulation of LH/FSH pulse amplitude. Elevated P4 not only diminished frequency of LH/FSH pulses but also converted an E2 increase into a negative feedback effect on LH/FSH pulse frequency leading to smaller F2 at deviation.

Exposure of rams in sexual rest to sexually activated males in spring increases plasma LH and testosterone concentrations

Eight stimulating rams, and twelve stimulated rams, were used to determine whether a similar endocrine response to the introduction of sexually active males in spring in a flock of ewes is observed in a flock of rams. The stimulating rams (n = 4) were induced into a sexually active state by exposure to 2 months of long days (16 h light/d) (15 December-15 February). At the end of the long-day period, rams were returned to the natural photoperiod. Control-stimulating rams (n = 4) were kept under the natural photoperiod. On April 20, stimulated rams were divided into 2 groups, and joined with activated (ACT; n = 6) or control stimulating rams (C; n = 6). On the day of ram introduction, stimulated rams were blood sampled for 8 h at 20-min intervals, from 4 h before to 4 h after ram introduction, and next day from 24 to 28 h after ram introduction, and analyzed for plasma LH concentrations, and 10, 20 and 30 days after ram introduction to measure plasma testosterone levels. Mean (±SEM) plasma LH concentrations (ng/ml) of stimulated rams were similar during the 4 h before stimulating-ram introduction (ACT: 0.59 ± 0.03; C: 0.53 ± 0.04; P > 0.05). The introduction of the photoperiod-treated stimulating rams increased LH concentrations of stimulated rams during the 4 h after their introduction (1.14 ± 0.37) compared with the C group (0.51 ± 0.03; P < 0.05), especially during the first hour (ACT: 0.93 ± 0.16; C: 0.49 ± 0.03; P < 0.05), and during the blood sampling period 24–28 h after ram introduction (0.75 ± 0.07 vs. 0.58 ± 0.04; P < 0.05). Before the introduction of stimulating rams, the LH pulse frequencies and amplitudes did not differ between groups; however, LH pulsatility was higher at 4 h (0.58 ± 0.11 pulses/h; P < 0.05), and had trend to be higher 24 h (0.50 ± 0.06) (P = 0.10) after the introduction of the photoperiod-treated stimulating rams compared with the control-stimulating rams (0.29 ± 0.08 and 0.29 ± 0.10, respectively). As for LH pulses, there was an effect of group (P < 0.05) on LH amplitude, which presented a trend to be higher in ACT rams 4 h after ram introduction (1.68 ± 0.30; P < 0.10) and higher 24 h (1.07 ± 0.08; P < 0.05) after ram introduction, compared with LH amplitudes of C rams (0.71 ± 0.06 and 0.82 ± 0.07, respectively). Plasma testosterone concentrations of rams exposed to photoperiod-treated activated rams were higher than those of rams exposed to control-stimulating rams, at 4 h, 20 and 30 days after ram introduction (P < 0.05). In conclusion, sexually active rams in spring are able to stimulate LH and testosterone secretion of other rams in sexual rest, a phenomenon we called “ram-to-ram effect".

252 Impact of Pre- and Postnatal Nutritional Extremes on Tonic Secretion of Gonadotropins and Feedback Responsiveness to Estradiol in Sexually Mature Heifers

Abstract Objectives were to test the hypotheses that nutritional extremes during prenatal development interact with postnatal nutrition during the juvenile period of heifers to impact 1) tonic secretion of gonadotropins, and 2) estradiol-17β (E2) negative and positive feedback responsiveness in adulthood. Heifers were selected from a larger population programmed nutritionally using a 3 x 2 factorial arrangement of pre- and postnatal diets. Beginning at 90 days of pregnancy, Bos indicus-influenced cows (n = 95) were fed to achieve body condition scores (BCS; 1–9 scale) of 3–3.5 (L; thin), 5.5–6 (M; moderate), or 7.5–8 (H; obese) by onset of the third trimester and maintained thereafter. Heifer offspring were weaned at 3–3.5 months of age and assigned to either a low- (L; 0.5 kg/day) or high-gain (H; 1.0 kg/day) diet until 8 mo. of age, then fed a common diet until puberty. Heifers (n = 18; 6/grp) representing HH, MH and LL combinations were ovariectomized postpubertally (17.1 mo. of age) and received E2 replacement. In Exp. 1, blood samples were collected at 10-min intervals to evaluate pulsatile secretion of LH and FSH for 5.5 hours. In Exp. 2, heifers received E2 (2.4 ug/kg I.M.), with blood sampling at 30-min to 1-h intervals for 30 h. Heifers in the MH (1.25 ± 0.11 ng/mL) group tended (P &amp;lt; 0.09) to have greater LH pulse amplitude compared to HH (0.91 ± 0.14 ng/mL) and LL (0.96 ± 0.09 ng/mL); otherwise, frequency, amplitude, and mean concentrations of LH and FSH did not differ among groups. Exogenous E2 (Exp. 2) suppressed (P &amp;lt; 0.0001) mean plasma concentrations of LH and FSH equally among groups, then stimulated equivalent surges of LH beginning at 14 ± 0.02 h, with only two FSH surges detected. Neither pre- nor postnatal nutrition affected E2 negative or positive feedback in this study.

Gonadotropin Response to Insulin and Lipid Infusion Reproduces the Reprometabolic Syndrome of Obesity in Lean Women

Introduction: The reprometabolic syndrome of obesity is associated with reduced gonadotropins and impaired LH and FSH response to gonadotropin releasing hormone (GnRH). We sought to reproductive the reprometabolic syndrome in normal weight, eumenorrheic women by infusing a combination of insulin and lipid. Materials and Methods: 15 women, mean age 32 (IQR 26,36) and BMI 21.9 (20.2, 22.9) were recruited with intent to perform early follicular phase, 6-hour infusions of insulin (20-40mg/mU/m2/min) and lipid (Intralipid) or saline infusion (controls); 12 women completed both intended studies and an additional 3 women completed only one of the two studies. The first 4 hours of each study assessed endogenous gonadotropins; at 4hrs, a 75 ng/kg GnRH bolus was administered and sampling continued until 6hrs. Linear mixed model analysis was used to determine differences between Intralipid versus saline on endogenous LH pulse amplitude (primary outcomes), mean FSH, and area under the curve (AUC) response to GnRH (secondary outcomes). Results: LH pulse amplitude, mean FSH, and both AUC responses to GnRH were all reduced by Intralipid/insulin; mean FSH (P=0.03) and AUC for LH (P=0.05) were at or near statistical significance. LH pulse amplitude and response to GnRH were significantly reduced (P=0.04 and 0.02) when one participant with very high LH and AMH levels was excluded. Discussion: Acute infusion of insulin/lipid to eumenorrheic, normal weight women recapitulated the reprometabolic syndrome of obesity. These findings imply that specific circulating factors in obese women contribute to their sub fertility and thus may be amenable to discovery and treatment.

Mutation of the GnRHR Proximal Promoter AP-1 Element in Mice Results in Suboptimal GnRH Induction of LH and an Abnormal Reproductive Phenotype

Reproduction is regulated by the gonadotropins, LH and FSH, which are synthesized and secreted by pituitary gonadotrophs in response to hypothalamic GnRH in a pulse frequency dependent manner. The gonadotroph decodes GnRH pulsatility via the GnRH receptor (GnRHR), which increases in expression and cell surface density before estrus and is responsible for downstream signaling cascades that differentially favor gonadotropin expression. The gonadotroph Gnrhr promoter contains a tripartite enhancer, including an AP-1 element that is necessary for full GnRH induction of Gnrhr expression in vitro. We previously generated an AP-1 knock-in (KI) mouse model with a single point mutation (C-269T) in the Gnrhr promoter AP-1 binding motif that resulted in an abnormal reproductive phenotype in female mice. Compared to wildtype (WT) littermates, female KI mice had a significant delay in first estrus, disrupted estrous cyclicity, fewer corpora lutea, and smaller litters. Males had no apparent reproductive phenotype. Basal serum gonadotropin levels were similar between WT and KI mice, but gonadectomy induced a significantly lower rise in serum LH levels of KI mice relative to WT mice, concomitant with significantly lower pituitary Gnrhr, Lhb, and Fshb mRNA levels in both sexes. We have now extended the characterization of these mice by measuring LH pulsatility and assessing GnRH induction of LH in vivo and in vitro. The frequency and amplitude of LH pulses over three hours were similar in ovariectomized WT and KI mice; however, KI mice had significantly reduced LH secretion, as measured by area under the curve. Similarly, GnRH treatment induced a diminished LH response in intact KI compared to WT males. In vitro cultures of hemi-pituitaries from gonadectomized WT and KI males were exposed to 0.01 nM GnRH and LH secretion into culture media was measured by ELISA at 0, 0.5, 1, 2, and 4 hours. There was no difference in basal LH secretion between WT and KI pituitaries but GnRH induction of LH was significantly lower in cultures from AP-1 mutant mice, indicating a direct impairment of GnRH action at the level of the pituitary. Taken together, these data indicate that the gonadotroph Gnrhr AP-1 promoter motif is critical for normal reproductive function. Prevention of AP-1 binding to the Gnrhr proximal promoter element decreases GnRH-induced Gnrhr, Lhb, and Fshb levels, impairs GnRH-stimulated LH secretion, and disrupts pubertal development and reproductive cyclicity in female mice.

Resolution of Abnormal Gonadotropin Secretion After Surgical Cure in Men With ACTH-Dependent Cushing Syndrome

Background: Hypothalamic-pituitary-gonadal (HPG) dysfunction is known to occur in male patients with Cushing Syndrome (CS) but the underlying mechanisms of HPG dysfunction remain unclear. We performed frequent blood sampling and GnRH stimulation tests to assess gonadotropin secretion in males before and after surgical cure of CS. Methods: We evaluated eight males age 32.5±12 years (mean ± SD) enrolled for surgical cure of ACTH-dependent CS at a tertiary care research center. Urinary free cortisol (UFC) was measured before and after surgery. Blood was sampled every 20 minutes for 24-hours with measurement of LH and FSH, before and 2 weeks to 160 weeks (median 80 weeks) after surgery. Patients received 100 μg of gonadotropin releasing hormone (GnRH) iv with measurement of LH and FSH at 0, 10 and 20 min before and 15, 30, 45, 60, 120 and 180 min after administration. Non-normative data was log-transformed before analysis using paired two-tail t-tests. A p-value of <0.05 was considered significant. Gonadotropin values are expressed in U/L as equivalents of the 2nd International Preparation of human menopausal gonadotropins with normal adult male ranges of 6-26 and 5-25 IU/L for LH and FSH, respectively. Results: Mean UFC was elevated ~ 6 times above the upper normal limit before surgery and returned to the reference range after surgery. 24-hour LH rose from 7.82 ± 1.48 preoperatively to 13.07 ± 2.96 IU/L (mean ± sem) after surgery (p=0.026) while mean FSH was unchanged (8.48 ± 1.51 vs 6.92 ± 1.29, p=0.37). LH pulse frequency, a marker of pulsatile GnRH secretion, recovered from a subnormal value of 6.88 ± 0.55 pulses/24 hr before to 12.13 ± 0.72 pulses/24 hr after surgery (p<0.0001) while LH pulse amplitude did not change (6.47 ± 1.21 vs 4.76 ± 0.88 IU/L, p=0.21). In response to GnRH there was a robust increase in LH that was not affected by curative surgery for CS (27.0 ± 7.2 vs 21.2 ± 7.6 IU/L; p=0.14) as was also the case for FSH (3.7 ± 1.2 vs 2.3 ± 0.5 IU/L; p=0.48), consistent with the absence of an effect of CS on gonadotroph responsiveness to LHRH. Conclusion: In men with Cushing Syndrome, hypogonadism is associated with inhibition of endogenous GnRH secretion but preservation of the pituitary response to GnRH. Hypothalamic suppression of the HPG axis is reversible after cure of CS.

Differing planes of pre- and postweaning phase nutrition in Holstein heifers: II. Effects on circulating leptin, luteinizing hormone, and age at puberty

The objectives were to investigate the effects of differing planes of pre- and postweaning nutrition on prepubertal serum leptin concentrations, LH profiles, and age at puberty in Holstein heifers. Thirty-six Holstein calves were randomly assigned to either a low (5 L) or high (10 L of whole milk/d) preweaning plane of nutrition from 1 to 7 wk of age, a transition diet (a dry total mixed ration with 85% of concentrate) from wk 8 to 10, and to either a low (70%) or high (85% of concentrate dry total mixed ration) postweaning plane from 11 to 25 wk of age. Serum leptin concentrations were measured every 2 wk from 1 to 25 wk of age, and LH profiles were determined both at wk 15 and 25 based on sequential blood samples taken every 12 min over 10 h. Starting at 26 wk of age, ovaries were examined weekly by transrectal ultrasonography until first ovulation (puberty) was confirmed. Heifers that received the high preweaning plane diet had greater mean (± standard error; SE) leptin concentrations during the preweaning phase than those fed the low plane (2.9 ± 0.1 vs. 2.6 ± 0.1 ng/mL). During the postweaning phase, mean circulating leptin was greater in heifers fed the high compared with the low postweaning diet [3.2 ng/mL (95% confidence interval; CI = 2.7 to 3.4) vs. 2.5 ng/mL (95% CI = 2.3 to 2.8)]. Mean (± SE) amplitude (2.1 ± 0.1 vs. 1.7 ± 0.1 ng/mL), peak (2.2 ± 0.1 vs. 1.8 ± 0.1 ng/mL), and duration (35.6 ± 2.1 vs. 28.7 ± 2.0 min) of LH pulses assessed at wk 15 were greater in heifers offered the high than those offered the low preweaning plane, but no nutritional effects were observed on LH pulses at wk 25. Mean (± SE) age at puberty was 250 ± 9 d and was not influenced by planes of nutrition. However, the likelihood of attaining puberty by 30 wk of age was greater (hazard ratio = 3.8; 95% CI = 1.0 to 14.4) in heifers fed the high postweaning plane compared with the low plane. Heifers achieving puberty by 30 wk also had greater leptin concentrations from wk 1 to 25, whereas for every 1 ng/mL increase in circulating leptin at wk 15 and 25, days to puberty were estimated to decrease by 22 d (95% CI = 1 to 44) and 13 d (95% CI = 1 to 24), respectively. Increasing the preweaning plane from 5 to 10 L/d of whole milk increased serum leptin concentrations at wk 1, 3, and 5 and LH pulse amplitude, peak, and duration at wk 15. Increasing the postweaning plane from 70 to 85% of concentrate resulted in greater circulating leptin concentrations, which may be linked to an earlier onset of puberty.

SUN-LB54 Nutritionally-Induced Alteration in KNDy Neuronal Expression in the Arcuate Nucleus of Ewes

Kisspeptin, neurokinin B, and dynorphin are imperative for GnRH/LH pulsatility and reproductive cyclicity. Neurons co-expressing these neuropeptides, KNDy neurons, within the arcuate nucleus of the hypothalamus (ARC) are positioned to integrate energy balance signals from neuronal and glial cells. Energy balance mediates neurokinin B expression in the ARC and LH pulse amplitude. Dynorphin mediates progesterone negative feedback on GnRH neurons. The hypothesis that the number of KNDy-expressing neurons in the ARC of ewes during the luteal phase of the estrous cycle is influenced by energy balance was tested using ovary-intact, mature ewes fed to lose, maintain, or gain body weight. Fluorescent multiplex immunohistochemistry was employed to identify and quantify neurons expressing a single neuropeptide and co-expressing kisspeptin, neurokinin B, and dynorphin in the ARC. Kaminski et al. (1) reported previously that concentrations of insulin and leptin differed between ewes fed to achieve different body weights and that ewes fed to gain body weight had increased concentrations of progesterone in the luteal phase of the estrous cycle. Moreover, tanycyte density and cellular penetration into the ARC are increased in ewes fed to gain body weight (2). Number of neurons in the ARC expressing kisspeptin (14.9 ± 2.7 neurons, 20.9 ± 3.6 neurons, and 51.5 ± 3.3 neurons in ewes fed to lose, maintain, and gain body weight, respectively), neurokinin B (21.5 ± 3.2 neurons, 31.3 ± 4.3 neurons, and 56.0 ± 3.9 neurons in ewes fed to lose, maintain, and gain body weight, respectively), and dynorphin (10.1 ± 2.4 neurons, 14.9 ± 3.2 neurons, and 33.1 ± 2.9 neurons in ewes fed to lose, maintain, and gain body weight, respectively) protein was increased (P < 0.0001) in ewes fed to gain body weight. Number of KNDy neurons in the ARC expressing kisspeptin, neurokinin B, and dynorphin protein was decreased in ewes fed to lose body weight (1.0 ± 0.5 neurons; P = 0.01) and increased in ewes fed to gain body weight (6.7 ± 0.6 neurons; P = 0.0005) when compared to ewes fed to maintain body weight (3.3 ± 0.7 neurons). These findings suggest that expression of kisspeptin, neurokinin B, and dynorphin protein in the ARC during the luteal phase of the estrous cycle may be influenced by nutritionally-induced alterations in circulating concentrations of progesterone that drive changes in morphology and density of tanycytes. Moreover, these results demonstrate that changes in KNDy neurons within the ARC occur as an adaptation to energy balance, potentially regulated divergently by metabolic milieu.

Central obestatin administration affect the LH and FSH secretory activity in peripubertal sheep

Obestatin – a 23 amino acid peptide is synthesized as another product of the ghrl gene and its synthesis occurs mainly in gastric mucosa cells. This hormone is involved in complex gut-brain neurohormonal networks, thereby can participates in the modulation of gonadotrophic axis activity.The aim of this study was to investigate the consequence of intracerebroventricular infusions of obestatin on LH and FSH pituitary cells secretory activity in peripubertal female sheep. Animals were randomly divided into two groups: the control group (n = 14) received intracerebroventricular infusions of Ringer-Lock solution (120 μL h−1), and the obestatin group (n = 14) was infused with obestatin (25 μg/120 μL h−1) diluted in Ringer-Lock solution. A series of four infusions was performed on three consecutive days. Blood samples were collected on day 0 and day 3. The sheep were slaughtered immediately after the end of the experiment. For molecular biological analysis, pituitaries from 7 sheep from each group (n = 7 + 7) were prepared and frozen in liquid nitrogen immediately after collection and then stored at −80 °C until Real Time RT-qPCR and RIA analyzes. For immunohistochemical analysis, pituitary tissues from the remaining animals (n = 7 + 7) was fixed in situ for further examination.Real-Time qPCR and immunohistochemistry analyses revealed substantial changes in the LH and FSH pituitary cells secretory activity in obestatin-infused sheep. Exogenous obestatin administration reduced LHβ mRNA expression and increased the accumulation of immunoreactive LH in gonadotrophic cells of the adenohypophysis. These changes were accompanied by a decrease in the mean LH concentration in the peripheral blood resulting from the lower LH pulse amplitude. Moreover, an increase in both FSHβ mRNA expression and FSH immunoreactivity and amount in pituitary cells were noted, while mean blood FSH concentration remained unchanged after obestatin treatment.The obtained results showed that exogenous obestatin affected LH secretory activity at the level of protein synthesis, accumulation and release as well as obestatin increase FSHβ mRNA expression and accumulation of this hormone but at the same time have no effect on FSH release to blood. Thus, obestatin can participate in the neuroendocrine network, which modulates gonadotrophic axis activity in sheep.

OR15-4 Long-Term Follow-Up of a Female with a Mutation in the Estrogen Receptor Alpha (ESR1) Gene

We previously reported the first female with a mutation in the ESR1 gene encoding estrogen receptor-α (ER-α), which dramatically reduced estrogen signaling in vitro. She presented at 179/12 years with absent breast development, markedly elevated serum estradiol and estrone levels, modestly high gonadotropins, and multicystic ovaries by ultrasound. The natural history of estrogen insensitivity in women is unknown. The purpose of this report is to present: 1) the neuroendocrine phenotype; 2) studies to identify a ligand to stimulate the mutant receptor; and 3) the effect of targeted treatment. We now provide long-term clinical follow up and a detailed hormonal analysis of the patient at age 234/12 years. On no treatment, serum FSH and LH were drawn every 10 minutes for 8 hours, and estradiol and estrone levels were measured every hour during the study. Various compounds that are potential for treatment were used in transient transfection assays to determine if estrogen signaling could be induced. A personalized medicine approach was tailored to our patient, in which she received increasing doses of diethylstilbestrol (DES) with the primary endpoints being increased vaginal discharge and breast development. Follow up exams, labs, pelvic ultrasounds, DXA, and bone age X-rays were completed at various intervals up to age 234/12 years. The Bioethics Board approved this treatment. During the frequent sampling study, E2 was 604 pg/mL, E1 was 596 pg/mL, FSH was 10.0+/-0.1 IU/L (mean+/-SE), and LH was 6.7+/-0.2 IU/L. There were 3 pulses in 8 hr with an LH pulse amplitude of 2.3+/-0.5 IU/L. Although DES was shown to successfully transactivate the mutant ESR1in vitro, both in HEK293 cells and in her lymphoblastoid cells, the patient had no clinical response. After 2.5 years of treatment with increasing doses of DES, there was no vaginal discharge and breasts remained at Tanner 1. At 234/12 years, LH, FSH and E2 were unchanged. A vaginal maturation index showed a predominance of parabasal cells consistent with a lack of estrogen effect. Ultrasound showed bilateral ovarian cysts. At chronologic age 234/12 years, her bone age was 17 years and the epiphysis remained open in the ulnar bone. DXA revealed a Z-score of -2.9 at the femoral neck and -4.5 at the lumbar spine. Our findings in this severely estrogen insensitive female indicate elevated gonadotropins with normal amplitude and low frequency LH pulse dynamics in the presence of markedly increased estrogens. Treatment with DES, which stimulated estrogen signaling in vitro, did not induce secondary sexual characteristics in our patient. She remains hypoestrogenic despite the presence of ovarian cysts with a hypoestrogenic vaginal smear, absent breast development, and low bone mineral mass. Future studies will be directed towards further identification of other potential signaling mechanisms and activators of this mutant receptor.

Regulation of gonadotrophin-releasing hormone secretion by testosterone in male sheep

In males, including the ram, testosterone, acting via its primary metabolites oestradiol and dihydrotestosterone (DHT), suppresses circulating LH concentrations. This effect is due primarily, although not totally, to decreased frequency of gonadotrophin-releasing hormone (GnRH) pulses. The arcuate-ventromedial region (ARC-VMR) of the mediobasal hypothalamus and possibly the medial preoptic area (mPOA) are sites at which oestradiol acts to suppress GnRH, but the site of DHT action is not known. Given that native GnRH neurones appear to contain few or no oestrogen or androgen receptors, the effects of testosterone metabolites probably are exerted by modulating activity of inhibitory interneurone systems such as beta-endorphin, dopamine, and gamma-aminobutyric acid (GABA). Although beta-endorphin clearly inhibits GnRH secretion, the observation that testosterone treatment during a long-day photoperiod reduced proopiomelanocortin (POMC) mRNA in the arcuate nucleus while coincidentally suppressing GnRH release indicates that beta-endorphin does not mediate the inhibitory effect of testosterone on GnRH. Activation of GABAA receptors in either the mPOA or ARC-VMR suppressed LH, whereas activation of GABAB receptors in the ARC-VMR increased LH pulse amplitude. Therefore, it is suggested that GABA acts in both regions to regulate LH. Whereas testosterone affects GABA metabolism in the rat hypothalamus, its effect in the ram hypothalamus is yet to be determined. Testosterone treatment activated dopaminergic cells in the retrochiasmatic A15 area in the same animals in which it suppressed POMC mRNA in the arcuate nucleus. This dopaminergic system may partially mediate the negative feedback effect of testosterone in the ram analogous to its role in partially mediating the negative effect of oestrogen in the ewe. Future studies must concentrate on determining how these and other putative inhibitory neuronal systems interact and how they in turn are regulated by environmental factors such as photoperiod.

Functional Hypogonadotropic Hypogonadism in Men: Underlying Neuroendocrine Mechanisms and Natural History.

After completion of puberty a subset of men experience functional hypogonadotropic hypogonadism (FHH) secondary to excessive exercise or weight loss. This phenomenon is akin to hypothalamic amenorrhea (HA) in women, yet little is known about FHH in men. To investigate the neuroendocrine mechanisms, genetics, and natural history underlying FHH. Retrospective study in an academic medical center. Healthy postpubertal men presenting with symptoms of hypogonadism in the setting of excessive exercise (>10 hours/week) or weight loss (>10% of body weight). Healthy age-matched men served as controls. Clinical assessment, biochemical and neuroendocrine profiling, body composition, semen analysis, and genetic evaluation of genes known to cause isolated GnRH deficiency. Reproductive hormone levels, endogenous GnRH-induced LH pulse patterns, and rare genetic variants. Ten men with FHH were compared with 18 age-matched controls. Patients had significantly lower body mass index, testosterone, LH, and mean LH pulse amplitudes yet normal LH pulse frequency, serum FSH, and sperm counts. Some patients exhibited nocturnal, sleep-entrained LH pulses characteristic of early puberty, and one FHH subject showed a completely apulsatile LH secretion. After decreased exercise and weight gain, five men with men had normalized serum testosterone levels, and symptoms resolved. Rare missense variants in NSMF (n = 1) and CHD7 (n = 1) were identified in two men with FHH. FHH is a rare, reversible form of male GnRH deficiency. LH pulse patterns in male FHH are similar to those observed in women with HA. This study expands the spectrum of GnRH deficiency disorders in men.

Insulin Resistance and Polycystic ovary Syndrome: A Review

Polycystic Ovary Syndrome (PCOS) is the most common, yet complex, endocrine disorder affecting women in their reproductive years and is a leading cause of infertility. This disease appears to be multifactorial and polygenic in nature involving multisystem dysfunction, namely reproduction, endocrine and metabolic. Hyperandrogenism and insulin resistance appear to be central cause to the pathophysiology of the disease. The glucose and insulin metabolism pathways have been studied and debated to understand whether Insulin Resistance is due to a defect in insulin action or a primary defect in β-cell function or decreased hepatic clearance of insulin, or a combination of all these factors. Numerous studies have demonstrated that obese, normal weight and thin women with PCOS have a form of insulin resistance that is unique and intrinsic to the disorder. Moreover obese women with PCOS possess an additional burden of insulin resistance resulting from their excess adiposity. Hyperinsulinemia leads to increase in androgen production directly by acting as a co-gonadotropin, augmenting Luteinizing Hormone activity within the ovary, and indirectly by increasing serum LH pulse amplitude. Whereas Androgens may in turn contribute at least partially to the insulin resistance state linked with PCOS. In this review, we will briefly study the role of insulin resistance in polycystic ovary syndrome.&#x0D; Keywords: Polycystic ovary syndrome, insulin resistance, Hyperandrogenism.

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.

Resistin regulates reproductive hormone secretion from the ovine adenohypophysis depending on season

Work in cattle and rodents has shown that resistin, in addition to its roles in insulin resistance and inflammation, is involved in the regulation of gonadal steroidogenesis. However, the role of resistin in the regulation of reproductive processes in other species, such as seasonally breeding sheep, is completely unknown. Herein, we tested the hypothesis that resistin can influence the secretion of anterior pituitary hormones and that its effect in ewes is dependent on the day length. Thirty Polish Longwool ewes, a breed that exhibits a strong seasonal reproductive pattern, were ovariectomized with estrogen replacement using subcutaneously inserted estradiol implants. Ewes were fed ad libitum and housed under a natural photoperiod (longitude: 19°57′ E, latitude: 50° 04′ N). Intravenous treatments consisted of control or recombinant bovine resistin (rbresistin) in saline: (1) control (saline; n = 10), (2) low resistin dose (1.0 μg/kg BW; n = 10), and (3) high resistin dose (10.0 μg/kg BW; n = 10). Experiments were conducted during both short-day (SD) and long-day (LD) seasons using 5 sheep per group within each season. Blood samples were collected every 10 min over 4 h. Blood plasma concentrations of FSH, LH, and prolactin (PRL) were assayed using RIA. A season × dose interaction was observed for all hormonal variables measured. Greater concentrations (P < 0.001) of LH and FSH were observed during SDs than during LDs in all groups. During SDs, the high dose (10 μg/kg BW) decreased (P < 0.001) basal LH levels and amplitude (P < 0.05) of LH pulses and increased (P < 0.001) circulating concentrations of FSH. However, the low dose of resistin decreased (P < 0.001) FSH concentrations compared to those of controls. During LDs, both the low and high resistin doses increased mean concentrations of LH (P < 0.001 and P < 0.05, respectively) and FSH (P < 0.001). A high dose of rbresistin increased (P < 0.001) the mean circulating concentrations of PRL during both seasons. However, in all groups, concentrations of PRL were greater during LDs than SDs. These results demonstrate for the first time that resistin is involved in the regulation of pituitary hormone secretion and that this effect is differentially mediated during LDs and SDs.