Mean Luteinizing Hormone Concentrations Research Articles

Central δ/κ opioid receptor signaling pathways mediate chronic and/or acute suckling-induced LH suppression in rats during late lactation.

In mammals, secretion of tonic (pulsatile) gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) is often suppressed during lactation. Suppression of GnRH/LH pulses in lactating dams is assumed to be caused by suckling stimuli and a chronic negative energy balance due to milk production. The present study aimed to investigate whether the central enkephalin-δ opioid receptor (DOR) signaling mediated the suppression of LH secretion by acute suckling stimuli and/or chronic negative energy balance due to milk production in rats during late lactation when dams were under a heavy energy demand. On postpartum day 16, the number of Penk (enkephalin mRNA)-expressing cells in the arcuate nucleus was significantly higher in lactating rats than in non-lactating control rats. Pulsatile LH secretion was suppressed in rats with chronic suckling or acute 1-h suckling stimuli 6 h after pup removal on day 16 of lactation. Central DOR antagonism significantly increased the mean LH concentrations and the baseline of LH pulses in rats with chronic suckling but not with acute suckling stimuli on day 16 of lactation. Besides, central κ opioid receptor (KOR) antagonism increased the amplitude of LH pulses in rats with the acute suckling stimuli on day 16 of lactation. These results suggest that central DOR signaling mediates the suppression of LH secretion caused by a negative energy balance in rats receiving chronic suckling during late lactation. On the other hand, central KOR signaling likely mediates acute suckling stimuli-induced suppression of LH secretion in rats during late lactation.

Chemogenetic Activation of RFRP Neurons Reduces LH Pulse Frequency in Female but not Male Mice.

The neuropeptide RFRP-3 (RFamide-related peptide-3) is thought to play a role in the negative regulation of fertility. However, the exogenous administration of RFRP-3 yields varying results depending on the dose and route of administration, sex of the subject, and many other variables. Manipulation of in vivo neuronal activity using DREADDs (designer receptor exclusively activated by designer drugs) technology enables investigation of cell type-specific neuronal activation in a manner that better reflects endogenous neuronal activity. To test the effects of RFRP neuronal activation on pulsatile luteinizing hormone (LH) secretion. We generated mice expressing the stimulatory hM3Dq designer receptor exclusively in RFRP cells using 2 different Cre-loxP-mediated approaches: (1) we bred mice to express hM3Dq in all Rfrp-Cre-expressing cells, including some that transiently expressed Rfrp-Cre neonatally (RFRP × hM3Dq mice), and (2) we stereotaxically injected Cre-dependent hM3Dq into the dorsomedial nucleus of RFRP-Cre mice to drive hM3Dq expression exclusively in a subpopulation of adult Rfrp-Cre neurons (RFRP-AAV-hM3Dq mice). We then investigated the effects of acute hM3Dq activation on LH pulse frequency in RFRP × hM3Dq mice, RFRP-AAV-hM3Dq mice, and their respective controls. In both female RFRP × hM3Dq and RFRP-AAV-hM3Dq mice, chemogenetic activation of Cre-driven hM3Dq led to a significant 35% to 50% reduction in LH pulse frequency compared with controls, while no differences in pulse amplitude or mean LH concentration were observed. In marked contrast, RFRP activation did not cause any changes to LH pulse dynamics in male mice. These data show for the first time that activation of neurons that have expressed Rfrp, or of a subset of adult RFRP neurons, can independently suppress LH pulsatility in female, but not male mice.

294 KISS1 Knockout Boars Have Decreased Concentrations of Gonadotropins Leading to Smaller Testes and Reduced Skatole in Backfat

Abstract In swine production, boars are castrated to prevent boar taint from testicular androgens and skatole accumulation in fat. Kisspeptin (KISS1) regulates secretion of gonadotropins and gonadal steroids. We used boars from our line of KISS1-/-pigs (10.3389/fgene.2022.1078991) to understand how loss of KISS1 function affects compounds causing boar taint. Boars were fed ad libitum during development. Body weights and testicular volume (TV) were collected at weaning, 40, 70, 100, 130, 160, and 190 d of age using TV = 4/3*π *a*b2*2 and subtracting scrotal skin thickness measured by a Harpenden skinfold caliper. Average daily gain (ADG) was calculated by subtracting the beginning from ending body weight at each age range and dividing by the number of days. At each age, a blood serum sample was collected by jugular venipuncture to quantify changes in hormones during development. Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone were quantified with radioimmunoassay and analyzed as repeated measures with genotype (KISS1+/+; KISS1+/-; KISS1-/-; n = 8 – 20 animals per genotype) and age as the fixed effects. Concentrations of androstenone and skatole in backfat (quantified using ELISA and HPLC, respectively) were log transformed and analyzed with ANOVA. Body weight did not differ among KISS1 genotypes but increased normally with age (P < 0.0001). During development, KISS1+/+ and KISS1+/- boars grew at similar rates. Although the ADG of KISS1-/-boars was similar to other genotypes up to 160 d of age, ADG was reduced at 190 d of age (age x genotype; P < 0.05). Mean concentrations of LH and FSH in KISS1+/+ and KISS1+/- boars were similar throughout development (LH, 1.16 ±0.11 and 1.34 ± 0.17 ng/mL; FSH, 0.88 ± 0.06 and 0.89 ± 0.05 ng/mL, respectively), and were greater than KISS1-/- boars (LH, 0.38 ± 0.23 ng/mL; FSH, 0.56 ± 0.07 ng/mL; P < 0.05). TV of KISS1+/+ and KISS1+/- boars did not differ during development and was greater than KISS1-/- boars (P < 0.05), for which testes could not be palpated after 100 d of age. Testosterone concentrations in KISS1+/+ and KISS1+/- increased 40-fold during development but were below detectable limits in KISS1-/- boars (age x genotype; P = 0.06). Androstenone and skatole in KISS1-/- boars (0.30 ± 1.24 µg/g and 12.6 ± 1.6 ng/g, respectively) at 190 d of age was less (P < 0.0001) than in KISS1+/+ and KISS1+/- boars (androstenone, 4.33 ± 1.16 µg/g; skatole, 69.2 ± 1.2 ng/g), which did not differ from one another. Thus, a single functional allele of KISS1 is sufficient to confer normal secretion of reproductive hormones, testicular growth, and skatole in boars. Moreover, knocking out the KISS1 gene will prevent boar taint, but might slightly reduce growth in later stages of development.

Assessment of pubertal onset and disorders of puberty in Indian children and youth with type-1 diabetes.

Disorders of pubertal development are enlisted as associated conditions in children and adolescents with type-1 diabetes (T1D). We conducted this study with objective (1) To estimate the median age at onset of puberty and luteinizing hormone (LH) and sex-steroid concentrations in Indian adolescents with T1D and (2) To assess the impact of puberty on glycemic control and insulin resistance (IR). This cross-sectional study included 399 children and youth aged 6-23 years with T1D. Demographic, anthropometric, biochemical and pelvic ultrasound data were collected using standard protocols. IR was calculated using estimated glucose disposal rate and puberty was assessed using Tanner staging. Median age at onset of thelarche, pubarche and menarche were 11.3, 11.4 and 12.8 years in girls and that of gonadarche and pubarche were 10.6 and 12.7 years for boys. The mean LH and sex-steroid concentrations of subjects with T1D were similar to healthy subjects at each stage of puberty. The cut-offs of LH and sex-steroids derived from healthy Indian children yielded high sensitivity and specificity in determining pubertal onset. The prevalence of precocity, delayed puberty, ovarian cysts and polycystic ovaries was 0.9 , 5.1, 5.1 and 8.6 %, respectively. Glycaemic control and insulin sensitivity was poor in pubertal subjects. The age at onset of puberty, LH, and sex-steroid concentrations in subjects with T1D were like otherwise healthy Indian children with poor glycemic control and IR in pubertal subjects. Although most complications of T1D are associated with poor glycemic control, pubertal disorders were significantly low despite the less-than-optimal glycemic control.

An automated system for cattle reproductive management under the IoT framework. Part II: Induction of luteinizing hormone release after gonadotropin releasing hormone analogue delivery with e-Synch

Technologies for automating animal management and monitoring tasks can improve efficiency and productivity of livestock production. We developed the e-Synch system for automated control and monitoring the estrous cycle of cattle through intravaginal hormone delivery and sensing. Thus, our objective was to evaluate luteinizing hormone (LH) concentrations after intravaginal instillation of the Gonadotropin-releasing hormone (GnRH) analogue Gonadorelin with the e-Synch system. This system consists of an intravaginal electronically controlled automated hormone delivery and sensing device integrated with an IoT platform. Lactating Holstein cows with their estrous cycle synchronized were used in two experiments (Exp). In Exp 1, at 48 h after induction of luteolysis, cows (n=5-6 per group) were randomized to receive 100 µg of Gonadorelin through intramuscular (i.m.) injection, 100 µg of Gonadorelin in a 2 mL solution delivered with e-Synch, and an empty e-Synch device. In Exp 2, at 48 h after induction of luteolysis cows (n=6-7 per group) were randomized to receive 100 µg of Gonadorelin i.m., or an intravaginal treatment with e-Synch consisting of 100 or 1,000 µg of Gonadorelin in 2 or 10 mL of solution containing 10% citric acid as absorption enhancer. Circulating concentrations of LH were analyzed with linear mixed models with or without repeated measurements. In Exp 1, cows in the i.m. Gonadorelin treatment had a surge of LH whereas cows in the other two treatments did not have a surge of LH for up to 8 h after treatment. In Exp 2, the 1,000 µg dose of Gonadorelin elicited more LH release than the 100 µg dose, regardless of solution quantity. The overall LH response as determined by area under the curve, mean, and maximum LH concentrations was similar between cows receiving 1,000 µg of Gonadorelin delivered with e-Synch and 100 μg of Gonadorelin i.m. Increasing volume of solution for delivering the same dose of Gonadorelin partially increased LH release only for the 100 µg dose. We conclude that the e-Synch system could be used to automatically release Gonadorelin in a dose and volume that induces a surge of LH of similar magnitude than after i.m. injection of 100 μg of Gonadorelin. Also, the dose of Gonadorelin delivered by e-Synch is more critical than the volume of solution used.

Effect of ovariohysterectomy on queen myocardial function: echocardiographic evidence.

Ovariohysterectomy (OHE) is one of the most common surgeries in veterinary medicine. Although this surgical method has several advantages, it can alter the function of various organs such as heart. The present study investigated the effect of OHE on cardiac functions using M-mode echocardiography. A total of 10 healthy adult domestic short-haired cats were enrolled in the current study. Fractional shortening (FS) and ejection fraction (EF) percentages along with cardiac output (CO) were measured through the right parasternal approach in papillary muscle level view. Moreover, the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured by enzyme-linked immunosorbent assay before OHE (D0) as well as 10 (D10), 20 (D20) and 30 (D30) days after OHE. The results of this study showed that the mean FS and EF decreased on all days of the study. The FS reduction was significant between D10 and D30 and EF changes were significant between D10 and D20. The means of CO increased significantly on D0 compared to the D10. After D20, CO reduced until the end of the study. Mean concentrations of LH and FSH increased on all research days; but, the changes were significant until D20. Despite the negative effects of OHE on myocardial function, there was no significant correlation between hormonal levels and echocardiographic findings after OHE in this study.

Induction of ovulation after artificial insemination in rabbits: Intramuscular injection of gonadotropin-releasing hormone (GnRH) agonist vs. intravenous administration of mated doe serum

BACKGROUND: Rabbits are reflex ovulators with the ovulatory process triggered by neuro-hormonal impulses generated during natural mating. When applying artificial insemination (AI), an array of biostimulation techniques and/or exogenous hormones (e.g., gonadotropin-releasing hormone (GnRH) or its analogues) must be used to induce ovulations in female rabbits. However, the effectiveness of various biostimulation techniques and exogenous hormones is not always satisfactory and the use of GnRH analogues is additionally associated with high production costs. Therefore, developing an alternative inexpensive, efficient and safe treatment for the induction of ovulation in artificially inseminated does is urgently needed. OBJECTIVES: In the present study, we examined and compared the effects of mated doe serum (MDS) and GnRH analogue (Gonadorelin) administered immediately after artificial insemination on circulating concentrations of luteinizing hormone (LH) and fertility in New Zealand does. METHODS: Forty artificially inseminated does were allocated to four equinumerous groups that received: 0.2 ml of saline i.m. (Control G), 0.8 µg of Gonadorelin dissolved in 0.2 ml of saline i.m. (Treatment G), 2.5 ml of mixed sex normal rabbit serum i.v. (Control M) or 2.5 ml of mated doe serum (MDS)/doe i.v. (Treatment M). RESULTS: A peak in systemic LH concentrations occurred earlier in Treatment M compared with Treatment G does (71 vs. 107 min post-AI, respectively; P≤ 0.05); mean LH concentrations did not vary (P≤ 0.05) from the pre-AI values in both control groups. Serum LH concentrations remained higher (P≤ 0.05) in Treatment M compared with Treatment G does from 30 to 90 min post-AI but they were greater (P≤ 0.05) in Treatment G than in Treatment M group at 120 and 160 min after AI. Gonadorelin and MDS injections both resulted in the same kindling rate (80%) at each of the four consecutive AI’s (initiated 30 days post-partum) and they were significantly greater than those recorded in control animals (20%). CONCLUSION: It can be concluded that MDS administration is an effective treatment to induce ovulations in rabbits, with the repeatability like that achieved with a GnRH analogue.

The Effect of Estetrol/Drospirenone on Ovarian Function Is Similar to a Well-Established Combined Oral Contraceptive: Results From a Phase 2 Study

Background: Combined oral contraceptives (COCs) often contain ethinylestradiol (EE), an estrogen known to be associated with several side effects including venous thromboembolism. Estetrol (E4) is a native estrogen synthesized by the human fetal liver during pregnancy. Results from a phase 2 dose-finding study showed that E4 15 mg in combination with drospirenone 3 mg (E4/DRSP) resulted in a good bleeding profile and cycle control. Here, we present phase 2 results showing the effect of E4/DRSP on ovarian function. Study Design: A single-center, randomized, open-label, parallel study was conducted in healthy young volunteers with proven ovulatory cycles. Study subjects received either E4 15 mg/DRSP 3 mg (n=41) or EE 20 µg/DRSP 3 mg (n=41) in a 24/4-day regimen for three consecutive cycles. Both in cycle 1 and 3, serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH), estradiol and progesterone levels were determined, and follicular size and endometrial thickness were measured every three days using transvaginal ultrasound. Ovarian function was assessed using the Hoogland score, which considers follicular size and estradiol/progesterone levels. Return of ovulation was determined after treatment cessation. Safety and tolerability were assessed by monitoring adverse events (AEs), vital signs, physical and gynecological examination, clinical laboratory parameters, 12-lead electrocardiogram and echocardiogram. Results: No ovulations were reported during the use of E4/DRSP, while three ovulations occurred in two subjects in the EE/DRSP group. In both groups, most participants had no ovarian activity according to the Hoogland score. In cycle 1, Hoogland scores and follicular diameters were similar in both groups. In cycle 3, these parameters were slightly less suppressed in the E4/DRSP group when compared to EE/DRSP. While mean FSH and LH concentrations were less inhibited by E4/DRSP, mean estradiol and progesterone concentrations and endometrial thickness were similarly suppressed in both groups. Return of ovulation occurred on average 15.5 days after discontinuation of E4/DRSP intake. The number of frequently reported AEs considered to be related to study medication was similar for both treatment groups except for breast pain (11 subjects in the E4/DRSP group versus 4 subjects with EE/DRSP). Most related AEs were of mild or moderate intensity. Three subjects discontinued due to an AE, one with E4/DRSP (severe stress, emotional lability), and two with EE/DRSP (emotional lability, depressed mood). Other safety assessments did not show significant abnormalities. No serious AEs were reported. Conclusions: The combination of E4 15 mg and DRSP 3 mg results in adequate ovulation inhibition and ovarian function suppression, which is similar to a well-established COC containing EE/DRSP. E4/DRSP is considered safe and well-tolerated.

104 Induction of cyclicity in seasonally anestrous recipient mares

Anestrous (AN) recipient mares’ natural cyclicity must be manipulated to produce foals from transferred embryos early in the year. One approach uses sulpiride to stimulate prolactin (PRL) that appears to have an important reproductive role effecting seasonality in mares. While previous research utilized sulpiride to induce ovulation (OV) in AN mares, few have transferred embryos after treatment. This study was designed to evaluate the effect of a treatment of estradiol cypionate (ECP) and sulpiride on inducing OV and subsequent pregnancy outcome in AN recipient mares of various reproductive status performed on a commercial embryo transfer farm. Anestrous recipient mares (n = 32) were randomly assigned to 4 treatment groups based on records of ovarian activity. Transitional (T, n = 8), AN (n = 11), and progesterone pre-treated (P, n = 9, received 4 mg altrenogest IM 3 wk prior) groups were treated January 20 with 50 mg of ECP IM and 3 g of sulpiride IM 24 h later. A control group (C, n = 4) of AN mares received the carrier. Jugular blood samples were collected at 0, 3, 6, 12, and 24 h post-treatment followed by daily collection for 10 d and collection every other day for 32 d thereafter and analyzed for luteinizing hormone (LH) and PRL. Ovaries were transrectally scanned every 2 to 3 d for 60 d. Embryos were transferred to 10 mares (5 T, 3 P, 2 AN) after at least the second estrous cycle up to the sixth cycle depending on their synchrony to the donor. Binomial data was analyzed via PROC GLIMMIX of SAS, and number of days to OV and hormonal data were analyzed via PROC MIXED with repeated measures. Nonparametric data were natural log-transformed and analyzed if normality was improved. Differences of least squares means were determined using the PDIFF option in SAS with ∝ ≤ 0.05 used to declare significance. There was an effect of treatment on the number of mares that achieved first OV ( P < 0.001) with 23 of 28 (82.1%) treated mares having a first OV within 22 d of treatment; and second OV ( P = 0.018) with 15 of the 23 (65.2%) mares that had a first OV achieving a second OV within 43 d. The mean number of days to first OV of T, AN, and P groups together were 32 ± 5 d earlier ( P < 0.001) than C. The transitional group ovulated sooner ( P = 0.05) than AN and tended ( P = 0.07) to ovulate sooner than P. There was an effect of the treatment-by-day interaction on plasma mean PRL ( P < 0.001) and LH ( P = 0.021) concentrations. Of the 10 embryos that were transferred, 7 (70%) resulted in a confirmed pregnancy at 60 d. Overall, the treatment protocol using ECP and sulpiride appeared to hasten OV and resulted in sustained pregnancy after embryo transfer.

Influences of Serotonin Hydrochloride on Adiponectin, Ghrelin and KiSS1 Genes Expression.

Background: Serotonin and kisspeptin stimulates gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) release while ghrelin and adiponectin inhibit them. In the present experimental study, the effects of central injection of serotonin were investigated on LH concentration, KiSS1, adiponectin, and ghrelin genes expression. Materials and Methods: Fifteen Wistar male rats in three groups received saline or serotonin hydrochloride via the third cerebral ventricle. Blood samples were collected via the tail vein. Serum LH concentration and relative gene expression were evaluated by radioimmunoassay and real-time polymerase chain reaction method, respectively. Results: Serotonin significantly increased the mean serum LH concentration and KiSS1 gene expression levels compared to the saline group. Serotonin significantly decreased the mean ghrelin and adiponectin genes expression levels compared to the saline group. Conclusion: The serotonergic pathway may have stimulatory effects on hypothalamic kisspeptin synthesis, partly via inhibiting hypothalamic ghrelin and adiponectin neural activity.

Influences of Serotonin Hydrochloride on Adiponectin, Ghrelin and KiSS1 Genes Expression

Background: Serotonin and kisspeptin stimulates gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) release while ghrelin and adiponectin inhibit them. In the present experimental study, the effects of central injection of serotonin were investigated on LH concentration, KiSS1, adiponectin, and ghrelin genes expression. Materials and Methods: Fifteen Wistar male rats in three groups received saline or serotonin hydrochloride via the third cerebral ventricle. Blood samples were collected via the tail vein. Serum LH concentration and relative gene expression were evaluated by radioimmunoassay and real-time polymerase chain reaction method, respectively. Results: Serotonin significantly increased the mean serum LH concentration and KiSS1 gene expression levels compared to the saline group. Serotonin significantly decreased the mean ghrelin and adiponectin genes expression levels compared to the saline group. Conclusion: The serotonergic pathway may have stimulatory effects on hypothalamic kisspeptin synthesis, partly via inhibiting hypothalamic ghrelin and adiponectin neural activity.[GMJ.2020;9:e1767]

Impaired prolactin transport into the brain and functional responses to prolactin in aged male mice.

Ageing is related to changes in a number of endocrine systems that impact on the central actions of hormones. The anterior pituitary hormone prolactin is present in the circulation in both males and females, with widespread expression of the prolactin receptor throughout the forebrain. We aimed to investigate prolactin transport into the brain, as well as circulating levels of prolactin and functional responses to prolactin, in aged male mice (23months). Transport of 125 I-labelled prolactin (125 I-prolactin) from the peripheral circulation into the brain was suppressed in aged compared to young adult (4months) male mice, with no significant transport into the brain occurring in aged males. We subsequently investigated changes in the negative-feedback regulation of prolactin secretion and prolactin-induced suppression of luteinising hormone (LH) pulsatile secretion in aged male mice. Feedback regulation of prolactin secretion appeared to be unaffected in aged males, with no change in levels of circulating prolactin, and normal prolactin-induced phosphorylated signal transducer and activator of transcription 5(pSTAT5) immunoreactivity in tuberoinfundibular dopaminergic (TIDA) neurones in the arcuate nucleus. There were, however, significant impairments in the ability of prolactin to suppress LH pulsatile secretion in aged males. In young adult males, acute prolactin administration significantly decreased LH pulses from 1.5±0.19 pulses of LH in 4hours to 0.5±0.27 pulses. In contrast, prolactin did not suppress LH pulse frequency in aged males, with prolactin leading to an increase in mean LH concentration. These data demonstrate the emergence of impairments in prolactin transport into the brain and deficits in specific functional responses to prolactin with ageing.

Verification of a ZBTB16 variant in polycystic ovary syndrome patients

Research questionThis study investigated whether rs1784692 is a risk factor for polycystic ovary syndrome (PCOS) in Han Chinese women. DesignA case-control study was conducted in Han Chinese women, involving 526 PCOS patients and 522 control participants. A TaqMan MGB probe assay was used to genotype the variant rs1784692. Dominant and additive models were employed for genotype–phenotype association analysis in the PCOS and control samples. ResultsThe minor allele C of rs1784692 is protective against PCOS (odds ratio [OR] 0.556, 95% confidence interval [CI] 0.408–0.759, P = 1.83 × 10–4), even after adjustment for body mass index (BMI) and age (ORadj 0.539, 95% CI 0.391–0.743, Padj= 1.62 × 10–4). Genotype–phenotype analysis of the dominant model showed that mean BMI in the CC+CT group was higher than in the TT group in the PCOS group (27.12 ± 5.82 versus 24.57 ± 4.52, P = 1.0 × 10–3), but not in the control groups, indicating that the minor allele C of rs1784692 associates with BMI in women with PCOS. The mean LH (luteinizing hormone) concentration in the CC+CT group was lower than in the TT group in PCOS and control participants (9.33 ± 5.08 versus 10.93 ± 5.91, P = 0.036; 4.39 ± 1.66 versus 4.89 ± 2.07, P = 0.021). Genotype–phenotype analysis of additive model showed that mean BMI in TC group was higher than in the TT group in PCOS patients compared with control participants (27.14 ± 5.81 versus 24.57 ± 4.52, P = 3.06 × 10–3). ConclusionsThe SNP rs1784692 in gene ZBTB16 is protective against PCOS but is associated with increased BMI in Han Chinese women with PCOS.

SUN-257 Evidence that Urocortin 2 Contributes to the Suppressive Effect of Metabolic Stress on LH Secretion in Female Mice

Pulsatile luteinizing hormone (LH) secretion is disrupted by numerous stimuli including metabolic stress. Insulin-induced hypoglycemia is a model of metabolic stress that suppresses LH secretion in numerous species including mice. Our recent work provides evidence that this inhibition of LH secretion occurs via suppression of neurons that contain kisspeptin (Kiss1), neurokinin B (NKB) and dynorphin (Dyn) in the arcuate (ARC) nucleus (KNDy cells). Thus, our current objective is to identify the neural components responsible for the suppression of KNDy cells during metabolic stress. Several lines of evidence support the hypothesis that the neuropeptide urocortin 2 (UCN2) has a key role in the inhibition of LH during stress in rats. First, ICV injection of UCN2 suppresses LH secretion. Second, an antagonist to the receptor for UCN2 reverses the suppression of LH during metabolic stress. Finally, restraint and osmotic stress increase UCN2 mRNA abundance in the paraventricular nucleus (PVN). To determine if UCN2 neurons in the PVN are activated during metabolic stress we performed immunohistochemistry for UCN2 and c-Fos in tissue collected 120 min after saline or insulin (0.75mU/kg) injection (n = 2/group, ovariectomized, adult, C57/BL6). Insulin significantly increased both the number of UCN2 cells (saline: 109.0 ± 8.5, insulin: 156.3 ± 10.8 cells) and the percentage of UCN2 cells that expressed c-Fos (saline: 13.1 ± 2.5%, insulin: 31.2 ± 0.8%). Next, we administered UCN2 (7.23nmol) via ICV injection to determine if this molecule suppresses LH secretion and/or mRNA abundance of KNDy genes. LH was measured in serial blood samples collected from 60 min prior to and 30-90 min following injection. Tissue was collected 3 h after ICV injection to confirm injection site and quantify mRNA abundance in ARC micropunches. In saline-treated mice (n = 5 successful injections), mean LH concentration and the number of LH pulses did not differ across sampling periods (mean: 6.4 ± 0.4 ng/mL vs. 6.0 ± 0.4 ng/mL; pulses: 2.6 ± 0.2 vs. 3.0 ± 0.3, pre vs. post). In contrast, in mice with successful UCN2 injections (n = 4) there was a significant reduction in both mean LH and the number of LH pulses following UCN2 (mean: 5.0 ± 0.3 ng/mL vs. 1.4 ± 0.2 ng/mL; pulses: 3.0 ± 0.0 vs. 0.25 ± 0.25, pre vs post). UCN2-treated animals had a significant reduction in the abundance of mRNAs encoding Kiss1 (~35%) and NKB (~40%) compared to saline-treated animals; the abundance of Dyn mRNA did not differ between treatments. These data demonstrate that PVN UCN2 cells are activated during metabolic stress and that UCN2 is sufficient to suppress LH secretion and the expression of genes involved in stimulating LH pulses. These data support the hypothesis that UCN2 released from neurons in the PVN impairs KNDy cell function and LH secretion during acute stress.

The interrelationship between the occurrence of oversized follicles and the peripheral and intra-follicular concentrations of E2, P4, FSH, and LH in female dromedary camels

The present study aimed to clarify the phenomenon of presence of larger than normal follicles (OVGF) in female dromedary camels. Females with OVGF (n=125) were examined by manual palpation and ultrasonography. Accordingly, the OVGF were subdivided into those with thin walls and clear hypoechogenic content (OVGF-TH, n=18) and those with thick walls and fibrous trabeculae (OVGF-TK, n=107). Transvaginal follicle aspiration was performed in females with OVGF and from a control group with growing follicles (1-2 cm in diameter, GF group, n=5). Serum was collected at the same time of follicle aspiration and analyzed for Follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4) and estradiol-17β profiles (E2). The follicular fluid (FF) was analyzed for E2 and P4. The results showed that mean E2 concentration in FF and serum were lower in OVGF-TH and OVGHTK groups than in the GF group (P &lt; 0.05). Difference between OVGF-TH and OVGH-TK groups was not significant. P4 in FF did not significantly differ among groups. Positive correlation was found between E2 in FF and E2 in serum (r = 0.495, r = 0.03). Mean FSH concentration in serum was higher in OVGF-TH and OVGH-TK groups than in the GF group (P = 0.03). Mean LH concentration was non-significantly (P=0.1) greater in OVGF-TH and OVGH-TK groups than in the GF group. In conclusion, female dromedary camels with OVGF had endocrine characteristics differed from camels with no OVGF. It seems that the high FSH and/or LH concentration(s) stimulated the continuing growth of the developing follicles to reach these large sizes, suggesting that the phenomenon of OVGF in camels is a pathological finding.

SAT-407 Evidence for Impaired Kisspeptin Signaling During Metabolic Stress in Female Mice

Stress impairs reproduction, at least in part, by suppressing gonadotropin secretion. One common metabolic stressor is hypoglycemia, which occurs in a variety of pathologies and in healthy people, particularly athletes. Insulin-induced hypoglycemia (IIH) is a repeatable, reliable, and quantifiable acute model of metabolic stress that inhibits pulsatile luteinizing hormone (LH) secretion, though little is known about the underlying mechanism. We have observed that IIH robustly suppresses LH pulses in female mice, which could be mediated by either a pituitary or hypothalamic effect. In the present study, we performed a kisspeptin challenge to determine whether downstream cells involved in LH pulsatility are impaired during IIH. Adult mice (C57/BL6, n = 8/group) were ovariectomized and acclimated to handling for tail-tip blood collection and i.p. injection. On the day of each challenge, mice were fasted for 5 hr and then received an i.p. injection of either saline or insulin (0.75mU/kg). All animals then received an injection of kisspeptin (2μg/g), 45 min later. This time point represents a consistent period of LH pulse suppression following IIH. Frequent blood samples were collected for 30 min before and after kisspeptin injection. Two-weeks later, the experiment was repeated with each mouse receiving the alternate treatment (i.e. insulin or saline) in a cross-over design. Mean LH concentrations were significantly increased after kisspeptin injection in animals treated with saline (saline, pre: 3.8 ± 0.6 vs. post: 7.8 ± 0.5 ng/ml). Interestingly, the LH response to kisspeptin in animals treated with insulin was also markedly increased (insulin, pre: 3.2 ± 0.6 vs. post: 6.4 ± 0.3 ng/ml), but not significantly different from animals treated with saline. We conclude from this study that impaired GnRH or gonadotrope cell function is not the primary cause of reduced LH secretion in this model of metabolic stress. Since kisspeptin neurons in the arcuate nucleus likely form the GnRH/LH pulse generator, we next determined whether arcuate kisspeptin cells were inhibited during IIH. For this purpose, we used a mouse model expressing GFP in kisspeptin cells (Kiss1-Cre-GFP). Mice were fasted and injected with insulin or saline, as above, and fixed neural tissue was collected for assessment of c-Fos immunoreactivity in arcuate Kiss1 cells. Two hours following saline injection ~20% of Kiss1 neurons contained c-Fos. In contrast, only 6.6 ± 0.5% of Kiss1 neurons contained c-Fos in animals treated with insulin. Thus, reduced gonadotropin secretion in IIH may be mediated by an inhibition of the ARC kisspeptin cells. Together these data support the hypothesis that inhibition of LH pulses by metabolic stress is mediated by neural factors including arcuate kisspeptin cells. Future work will investigate kisspeptin cell afferents that convey the inhibitory signal initiated by metabolic stressors.

Luteinising hormone profiles in conception and non-conception natural cycles

Objective: The aim of this study was to assess whether luteinising hormone (LH) surge characteristics influenced the likelihood of conceiving naturally. Methods: This was a single-cycle, home-based, observational, case-controlled study. Volunteers collected daily urine samples for one menstrual cycle. LH was measured and the basal levels, surge day, peak day, peak concentration and magnitude of LH surges were examined. Predictive models using sociodemographic data, LH surge characteristics, and sociodemographic data combined with LH profile properties, were evaluated. Results: The surge profile did not differ between cycles with early or late ovulation and was not affected by age or body mass index (BMI). The mean LH surge day was day 16 for both groups. Mean LH surge and concentrations did not differ between groups (surge concentration 54.8 IU/l vs. 58.2 IU/l and peak concentration 82.0 IU/l vs. 81.6 IU/l for pregnant vs. non-pregnant volunteers, respectively). Non-pregnant volunteers were more likely, however, to have a raised or a reduced basal LH on day 6 or have an atypical LH profile. Sociodemographic characteristics were significant predictors of pregnancy, and sociodemographic variable-based models had the greatest predictive ability for conception, providing up to 65% predictive accuracy. Conclusions: Sociodemographic variables can be used to predict the likelihood of a woman conceiving naturally. Provided an LH surge is present, its profile does not relate to the likelihood of spontaneous pregnancy. The conception rate was significantly lower, however, in women with elevated or reduced basal levels of LH, suggesting that follicular maturation needs an optimal basal level of LH in natural conception cycles. Trial registration: ClinicalTrials.gov identifier: NCT01577147.

Association of vascular and inflammatory markers with metabolic disorders in women with polycystic ovary syndrome

Background/Aim. The prevalence of metabolic disorders, obesity and insulin resistance in women with polycystic ovary syndrome (PCOS) occur early in life and places this group at risk of cardiovascular disease. Hyperhomocysteinemia and increased C-reactive protein (CRP) activity have an effect on promoting atherosclerosis. This study was designed to evaluate whether high sensitivity (hs-CRP) and homocysteine (Hcy) are elevated in PCOS and to elucidate their possible relation to obesity, insulin resistance, or metabolic changes usually present in women suffering from PCOS. Methods. Serum concentration of hs-CRP and plasma levels of Hcy were evaluated in 73 PCOS women and 43 healthy women, together with clinical, anthropometric and hormonal parameters. Results. The mean of body mass index (BMI), waist circumference (WC), waist to hip ratio and mean concentration of luteinizing hormone (LH), testosterone, androstenedione, free androgen index, fasting insulin, homeostatic model assessment of insulin resistance (HOMA- IR), hs-CRP and Hcy were significantly higher in PCOS women compared to age-matched healthy women. There was a positive correlation between hs-CRP and BMI, WC, insulin, triglycerides (p &lt; 0.001) and significant negative correlation with LH, sex hormone binding protein (SHGB), HOMA-IR, high density lipoprotein cholesterol (HDL-C) (p &lt; 0.001). The Hcy concentration had a significant negative correlation with HDL-C level (p &lt; 0.05). The present study demonstrated increased mean concentration of Hcy in hs-CRP women with PCOS. Conclusion. Our results support the use of these biomarkers in the evaluation of potential risk for cardiovascular diseases and early prognosis and treatment implications.

Serum Progesterone, Oestradiol-17β, FSH and LH levels in Anestrus Surti buffaloes treated with Ovsynch alone and in combination with PRID

This study was carried out on 18 postpartum anestrus Surti buffaloes, divided into three equal groups (6 in each) to evaluate the serum progesterone, oestradiol-17β, FSH and LH concentrations (by ELISA) before, during and after Ovsynch and Ovsynch + PRID protocols. Six anestrus buffaloes each were treated with standard Ovsynch protocol (T1) and Ovsynch along with PRID (Progesterone releasing intravaginal device) protocol (T2) with fixed-time AI (FTAI). Six anestrus buffaloes were kept as untreated control (T3). Blood samples were obtained from the animals on day 0 (before treatment), day 4 (during treatment), day 8 (after PGF2α injection), day of induced estrus/FTAI and day 18 post-AI (after treatment). The mean serum progesterone level on day 4 was significantly (p less than 0.01) higher in the T2 group as compared to T1 and T3 groups, while the level on day 8 was significantly (p less than 0.01) higher in both the treatment groups (T1 and T2) as compared to control group, however there was no significant difference (p&gt;0.05) on the day of estrus between three groups. The mean serum oestradiol-17β and follicle stimulating hormone (FSH) levels on 8th day were significantly higher (p less than 0.01) in both the treatment groups (T1 and T2) as compared to control group (T3). However, there was no significant difference on day 0, 4, day of estrus and day 18 post-AI between groups. The mean serum luteinizing hormone (LH) levels of acyclic buffaloes were not found significantly different (p&gt;0.05) among days in any of the treatment and control groups. But the mean LH concentration on the day of estrus in the T1, T2 and T3 was significantly higher (p less than 0.01) and thereafter decreased markedly on 18th day post-AI in all the groups. The conception rates at FTAI/first estrus in three groups were 66.66%, 50.00% and 75.00%, respectively.

Role of progesterone concentrations during early follicular development in beef cattle: I. Characteristics of LH secretion and oocyte quality

Objective was to investigate the effect of different progesterone (P4) concentrations during early follicular development on luteinizing hormone (LH) secretion and oocyte characteristics in beef cows. Primiparous cows (n = 24) were estrous pre-synchronized and follicular ablation was performed (d 0) 6 days following the time of ovulation. At the time of follicular ablation, cows were assigned to either: 1) high P4 treatment - HiP4; a new CIDR was inserted on d 0 to supplement P4 from the existing corpus luteum [CL], or 2) low P4 treatment - LoP4; a previously-used CIDR and two doses of PGF 8 to 12 h apart were given on d 0. Concentrations of P4 were greater (P < 0.01) in the cows of the HiP4 than LoP4 group on d 1.5, 2.5, and 3.5. Peripheral concentrations of E2 were greater (P < 0.05) in the cows of the LoP4 than HiP4 group on d 2.5 and 3.5. Frequency of LH pulses was greater (P < 0.05) in the LoP4 than HiP4 group on d 2.5, but mean LH concentration and pulse amplitude did not differ between treatments. Number of follicles aspirated per cow, total oocytes recovered, recovery rate, percentage of oocytes graded 1 to 3, oocyte diameter, percentage BCB+ oocytes, and relative abundance of oocyte mRNA for FST did not differ (P > 0.10) between treatments. In conclusion, lower P4 concentrations during early follicular development resulted in increased LH pulse frequency and E2 concentrations, but did not affect characteristics of oocyte developmental competence.