GnRH Secretion Research Articles

CONTINUOUS SWIMMING TRAINING IN MODERATE INTENSITY DECREASED FOLLICLE STIMULATING HORMONE LEVEL IN FEMALE MICE ON A HIGH-CALORIE DIET

Background: The increase of calorie intake can increase the risk of obesity and diabetes mellitus. One of the efforts to reduce the risk is doing physical exercise. Types of physical exercise that can affect the system of female reproduction are not clearly defined. Purpose: To analyse the effect of moderate intensity continuous physical exercise on FSH levels in female rats exposed to a high-calorie diet. Methods: Laboratory experimental was research with Randomized Post test only Control Group Design. Sampel size was 27 rats (150-200, adults) in the estrus phase, then divided into 3 groups (negative control group, positive control group (high-calorie diet), and the Moderate Intensity Continuous Training group (MICT with high-calorie diet)). Results: The results showed that there was an significant effect of continuous physical exercise in moderate intensity on FSH level in female rats exposed to other group. The FSH level in the MICT group had the lowes level (16.78 ±4.83 mIU/ml). Conclusion: There is an effect of MICT on FSH level decreases in female rats exposed to a high diet calories. This can be caused, that high-calorie diet can inhibit GnRH secretion and continuous moderate-intensity physical exercise changes the rat energy metabolism

Physiological and pathological roles of locally expressed kisspeptin and KISS1R in the endometrium.

Kisspeptins, encoded by the KISS1 gene, are a family of polypeptides that bind the kisspeptin receptor (KISS1R) to perform biological functions. Produced mainly in the hypothalamus, these neuropeptides regulate the pulsatile secretion of GnRH and trigger the hypothalamus-pituitary-gonadal axis. Other peripheral organs also express kisspeptin, which inhibits metastasis. Kisspeptin and KISS1R are reportedly present in the endometrium and may play roles in limiting the migration and invasion of trophoblasts into the endometrium during pregnancy (decidua) to maintain endometrial homeostasis. A deficiency of kisspeptin and KISS1R in the endometrium can lead to pathological conditions such as endometriosis and endometrial carcinoma. Kisspeptin and KISS1R in the endometrium can also promote endometrial receptivity and decidualization. Overall, kisspeptin and KISS1R are important for maintaining the normal physiological functions of the endometrium. By summarizing the roles of kisspeptin and KISS1R in the endometrium, our review explores the regulatory roles in the peripheral reproductive system of this peptide family that plays broad and profound roles in many physiological processes.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12μg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48μg/kg.d; KTZ 3-12-48mg/kg.d). Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

Hypothalamic neuroglial plasticity is regulated by anti-Müllerian hormone and disrupted in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common reproductive-endocrine disorder affecting between 5 and 18% of women worldwide. An elevated frequency of pulsatile luteinizing hormone (LH) secretion and higher serum levels of anti-Müllerian hormone (AMH) are frequently observed in women with PCOS. The origin of these abnormalities is, however, not well understood. We studied brain structure and function in women with and without PCOS using proton magnetic resonance spectroscopy (MRS) and diffusion tensor imaging combined with fiber tractography. Then, using a mouse model of PCOS, we investigated by electron microscopy whether AMH played a role on the regulation of hypothalamic structural plasticity. Increased AMH serum levels are associated with increased hypothalamic activity/axonal-glial signalling in PCOS patients. Furthermore, we demonstrate that AMH promotes profound micro-structural changes in the murine hypothalamic median eminence (ME), creating a permissive environment for GnRH secretion. These include the retraction of the processes of specialized AMH-sensitive ependymo-glial cells called tanycytes, allowing more GnRH neuron terminals to approach ME blood capillaries both during the run-up to ovulation and in a mouse model of PCOS. We uncovered a central function for AMH in the regulation of fertility by remodeling GnRH terminals and their tanycytic sheaths, and provided insights into the pivotal role of the brain in the establishment and maintenance of neuroendocrine dysfunction in PCOS. INSERM (U1172), European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement n° 725149), CHU de Lille, France (Bonus H).

Circulating levels and the bioactivity of miR-30b increase during pubertal progression in boys.

Puberty marks the transition from childhood to adulthood and is initiated by activation of a pulsatile GnRH secretion from the hypothalamus. MKRN3 functions as a pre-pubertal break on the GnRH pulse generator and hypothalamic expression and circulating levels of MKRN3 decrease peri-pubertally. In rodents, microRNA miR-30b seems to directly target hypothalamic MKRN3 expression - and in boys, circulating levels of miR-30b-5p increase when puberty is pharmacologically induced. Similarly, miR-200b-3p and miR-155-5p have been suggested to inhibit expression of other proteins potentially involved in the regulation of GnRH secretion. Here we measure circulating levels of these three miRNAs as boys progress through puberty. Forty-six boys from the longitudinal part of the Copenhagen Puberty Study were included. All boys underwent successive clinical examinations including estimation of testis size by palpation. miR-30b-5p, miR-200b-3p, and miR-155-5p were measured in serum by RT-qPCR using a kit sensitive to the phosphorylation status of the miRNAs. Thirty-nine boys had miRNA levels measured in three consecutive samples (pre-, peri-, and post-pubertally) and seven boys had miR-30b-5p levels measured in ten consecutive samples during the pubertal transition. When circulating levels of miR-30b-5p in pre- and peri-pubertal samples were compared with post-pubertal levels, we observed a significant increase of 2.3 and 2.2-fold (p-value<6.0×10-4), respectively, and a larger fraction of miR-30b-5p appeared to be phosphorylated post-pubertally indicating an increase in its bioactivity. We also observed a negative correlation between circulating levels of miR-30b-5p and MKRN3. The inter-individual variation in circulating miR-30b levels was substantial and we could not define a clinical threshold for miR-30b-5p suggestive of imminent puberty. Also, miR-155-5p showed significantly increasing levels from the peri- to the post-pubertal stage (p=3.0×10-3), whereas miR-200b-3p did not consistently increase. Both circulating levels of miR-30b-5p and its bioactivity increase during the pubertal transition in boys supporting its role in the activation of the HPG axis at the onset of physiologically normal puberty.

Mechanism of LH release after peripheral administration of kisspeptin in cattle.

Kisspeptin modulates GnRH secretion in mammals and peripheral administration of 10-amino acid fragment of kisspeptin (Kp10) induces LH release and ovulation in cattle. Experiments were done to determine if iv administration of kisspeptin will activate GnRH neurons (i.e., after crossing the blood-brain barrier) and if pre-treatment with a GnRH receptor blocker will alter kisspeptin-induced LH release (from gonadotrophs) and ovulation. In Experiment 1, cows (n = 3 per group) were given human-Kisspeptin10 (hKp10; 3 x 15 mg iv at 60-min intervals) or normal saline and euthanized 150 min after treatment was initiated. Every 20th free-floating section (50 μm thickness) from the preoptic area to hypothalamus was double immunostained to colocalize GnRH- (DAB) and activated neurons (cFOS; Nickel-DAB). Kisspeptin induced plasma LH release from 15 to 150 min (P = 0.01) but the proportion of activated GnRH neurons did not differ between groups (5.8% and 3.5%, respectively; P = 0.11). Immunogold electron microscopy detected close contacts between kisspeptin fibers and GnRH terminals in the median eminence. In Experiment 2, pubertal heifers (n = 5 per group) were treated with 1) hKp10 iv, 2) Cetrorelix (GnRH antagonist; im) + hKp10 iv or 3) saline on Day 6 of the follicular wave under low-progesterone condition. A rise in plasma LH concentration was detected from 15 to 240 min in the hKp10 group but not in cetrorelix or control group (P<0.001). Ovulations were detected only in the hKp10 group (4/5; P = 0.02). Cetrorelix treatment was associated with regression of the preovulatory dominant follicle and emergence of a new follicular wave 3.4±0.75 days after the treatment in all five heifers. Results support the hypothesis that the effect of peripheral kisspeptin is mediated downstream of GnRH synthesis and does not involve GnRH-independent LH release from gonadotrophs. Peripheral kisspeptin may release pre-synthesized GnRH from the nerve terminals in areas outside the blood-brain barrier.

LBMON114 Enrichment Of Rare Sequence Variants In Genes That Communicate Metabolic Signals To The GnRH System In Hypothalamic Amenorrhea

Abstract Introduction Functional hypothalamic amenorrhea (HA) is commonly associated with increased exercise or decreased caloric intake and often with stress. We have previously demonstrated an increased burden of rare sequence variants (RSVs) in genes involved in GnRH ontogeny and upstream regulation in women with HA, but the role of metabolic and stress signaling to the GnRH neuronal system is poorly defined in this population. Methods The study included 100 women with a confirmed diagnosis of HA. The control cohort consisted of 468 women (aged 45­­-65 years) drawn from the NIH ClinSeq® Project. Exome sequencing was performed on peripheral blood genomic DNA. A subset of 72 genes was analyzed that have been shown to: 1) link metabolic or stress with reproductive phenotypes or 2) integrate metabolic and stress pathways with control of GnRH secretion. Joint genotyping of case and control samples was performed using the GATK GenotypeGVCFs function, locus-filtering using the VariantRecalibrator function, and genotype refinement using CalculateGenotypePosteriors with computation of median depths. Median depth positions &amp;lt;10, positions failing GATK VQSR or GATK genotype quality scores &amp;lt;20 were excluded. RSVs were identified by &amp;lt; 1% frequency in any subpopulation in gnomeAD for all-subjects (AS) and &amp;lt; 1% frequency in non-Finnish Europeans for Caucasians (CS). Data were analyzed for AS and for CS using a one-sided Fisher exact test for metabolism genes and stress genes. An additional regression analysis was conducted on the number of RSVs in a given gene as a predictor of HA vs. control. Comparisons with a p-value of &amp;lt; 0.1 are reported. Results HA patients exhibited an increased burden of RSVs in metabolism genes vs. controls (AS p=0. 043; CS p=0.105). The total number of RSVs per gene highlighted differences between HA and controls for the following genes: ADAMTSL1, GRINA, GRIN1, HCRTR1, TENM3, and NOS1 (AS p&amp;lt;0. 001, p=0. 032, p=0. 057, p=0. 082, p=0. 091, p=0. 095; CS p=0. 024, p=0. 044, p=0. 044, p NS, p=0. 086, p NS). Interestingly, RSVs in NOS1 and TENM3 appeared to be protective for HA (odds ratio &amp;lt;1 for both). In contrast, candidate stress genes were not significant in either the AS or CS (p=0.788, p=0.910). Conclusions These data suggest that RSVs in genes involved in phenotypes or signaling pathways that link metabolism to GnRH secretion may predispose to development of HA in the setting of decreased energy balance, but not for stress-related genes. GRINA and GRIN1 are important components of glutamate signaling that facilitate both appetite and GnRH secretion either directly or through kisspeptin. HCRTR1 plays a similar role in linking appetite and GnRH secretion while NOS1, which facilitates kisspeptin signaling, may be protective. This work highlights the need for further studies to understand the potential roles of ADAMTSL1 and TENM3 as risk factors for HA. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

RF10 | PMON215 Greater changes in metabolic hormones are associated with a smaller decrease in pulsatile LH secretion in response to short term moderate energy restriction in healthy, young women

Abstract Background Energy restriction may result in hypothalamic amenorrhea by inhibiting GnRH secretion and downstream pulsatile LH secretion. Energy restriction also leads to adaptive changes in metabolic hormones. We sought to determine whether metabolic changes in response to energy restriction predict or mitigate inhibition of GnRH secretion. Methods Nineteen healthy women, (mean age ± SD; 23.36 ± 2.08 yrs) with regular ovulatory cycles and no evidence of energy restriction or excessive exercise, underwent two 5-day dietary interventions with identical exercise in the early follicular phase of two menstrual cycles. A neutral energy availability (NEA; 45 kCal/kg*LBM/d) was followed by a deficient energy availability (DEA; 20 kCal/kg*LBM/d) diet. On day five of each intervention, body composition was analyzed (BodPod®), and blood was sampled between 0800 —1600 hours for LH, TSH and GH, every 10 min, cortisol every 30 min, T3, reverse T3 (rT3) and T4 every 60 min, free T3 (FT3), free T4 (FT4) and TBG at 0800 h and 1600 h. We correlated means of pulsatile LH pulse frequency (LHPF) with integrated metabolic hormone changes across the day in the subset of women with reduced or unchanged LHPF after DEA (n=10) using linear regression. Results In ∼50% of healthy young women, LHPF decreased or was unchanged in response to short-term moderate energy deprivation. BMI and bodyweight declined after DEA in both groups while % fat mass was unchanged. TSH, T3, leptin, insulin declined, and T4 increased from NEA to DEA while glucose, cortisol and GH were unchanged. In subjects with reduced/unchanged LHPF, % delta T3 correlated inversely with delta LHPF (r=-0.727, p=0.017). Similarly, non-fasting delta TSH correlated inversely with % delta LHPF (r=-0.643, p=0.045), and % delta AUC TSH (r=-0.642, p=0.045) correlated inversely with DEA LHPF. Percent delta insulin (r= -0.722, p=0.018) and % delta insulin AUC (r= – 0.688, p=0.028) correlated inversely with delta LHPF. Percent delta insulin-glucose ratio (r=-0.772, p=0.009) and % change in AUC insulin-glucose ratio (r=-0.759, p=0.011) also correlated inversely with delta LHPF. This inverse relationship was preserved when insulin was normalized for caloric intake (n-insulin) was correlated with % delta LHPF (r=-0.722, p=0.018) and % delta AUC n-insulin correlated with delta LHPF (r= -0.631 and p= 0.050). Delta AUC leptin (r=-0.684, p=0.029) and % delta AUC leptin (r=-0.670, p=0.034) also correlated inversely with delta LHPF. Mean cortisol at NEA correlated inversely with LHPF during DEA (r=-0.816, p=0.007), implying that higher baseline cortisol levels may predict a greater relative fall in LHPF. Cortisol AUC in DEA also correlated inversely with LHPF in DEA (r=-0.722, p=0.028). Conclusion Our data suggest that greater changes in metabolic hormones may protect against the inhibitory effect of moderate energy deprivation on GnRH secretion in healthy young women with normal reproductive function. Presentation: Saturday, June 11, 2022 1:30 p.m. - 1:35 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ODP407 Effects of Leptin and Ghrelin on Ovarian Steroidogenesis and Involvement of BMP Action in Rat Granulosa Cells.

Abstract Leptin and ghrelin are the two major hormones which regulate food intake and energy balance. They have also been reported to have pleiotropic functions, including the modulation of ovarian function via the hypothalamic-pituitary-ovarian system or in a direct manner to the ovaries. Blood leptin levels increase over the menstrual cycle and peak at the time of the luteinizing hormone (LH) surge. Leptin secreted from adipocytes acts on the hypothalamus and pituitary gland to stimulate the secretion of GnRH and gonadotropins. In the ovaries, high levels of leptin inhibit follicle-stimulating hormone (FSH)-induced steroid secretion and follicular development. Ghrelin, secreted by the stomach, has been reported to modulate GnRH secretion in the hypothalamus and to affect FSH and LH secretion, as well as steroidogenesis in the ovaries. Knockout of these hormones leads to impaired fertility. However, the mechanism of the direct effects of leptin and ghrelin on the ovaries has not been well elucidated. In the present study, we investigated the effects of leptin and ghrelin on the ovarian steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic protein (BMP) system, which plays an important role in follicular development and steroidogenesis and acts as a luteinizing inhibitor. The results showed that treatment with leptin (1 to 300 ng/mL) did not affect either estradiol (E2) or progesterone (P4) production, but enhanced FSH-induced P4 production at low concentrations (10 ng/mL). On the other hand, treatment with ghrelin (1 to 300 nM) did not affect E2 and P4 production, but enhanced FSH-induced P4 production in a concentration-responsive manner. The mRNA levels of the follicular steroid synthases (StAR, P450scc, 3βHSD, 20αHSD) were examined. Leptin treatment enhanced 3βHSD mRNA levels. Both leptin treatment and ghrelin treatment decreased FSH-induced 20αHSD mRNA levels. However, both treatments decreased FSH-induced cAMP production. In addition, leptin treatment tended to suppress the transcriptional activity of Id-1 induced by BMP-6. Collectively, these results indicate that both leptin and ghrelin enhance FSH-induced P4 production by suppressing BMP signaling in rat granulosa cells. It was thus suggested that leptin and ghrelin which regulate food intake and energy balance can also act as endogenous regulators for the modulation of P4 synthesis and luteinizing process by ovarian follicles. Presentation: No date and time listed

OR17-3 Immortalized AVPV Kisspeptin Neurons Transiently Express GnRH Receptors under Specific Sex Steroid Conditions in vitro

Abstract In female animals, ovarian estradiol (E2) acts as negative feedback inhibition of GnRH secretion and positive feedback stimulation at ovulation. Both E2-regulated mechanisms work via stimulation or repression of distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While E2 clearly stimulates AVPV KP neurons to increase kiss1 expression during the preovulatory surge, mechanisms required for optimal potentiation of maximal GnRH surge release remain incompletely characterized. We used two immortalized KP cell lines demonstrating increased kiss1 expression under high E2 (KTaV-3), or kiss1 suppression under low E2 (KTaR-1), to probe GnRH receptor (GnRHR) expression levels under E2 conditions of varying doses and times of exposure. Additionally, we have begun to investigate transcriptional mechanisms underlying gnrhr expression in KP neurons in vitro. AVPV-derived KTaV-3 cells were treated with either E2 alone (5-100pM) or E2 with progesterone (4-40nM) for varying durations (4-96h), either constitutively or under modulating levels approximating changes found during the murine estrous cycle. All treatments were undertaken in serum-free (SF) media (DMEM) following a 50% serum synchronization. After total RNA isolation, cDNAs were probed with mouse gnrhr primers. Results reveal that gnrhr expression, not observed in typical growth media conditions, is induced in KTaV-3 cells to varying degrees dependent on prior E2/P4 exposure. Following shorter treatment times (12-24h), proestrus levels (100pM E2; 4nM P4) of sex steroids noticeably induced gnrhr, while exposure of KTaV-3 cells to a 96h estrous cycle revealed maximal gnrhr expression at the diestrus II→proestrus transition, with exposure cycles concluding with declining E2/rising P4 exhibiting no to minimal gnrhr expression. These results suggest that maximal KP neuronal expression of gnrhr may occur prior to the E2 peak on proestrus, potentially preparing AVPV KP neurons for GnRH surge release 12-16h later. Similar steroid conditions also increased dlx3 expression, a transcription factor required for GnRHR in pituitary, only in KTaV-3 cells. In KTaR-1 cells, gnrhr expression was observed only following E2 decreases, while dlx3 was constitutively elevated. Since cFos/JunB complex formation on the gnrhr promoter has been implicated in gonadotrope GnRHR levels, we explored how transfection of these transcription factors might affect gnrhr expression in KTaV-3 cells. Preliminary results reveal that fos/jun co-transfection stimulates gnrhr expression maximally at intermediate (DII) E2 levels, while higher E2 levels are required with fos or jun expression alone. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these results suggest KP neurons may possess the capacity to respond to intrahypothalamic GnRH only in certain estrous cycle stages, by differentially modulating GnRH receptivity in the AVPV. These findings supplement the prevailing model of feed-forward KP regulation of GnRH with potential short-loop KP-GnRH positive feedback regulation, providing an increased understanding of feedback mechanisms required for normal neuroendocrine regulation of reproduction. Presentation: Sunday, June 12, 2022 11:30 a.m. - 11:45 a.m.

The influence of estro-progestin therapy on neurohormonal activity in functional hypothalamic amenorrhea

Background: Functional hypothalamic amenorrhea (FHA) is a chronic endocrine disorder caused by the abnormal pulsatile secretion of neurohormones in the hypothalamus. Secretion of GnRH is regulated by kisspeptin/neurokinin B/dynorphin (KNDy) neurons. These neurons produce, among other neurohormones, neurokinin B (NKB) which regulates the coordinated stimulation or inhibition of GnRH secreting neurons. Aim of the study: Assessment and comparison of serum NKB in patients with FHA at baseline, and following 6 months of estrogen-progestagen therapy. Materials and methods: Fifty-five patients with functional hypothalamic amenorrhea were included in the study group. Serum concentrations of neurokinin B (NKB), follicle stimulating hormone (FSH), luteinizing hormone (LH), 17-β-estradiol (E2), prolactin (PRL), cortisol, testosterone (T), dehydroepiandrosterone sulfate (DHEA-S), thyroid-stimulating hormone (TSH), free thyroxine (fT4), fasting glucose and insulin, as well as lipid profile were measured at baseline. At the time of diagnosis, patients with FHA were prescribed a course of 2 mg 17-β-estradiol and 10 mg dydrogesterone for duration of 6 months. Serum NKB was then reassessed following treatment at 6 months. Results: At baseline, the FHA group was found to have a decreased serum NKB concentration when compared to a healthy control group. Following 6 months of sequential estrogen-progestogen hormone therapy, this study did not find any statistically significant difference in serum NKB concentration in the treatment arm compared to baseline. Conclusions: For the first time, NKB secretion has been studied in patients with FHA. A significantly lower level of serum NKB was observed in these patients at baseline, when compared to a control group. After 6 months of combination estrogen-progesterone therapy, no significant changes in NKB levels were observed in these patients. These findings, for the first time in the literature, provide insight into the perceived benefit of HRT, calling into question its benefit in addressing the underlying etiopathogenetic contributors of FHA. These new findings may contribute to more targeted and appropriate treatment of such patients in the future.

The Kisspeptin and Kisspeptin receptor in follicular microenvironment: is that really necessary for oocyte maturation and fertilisation?

The aim of this study was to determine whether Kisspeptin and Kisspeptin receptor in the follicular microenvironment is necessary for human oocyte maturation and fertilisation. The cumulus cell (CC) and follicle fluids (FF) obtained from the first aspirated follicles (n = 52) from 32 patients were divided into three groups considering nuclear maturation and fertilisation results of oocytes: (1) Metaphase I or germinal vesicle stage oocytes (incomplete nuclear maturation, n = 10), (2) unfertilised metaphase II oocytes (incomplete cytoplasmic maturation, n = 16), and (3) fertilised metaphase II oocytes (completed nuclear-cytoplasmic maturation, n = 26). The gene expression levels were assessed by RT-PCR. The levels of Kisspeptin (KISS1) and Kisspeptin receptor (KISS1R) were measured by ELISA. There were no significant efficacy KISS1 and KISS1R gene expressions in cumulus cells in terms of oocyte nuclear maturation stage (Group 1, vs Group 2 + Group 3) (respectively p = .49; p = .45). In terms of the cytoplasmic maturation stage (Group 2, vs Group 3); KISS1 and KISS1R expressions in CCs were comparable (respectively p = .07; p = .08). In FFs, KISS1 and KISS1R concentrations were similar between all groups (respectively p = .86; p = .26). In conclusion, the relative KISS1 and KISS1R expressions in CC and also KISS1 and KISS1R level of FF were independent of oocytes nuclear and/or cytoplasmic maturation. Impact statement What is already known on this subject? It has been demonstrated that Kisspeptin is an essential regulator of reproductive function and plays a key role in the modulation of GnRH secretion and gonadotropin release. Still, no information is available about the link between gene expression or concentration in the follicular microenvironment and oocyte development. What do the results of this study add? The study has shown that the relative Kisspeptin (KISS1) and Kisspeptin receptor (KISS1R) and expressions in cumulus cell (CC) and also KISS1 and KISS1R levels of follicle fluids (FF) were independent of oocytes nuclear and/or cytoplasmic maturation. What are the implications of these findings for clinical practice and/or further research? Based on the findings, it is difficult to establish a concept that kisspeptin can directly induce oocyte maturation. Nevertheless, to confirm these findings, further studies with a larger sample size are needed.

The Congenital and Acquired Mechanisms Implicated in the Etiology of Central Precocious Puberty.

The etiology of central precocious puberty (CPP) is multiple and heterogeneous, including congenital and acquired causes that can be associated with structural or functional brain alterations. All causes of CPP culminate in the premature pulsatile secretion of hypothalamic GnRH and, consequently, in the premature reactivation of hypothalamic-pituitary-gonadal axis. The activation of excitatory factors or suppression of inhibitory factors during childhood represent the 2 major mechanisms of CPP, revealing a delicate balance of these opposing neuronal pathways. Hypothalamic hamartoma (HH) is the most well-known congenital cause of CPP with central nervous system abnormalities. Several mechanisms by which hamartoma causes CPP have been proposed, including an anatomical connection to the anterior hypothalamus, autonomous neuroendocrine activity in GnRH neurons, trophic factors secreted by HH, and mechanical pressure applied to the hypothalamus. The importance of genetic and/or epigenetic factors in the underlying mechanisms of CPP has grown significantly in the last decade, as demonstrated by the evidence of genetic abnormalities in hypothalamic structural lesions (eg, hamartomas, gliomas), syndromic disorders associated with CPP (Temple, Prader-Willi, Silver-Russell, and Rett syndromes), and isolated CPP from monogenic defects (MKRN3 and DLK1 loss-of-function mutations). Genetic and epigenetic discoveries involving the etiology of CPP have had influence on the diagnosis and familial counseling providing bases for potential prevention of premature sexual development and new treatment targets in the future. Global preventive actions inducing healthy lifestyle habits and less exposure to endocrine-disrupting chemicals during the lifespan are desirable because they are potentially associated with CPP.

Hypothalamic Kisspeptin Neurons: Integral Elements of the GnRH System.

Highly sophisticated and synchronized interactions of various cells and hormonal signals are required to make organisms competent for reproduction. GnRH neurons act as a common pathway for multiple cues for the onset of puberty and attaining reproductive function. GnRH is not directly receptive to most of the signals required for the GnRH secretion during the various phases of the ovarian cycle. Kisspeptin neurons of the hypothalamus convey these signals required for the synchronized release of the GnRH. The steroid-sensitive anteroventral periventricular nucleus (AVPV) kisspeptin and arcuate nucleus (ARC) KNDy neurons convey steroid feedback during the reproductive cycle necessary for GnRH surge and pulse, respectively. AVPV region kisspeptin neurons also communicate with nNOS synthesizing neurons and suprachiasmatic nucleus (SCN) neurons to coordinate the process of the ovarian cycle. Neurokinin B (NKB) and dynorphin play roles in the GnRH pulse stimulation and inhibition, respectively. The loss of NKB and kisspeptin function results in the development of neuroendocrine disorders such as hypogonadotropic hypogonadism (HH) and infertility. Ca2+ signaling is essential for GnRH pulse generation, which is propagated through gap junctions between astrocytes-KNDy and KNDy-KNDy neurons. Impaired functioning of KNDy neurons could develop the characteristics associated with polycystic ovarian syndrome (PCOS) in rodents. Kisspeptin-increased synthesis led to excessive secretion of the LH associated with PCOS. This review provides the latest insights and understanding into the role of the KNDy and AVPV/POA kisspeptin neurons in GnRH secretion and PCOS.

The Role of Kisspeptin in the Control of the Hypothalamic-Pituitary-Gonadal Axis and Reproduction.

The discovery of kisspeptin as a critical central regulatory factor of GnRH release has given people a novel understanding of the neuroendocrine regulation in human reproduction. Kisspeptin activates the signaling pathway by binding to its receptor kisspeptin receptor (KISS1R) to promote GnRH secretion, thereby regulating the hypothalamic-pituitary-gonadal axis (HPG) axis. Recent studies have shown that kisspeptin neurons located in arcuate nucleus (ARC) co-express neurokinin B (NKB) and dynorphin (Dyn). Such neurons are called KNDy neurons. KNDy neurons participate in the positive and negative feedback of estrogen to GnRH secretion. In addition, kisspeptin is a key factor in the initiation of puberty, and also regulates the processes of female follicle development, oocyte maturation, and ovulation through the HPG axis. In male reproduction, kisspeptin also plays an important role, getting involved in the regulation of Leydig cells, spermatogenesis, sperm functions and reproductive behaviors. Mutations in the KISS1 gene or disorders of the kisspeptin/KISS1R system may lead to clinical symptoms such as idiopathic hypogonadotropic hypogonadism (iHH), central precocious puberty (CPP) and female infertility. Understanding the influence of kisspeptin on the reproductive axis and related mechanisms will help the future application of kisspeptin in disease diagnosis and treatment. In this review, we critically appraise the role of kisspeptin in the HPG axis, including its signaling pathways, negative and positive feedback mechanisms, and its control on female and male reproduction.

Whole Transcriptome Sequencing of Peripheral Blood Shows That Immunity/GnRH/PI3K-Akt Pathways Are Associated With Opioid Use Disorder.

BackgroundOpioid use disorder (OUD), which is most commonly exhibited as addiction, is a persistent chronic disease that places a burden on families and society. Various peripheral traits have been linked to OUD in the past, but research on this topic is insufficient.MethodsSeven male patients with OUD and 7 male healthy controls with matched demographic and clinical data were enrolled in this study. Peripheral blood RNA was used to construct an rRNA-removed library and a small RNA library. The peripheral transcriptomic differences between the two groups were investigated using RNA-seq. Differentially expressed messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) were identified by bioinformatics methods, and functional enrichment analysis with differentially expressed RNAs was performed to investigate the potential biological mechanisms of OUD.ResultsA total of 229 mRNAs (115 upregulated, 114 downregulated), 416 lncRNAs (191 upregulated, 225 downregulated), 17 circRNAs (16 upregulated, 1 downregulated) and 74 miRNAs (42 upregulated, 32 downregulated) were differentially expressed between the OUD group and the healthy control group. Functional enrichment analysis with differentially expressed mRNAs showed that immunity, GnRH secretion, and PI3K-Akt signaling pathways were associated with OUD. Immunity-, JAK-STAT-, and insulin-related pathways were enriched in functional enrichment analysis of target genes predicted by differentially expressed miRNAs.ConclusionWe identified hundreds of differentially expressed genes that were enriched in immunity, GnRH secretion and PI3K-Akt signaling pathways. Some genes with significant changes might be used as potential biomarkers for progression and treatment of OUD.

HnRNP E1 Regulates HPV16 Oncogene Expression and Inhibits Cervical Cancerization.

hnRNP E1 (heterogeneous nuclear ribonucleoprotein E1) is an important RNA-binding protein (RBPs) that plays a vital role in tumor development. Human papillomavirus 16 (HPV16) contains numerous sites that can bind to RNA/DNA and may be modified by multiple RBPs, which contribute to HPV gene expression and HPV-associated cancer development. However, the effects of hnRNP E1 on HPV16 oncogenes in the development of cervical lesions remain unclear. A total of 816 participants with different grades of cervical lesions were enrolled in a community-based cohort established in Shanxi Province, China. The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were used to analyze the association between hnRNP E1 mRNA expression and cervical lesions. Cells with up_ and down_regulated hnRNP E1 were established. hnRNP E1 functions were evaluated using cell counting kit-8, flow cytometry analyses, and chromatin immunoprecipitation sequencing. Our results showed that hnRNP E1 expression was linearly dependent on the severity of the cervical lesions. Low expression of HPV16 E2, high expression of E6, and a low ratio of E2 to E6 could increase the risk of cervical lesions. hnRNP E1 expression was correlated with HPV16 oncogene expression. hnRNP E1-relevant genes were involved in the dopaminergic synapses, Wnt signaling pathway, gnRH secretion, and mTOR signaling pathway. hnRNP E1 significantly inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G0/G1 stage, and decreased HPV16 E6 expression. Our results indicate that hnRNP E1 could downregulate HPV16 E6 oncogene expression and inhibit cervical cancerization, which sheds new light on preventing the carcinogenicity of HPV across a range of diseases by regulating RNA-binding proteins.

Differences in Vitreous Protein Profiles in Patients With Proliferative Diabetic Retinopathy Before and After Ranibizumab Treatment.

Proliferative diabetic retinopathy (PDR) accounts for severe impact on vision, its mechanism is still poorly understood. To compare the differences of vitreous protein profiles in PDR patients before and after a complete anti-vascular endothelial growth factor (VEGF) loading dose with ranibizumab treatment. Twelve vitreous humor (VH) samples were collected from six PDR patients before (set as pre group) and after (set as post group) intravitreal injection of ranibizumab (IVR) treatment. LC–MS/MS and bioinformatics analysis were performed to identify differentially expressed proteins. Proteins were validated with targeted proteomics using parallel reaction monitoring (PRM) in a validation set consisting of samples from the above patients. A total of 2680 vitreous proteins were identified. Differentially expressed proteins were filtrated with fold change ≥2.0 (post group/ pre group protein abundance ratio ≥2 or ≤ 0.5) and p-value <0.05. 11 proteins were up-regulated and 17 proteins were down-regulated, while consistent presence/absence expression profile group contains one elevated protein and nine reduced proteins, among which seven proteins were identified as potential biomarkers for IVR treatment through PRM assays. Bioinformatics analysis indicated the up-regulated proteins were significantly enriched in “GnRH secretion” and “Circadian rhythm” signaling pathway. This report represents the first description of combined label-free quantitative proteomics and PRM analysis of targeted proteins for discovery of different proteins before and after IVR treatment in the same patient. IVR treatment may protect against PDR by promoting SPP1 expression through “GnRH secretion” and “Circadian rhythm” signaling pathway.

Developmental sex differences in the peri-pubertal pattern of hypothalamic reproductive gene expression, including Kiss1 and Tac2, may contribute to sex differences in puberty onset

The mechanisms regulating puberty still remain elusive, as do the underlying causes for sex differences in puberty onset (girls before boys) and pubertal disorders. Neuroendocrine puberty onset is signified by increased pulsatile GnRH secretion, yet how and when various upstream reproductive neural circuits change developmentally to govern this process is poorly understood. We previously reported day-by-day peri-pubertal increases (Kiss1, Tac2) or decreases (Rfrp) in hypothalamic gene expression of female mice, with several brain mRNA changes preceding external pubertal markers. However, similar pubertal measures in males were not previously reported. Here, to identify possible neural sex differences underlying sex differences in puberty onset, we analyzed peri-pubertal males and directly compared them with female littermates. Kiss1 expression in male mice increased over the peri-pubertal period in both the AVPV and ARC nuclei but with lower levels than in females at several ages. Likewise, Tac2 expression in the male ARC increased between juvenile and older peri-pubertal stages but with levels lower than females at most ages. By contrast, both DMN Rfrp expressionand Rfrp neuronal activation strongly decreased in males between juvenile and peri-pubertal stages, but with similar levels as females. Neither ARC KNDy neuronal activation nor Kiss1r expression in GnRH neurons differed between males and females or changed with age. These findings delineate several peri-pubertal changes in neural populations in developing males, with notable sex differences in kisspeptin and NKB neuron developmental patterns. Whether these peri-pubertal hypothalamic sex differences underlie sex differences in puberty onset deserves future investigation.

Psikoneuroimunologi Depresi pada Polycystic Ovary Syndrome (PCOS)

&lt;p&gt;&lt;strong&gt;&lt;em&gt;Introduction&lt;/em&gt;&lt;/strong&gt;&lt;em&gt;: Polycystic ovary syndrome (PCOS) is heterogeneous, complex endocrine disorder with unknown etiology, affecting 4%−18% of reproductive age women and associated with reproductive, metabolic and psychological dysfunction. Along with physical disorders, many mental disorders are also associated with PCOS, about 40% of women with PCOS have depression. This literature review aims to explore how PCOS can lead to depression in term of psychoneuroimmunology aspects.&lt;/em&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;em&gt;Method&lt;/em&gt;&lt;/strong&gt;&lt;em&gt;: Literature review was conducted through search engine from Google Scholar and Clinical Key with keywords “Polycystic ovary syndrome”, “depression”, “psychoneuroimmunology”, “distress”, and “inflammation” from 2015-2020.&lt;/em&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;em&gt;Result&lt;/em&gt;&lt;/strong&gt;&lt;em&gt;: The important role of the HPG axis in the pathophysiology of mood disorder has concluded that high comorbidity between depression and PCOS are caused by hyperandrogenism and insulin resistance mediated by low grade chronic inflammation. The activation of HPA by stress exerts an inhibitory effect on the female reproductive system. CRH can inhibit GnRH secretion from the hypothalamus. The anti-reproductive action of CRH in the ovaries of women with high psychosocial stress lead to early ovarian failure. In addition, the pathophysiology of depression and mental stress in PCOS is associated with various changes including high activity of pro-inflammation markers and the body's immune system during stress, it has also been linked to increased cortisol levels, increased sympathetic activity and decreased serotonin levels in the central nervous system.&lt;/em&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;em&gt;Result&lt;/em&gt;&lt;/strong&gt;&lt;em&gt;: There’s close relationship between PCOS and depression in terms of psychoneuroimmunology aspects, by HPG axis role. Comprehensive management of depression is improving outcome strategy in women with PCOS by involving CLP.&lt;/em&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;em&gt;Keywords&lt;/em&gt;&lt;/strong&gt;&lt;em&gt;: &lt;strong&gt;Polycystic ovary syndrome (PCOS), Depression, Psychoneuroimmunology, HPG axis&lt;/strong&gt;&lt;/em&gt;&lt;/p&gt;