GnRH Secretion Research Articles

Impact of Cushing's syndrome on fertility and pregnancy

Cushing's syndrome is defined by an endogenous or exogenous hypercortisolism. Increased cortisol, as well as increased androgens will have a negative impact on the pulsatile secretion of GnRH, thus leading to an increased risk of infertility. However, pregnancy can occur in a woman with Cushing's syndrome: it is a challenging situation, because of the numerous consequences which can be observed in the mother (increased risk of gestational diabetes, hypertension, eclampsia… in addition to the specific complications of hypercortisolism) and in the fetus (intrauterine growth retardation, prematurity). In contrast, Cushing's syndrome can also appear during pregnancy. It is a very rare situation: the diagnosis is challenging because of the numerous hormonal changes induced by pregnancy on cortisol levels. The objective of this brief review will be to detail the mechanisms of infertility due to hypercortisolism, the diagnostic methods of Cushing's syndrome during pregnancy, the maternal and fetal consequences of hypercortisolism during pregnancy, and finally the potential means of contraception that can be proposed.

Maternal DBP exposure promotes synaptic formation in offspring by activating astrocytes via the AKT/NF-κB/IL-6/JAK2/STAT3 signaling pathway

It has been demonstrated that activated astrocytes in the hypothalamus could disrupt GnRH secretion in offspring after maternal di-n-butyl phthalate (DBP) exposure, indicating that the effect of DBP on astrocyte activation and crosstalk between astrocytes and neurons is still worthy of further investigation. In this study, pregnant mice were intragastrically administered DBP dissolved in corn oil from gestational days (GD) 12.5–21.5. Maternal DBP exposure resulted in hippocampal astrocyte activation, abnormal synaptic formation, and reduced autonomic and exploratory behavior in offspring on postnatal day (PND) 22. Further studies identified that mono-n-butyl phthalate (MBP) induced astrocyte activation and proliferation by activating the AKT/NF-κB/IL-6/JAK2/STAT3 signaling pathway. Moreover, upregulated thrombospondin 1 (TSP1) in activated astrocytes regulated synaptic-related protein expression. This study highlights the neurotoxicity of maternal DBP exposure to offspring, which provides new insights into identifying potential molecular targets for the treatment of diseases related to neurological development disorders in children.

Lactational Amenorrhea: Neuroendocrine Pathways Controlling Fertility and Bone Turnover.

Lactation is a physiological state of hyperprolactinemia and associated amenorrhea. Despite the fact that exact mechanisms standing behind the hypothalamus–pituitary–ovarian axis during lactation are still not clear, a general overview of events leading to amenorrhea may be suggested. Suckling remains the most important stimulus maintaining suppressive effect on ovaries after pregnancy. Breastfeeding is accompanied by high levels of prolactin, which remain higher than normal until the frequency and duration of daily suckling decreases and allows normal menstrual function resumption. Hyperprolactinemia induces the suppression of hypothalamic Kiss1 neurons that directly control the pulsatile release of GnRH. Disruption in the pulsatile manner of GnRH secretion results in a strongly decreased frequency of corresponding LH pulses. Inadequate LH secretion and lack of pre-ovulatory surge inhibit the progression of the follicular phase of a menstrual cycle and result in anovulation and amenorrhea. The main consequences of lactational amenorrhea are connected with fertility issues and increased bone turnover. Provided the fulfillment of all the established conditions of its use, the lactational amenorrhea method (LAM) efficiently protects against pregnancy. Because of its accessibility and lack of additional associated costs, LAM might be especially beneficial in low-income, developing countries, where modern contraception is hard to obtain. Breastfeeding alone is not equal to the LAM method, and therefore, it is not enough to successfully protect against conception. That is why LAM promotion should primarily focus on conditions under which its use is safe and effective. More studies on larger study groups should be conducted to determine and confirm the impact of behavioral factors, like suckling parameters, on the LAM efficacy. Lactational bone loss is a physiologic mechanism that enables providing a sufficient amount of calcium to the newborn. Despite the decline in bone mass during breastfeeding, it rebuilds after weaning and is not associated with a postmenopausal decrease in BMD and osteoporosis risk. Therefore, it should be a matter of concern only for lactating women with additional risk factors or with low BMD before pregnancy. The review summarizes the effect that breastfeeding exerts on the hypothalamus–pituitary axis as well as fertility and bone turnover aspects of lactational amenorrhea. We discuss the possibility of the use of lactation as contraception, along with this method’s prevalence, efficacy, and influencing factors. We also review the literature on the topic of lactational bone loss: its mechanism, severity, and persistence throughout life.

Congenital hypogonadotropic hypogonadism complicated by neuroblastoma

.A 3-mo-old male infant was referred to our hospital with micropenis. Since his serum LH, FSH, and testosterone levels were low (< 0.3 mIU/mL, 0.08 mIU/mL, and < 0.03 ng/mL, respectively), Kallmann syndrome/normosmic hypogonadotropic hypogonadism was suspected. In the process of searching for complications of Kallmann syndrome/normosmic hypogonadotropic hypogonadism, a right adrenal gland tumor was incidentally discovered. The patient was diagnosed with stage 1 neuroblastoma. A homozygous p.P147L (c.C440T) mutation in the KISS1R gene was detected as a cause of the congenital hypogonadotropic hypogonadism. KISS1-KISS1R signaling, which is essential for GnRH secretion, exhibits anti-metastatic and/or anti-tumoral roles in numerous cancers. High KISS1 expression levels reportedly predict better survival outcomes than low KISS1 expression levels in neuroblastoma. Therefore, decreased KISS1-KISS1R signaling may have played a role in the neuroblastoma in this patient.

The m6A mRNA demethylase FTO regulates GnRH secretion in Mn-induced precocious puberty

The GABAA receptor (GABAAR) plays important roles in the regulation of Mn-induced GnRH secretion in immature female rats. However, the underlying molecular mechanisms remain unknown. Here, we assessed whether FTO and its substrate m6A are correlated with GABAAR expression in GnRH neurons after treatment with Mn in vitro and in vivo. Our study indicated that Mn treatment increased the expression of GnRH mRNA and decreased the levels of GABAAR protein but had no effect on GABAAR mRNA. Moreover, Mn upregulated the levels of FTO and inhibited global cellular m6A levels and GABAAα2 mRNA m6A levels. Knockdown of FTO increased the expression of GABAAR protein and GABAAα2 mRNA m6A levels. Data from rat models further demonstrate that inhibition of FTO suppressed GABAAR protein expression in the hypothalamus, causing delayed puberty onset. Collectively, our findings suggest that FTO-dependent m6A demethylation plays a critical role in regulating GABAAR mRNA processing in GnRH neurons.

Functional analysis of the emerging roles for the KISS1/KISS1R signaling pathway in cancer metastasis

Cancer metastasis, a process that primary tumor cells disseminate to secondary organs, is the most lethal and least effectively treated characteristic of human cancers. Kisspeptins are proteins encoded by the KISS1 gene that was originally described as a melanoma metastasis suppressor gene. Then, Kisspeptins were discovered as the natural ligands of the G-protein-coupled receptor 54 (GPR54) that is also called KISS1R. The KISS1/KISS1R signaling is essential to control GnRH secretion during puberty and to establish mammalian reproductive function through the hypothalamic-pituitary-gonadal (HPG) axis. Although KISS1 primarily plays a suppressive role in the metastasis progression in several cancer types, emerging evidence indicates that the physiological effect of KISS1/KISS1R in cancer metastasis is tissue context-dependent and still controversial. Here, we will discuss the epigenetic mechanism involved in the regulation of KISS1 gene expression, the context-dependent role of KISS1/KISS1R, prometastasis/anti-metastasis signaling pathways of KISS1/KISS1R, and the perspective anticancer therapeutics via targeting KISS1/KISS1R.

Pathogenic and Low-Frequency Variants in Children With Central Precocious Puberty.

BackgroundCentral precocious puberty (CPP) due to premature activation of GnRH secretion results in early epiphyseal fusion and to a significant compromise in the achieved final adult height. Currently, few genetic determinants of children with CPP have been described. In this translational study, rare sequence variants in MKRN3, DLK1, KISS1, and KISS1R genes were investigated in patients with CPP.MethodsFifty-four index girls and two index boys with CPP were first tested by Sanger sequencing for the MKRN3 gene. All children found negative (n = 44) for the MKRN3 gene were further investigated by whole exome sequencing (WES). In the latter analysis, the status of variants in genes known to be related with pubertal timing was compared with an in-house Cypriot control cohort (n = 43). The identified rare variants were initially examined by in silico computational algorithms and confirmed by Sanger sequencing. Additionally, a genetic network for the MKRN3 gene, mimicking a holistic regulatory depiction of the crosstalk between MKRN3 and other genes was designed.ResultsThree previously described pathogenic MKRN3 variants located in the coding region of the gene were identified in 12 index girls with CPP. The most prevalent pathogenic MKRN3 variant p.Gly312Asp was exclusively found among the Cypriot CPP cohort, indicating a founder effect phenomenon. Seven other CPP girls harbored rare likely pathogenic upstream variants in the MKRN3. Among the 44 CPP patients submitted to WES, nine rare DLK1 variants were identified in 11 girls, two rare KISS1 variants in six girls, and two rare MAGEL2 variants in five girls. Interestingly, the frequent variant rs10407968 (p.Gly8Ter) of the KISS1R gene appeared to be less frequent in the cohort of patients with CPP.ConclusionThe results of the present study confirm the importance of the MKRN3-imprinted gene in genetics of CPP and its key role in pubertal timing. Overall, the results of the present study have emphasized the importance of an approach that aligns genetics and clinical aspects, which is necessary for the management and treatment of CPP.

A dual kisspeptin-GnRH immunogen for reproductive immunosterilization

GnRH immunogens have been extensively employed in immunocontraception of animals. While they are effective, they are not 100% efficacious and of limited duration. GnRH secretion is dependent on upstream stimulation by kisspeptin. We therefore hypothesised that a dual immunogen combining GnRH and kisspeptin may be more efficacious through targeting two levels of the axis. We have previously shown GnRH immunogen elicits permanent sterilisation when sheep are vaccinated neonatally suggesting that the efficacy of GnRH immunisation may be dependent on the stage of reproductive development. We have now studied over 300 days the efficacy of immunisation with a dual immunogen comprising GnRH linked to kisspeptin via a hepatitis B T helper peptide sequence (GKT) administered to male and female rats prepubertally, pubertally and as adults. At all stages of development all immunised animals produced antibodies to GnRH, kisspeptin and GKT but differentially in titre with respect to sex and stage of development. In immunised adult, prepubertal and pubertal males testosterone and testes length was markedly reduced by 60 days and remained at low levels until day 150. Thereafter, testosterone recovered to pre immunisation levels and testes length increased to a maximum of about 40% of controls. 80% of males were infertile in three matings over 250 days. In prepubertal and pubertal female rats a single immunisation at day 0 reduced estradiol to low levels by day 60 which remained low until termination of the experiment on day 300. In matings of these females with fertile males on days 90, 120 and 250, 74% of prepubertal females were infertile and impressively, 100% (10/10) of pubertal females were infertile after a single immunisation on day 0. These findings set the scene for exploration of immunosterilisation of wild and domestic animals after a single immunisation.

Epigenomic and transcriptomic analyses reveal early activation of the HPG axis in in vitro-produced male dairy calves.

In cattle, several calves born after IVP ("in vitro" embryo production) present similar birthweight to those generated after MOET (multiple ovulation and embryo transfer). However, the underlying molecular patterns in organs involved in the developmental process are unknown and could indicate physiological programming. The objectives of this study were: (1) to compare epigenomic and transcriptomic modifications in the hypothalamus, pituitary, gonadal and adrenal organs between 3months old ovum pick-up-IVP and MOET male calves (n=4 per group) and (2) to use blood epigenomic data to proxy methylation of the inner organs. Extracted gDNA and RNA were sequenced through whole-genome bisulfite sequencing and RNA sequencing, respectively. Next, bioinformatic analyses determined differentially methylated cytosines (DMC) and differentially expressed genes (DEG) (FDR<0.05) in IVP versus MOET samples and the KEGG pathways that were overrepresented by genes associated with DMC or DEG (FDR<0.1). Pathways related to hypothalamus, pituitary, gonadal (HPG) axis activation (GnRH secretion in the hypothalamus, GnRH signaling in the pituitary, and steroidogenesis in the testicle) were enriched in IVP calves. Modeling the effect of the methylation levels and the group on the expression of all the genes involved in these pathways confirmed their upregulation in HPG organs in IVP calves. The application of the DIABLO method allowed the identification of 15 epigenetic and five transcriptomic biomarkers, which were able to predict the embryo origin using the epigenomic data from the blood. In conclusion, the use of an integrated epigenomic-transcriptomic approach suggested an early activation of the HPG axis in male IVP calves compared to MOET counterparts, and the identification of potential biomarkers allowed the use of blood samples to proxy methylation levels of the relevant internal organs.

Identification of Photoperiod-Induced LncRNAs and mRNAs in Pituitary Pars Tuberalis of Sheep.

The pituitary pars tuberalis (PT) is the regulating center of seasonal reproduction, which can sense the melatonin signal and eventually cause downstream changes of GnRH secretion through TSHβ. Recently, lncRNAs have been identified in animal reproductive-related tissues, and they play important roles in reproductive regulation. Therefore, in this study, we expect to identify photoperiod-induced lncRNAs and genes in pituitary PT of sheep by comparison of expression profiles between short photoperiod (SP) and long photoperiod (LP). Through RNA-Seq, a total of 55,472 lncRNAs were identified in pituitary PT of Sunite ewes. The number of differentially expressed (DE) genes and lncRNAs between SP and LP increased gradually with the extension of LP (from LP7 to LP42). The notable LP-induced candidate genes included EYA3, TSHB, SIX1, DCT, VMO1, AREG, SUV39H2, and EZH2, and SP-induced genes involved ENSOARG00000012585, CHGA, FOS, SOCS3, and TH. In enriched pathways for DE genes and lncRNA target genes between SP and LP, the reproduction- and circadian-related pathways were highlighted. In addition, the interactome analysis of lncRNAs and their targets implied that MSTRG.209166 and its trans-target TSHB, MSTRG.288068 and its cis-target SIX1, and ENSOARG00000026131 and its cis-target TH might participate in regulation of seasonal reproduction. Together, these results will help to determine important photoperiod-induced lncRNAs and genes and give us some new insights into the epigenetic regulation of seasonal reproduction in sheep.

Functional Hypothalamic Amenorrhea: A Stress-Based Disease

The aim of the study is to present the problem of functional hypothalamic amenorrhea, taking into account any disease and treatment, diagnosis, and consequences of this disease. We searched PubMed (MEDLINE) and included 38 original and review articles concerning functional hypothalamic amenorrhea. Functional hypothalamic amenorrhea is the most common cause of secondary amenorrhea in women of childbearing age. It is a reversible disorder caused by stress related to weight loss, excessive exercise and/or traumatic mental experiences. The basis of functional hypothalamic amenorrhea is hormonal, based on impaired pulsatile GnRH secretion in the hypothalamus, then decreased secretion of gonadotropins, and, consequently, impaired hormonal function of the ovaries. This disorder leads to hypoestrogenism, manifested by a disturbance of the menstrual cycle in the form of amenorrhea, leading to anovulation. Prolonged state of hypoestrogenism can be very detrimental to general health, leading to many harmful short- and long-term consequences. Treatment of functional hypothalamic amenorrhea should be started as soon as possible, and it should primarily involve lifestyle modification. Only then should pharmacological treatment be applied. Importantly, treatment is most often long-term, but it results in recovery for the majority of patients. Effective therapy, based on multidirectional action, can protect patients from numerous negative impacts on fertility, cardiovascular system and bone health, as well as reducing mental morbidity.

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.

Chronic Undernutrition Inhibits KNDy Neurons in Ovariectomized Ewe Lambs

Undernutrition negatively impacts reproductive success, at least in part, through a central suppression of GnRH secretion. Given that GnRH neurons are devoid of receptors for peripheral metabolic hormones such as leptin and insulin, nutritional regulation of GnRH secretion must be through afferent input. Neurons which co-express kisspeptin, neurokinin B (NKB), and dynorphin, termed KNDy neurons, are a unique population of cells in the arcuate nucleus (ARC) of the hypothalamus and are believed to play a critical role in GnRH/LH pulse generation. With our recent evidence that chronic feed restriction reduced kisspeptin and NKB in young, castrated male sheep, we hypothesized that nutrient restriction would inhibit expression of the KNDy neuron peptides kisspeptin and NKB, while increasing dynorphin expression in young, ovariectomized female sheep. Fifteen ewe lambs were ovariectomized and were fed to maintain body weight (n=7; Fed) or feed-restricted to lose 20% of pre-study body weight (FR; n=8). Blood samples were taken weekly every 12 minutes for 4.5 hours via jugular venipuncture and plasma was stored at -20°C until assessment of LH using radioimmunoassay. Body weights were recorded weekly and feed amounts were adjusted to achieve desired body weights. Following blood collection at Week 13, animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and tissue containing the hypothalamus was collected for assessment of KNDy neuropeptide mRNA abundance by in situ hybridization (RNAscope). At Week 13, the average percent change in body weight was clearly evident (Fed, 7.35 +/- 2.2% vs FR, -21.36 +/- 0.9%), and mean LH concentrations were lower in FR ewes (10.70 +/- 3.1 ng/ml) compared to Fed controls (20.98 +/- 3.8 ng/ml). Data analyzed to date for kisspeptin, NKB, and dynorphin in the ARC show that feed restriction reduced the number of kisspeptin mRNA-expressing cells (Fed, 165 +/- 25 vs FR, 9.25 +/- 6), the number of NKB mRNA-expressing cells (Fed, 141 +/- 28 vs FR, 24 +/- 5), and the number of dynorphin mRNA-expressing cells (Fed, 109 +/- 41 vs FR, 29.5 +/- 24). Together, these findings demonstrate that chronic feed restriction suppresses KNDy neurons and supports a role for these key reproductive neurons in the central mechanism governing GnRH/LH secretion during undernutrition in female sheep.

Acute Ischaemic Stroke With Hyperprolactinemia:A Case Report

Background: Macroincidentalomas were reported in 0.2% of patient underwent imaging (CT scans) for central nervous symptoms (1). In acute ischaemic stroke with hyperprolactinemia, the diagnosis of a double pathology of ischemic stroke and sellar tumour especially prolactinoma need to be considered. Hyperprolactinemia itself may be considered as a risk factor for ischemic stroke due to its thrombogenic effect (3). Clinical Case: A 47-year old man underlying hypertension and diabetes mellitus for 5 years presented with sudden onset of right sided body weakness associated with facial asymmetry and aphasia. No history of fever or trauma. Asymptomatic of hyperprolactinemia previously. On general examinations Glasgow Coma Scale 11/15 Eye 4 Verbal 1 Motor 6, blood pressure was unstable with readings of systolic 244mmHg and diastolic 142mmHg. Neurological examinations showed expressive aphasia, right hemianopia, right facial nerve palsy and absence of gag reflex. Cerebellar signs were negative. Motor function examinations of right upper and lower limbs showed hypertonia, reduce power of 2/5, normal reflexes and up going plantar response. Sensory functions of right upper and lower limbs were reduced. Clinically diagnosed as stroke with hypertensive emergency. CT brain showed multiple hypodensities due to recent infarct and incidental finding of an aggressive sellar mass. MRI brain showed left Middle Cerebral Artery territory infarct and an aggressive sphenoid sinus mass with suprasellar and bilateral cavernous sinus extension possibility of a macroadenoma. Serum prolactin level showed markedly hyperprolactinemia (21146 ng/ml, n 4.04 – 15.2 ng/ml) which level of 500ng/ml or greater is diagnostic of a macroprolactinoma (2). FSH level (0.929 IU/L, n 1.5-12.4 IU/L) and LH level (1.11 IU/L, n 1.7-8.6 IU/L) were low in this patient due to suppression of GnRH secretion from hypothalamus by prolactin. Testosterone level (0.15 nmol/L, n 8.64-29.0 nmol/L) was low secondary to low LH. Serum cortisol, growth hormone and TSH were normal. Platelet count and coagulation profiles were normal. The patient was treated conservatively in ward for acute ischaemic stroke and later was started on dopamine agonist cabergoline for hyperprolactinemia. Conclusion: This is a case report of acute ischaemic stroke with markedly hyperprolactinemia secondary to incidentaloma macroprolactinoma. Reference: (1)Freda PU, Beckers AM, Katznelson L, et al. Pituitary incidentaloma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2011; 96 (4): 894-904.(2)Abha Majumdar and Nisha Sharma Mangal. Hyperprolactinemia. J Hum Reprod Sci. 2013 Jul-Sep; 6(3): 168–175.(3)Sankalp Kumar Tripathi, Pallavi Kamble, M.G. Muddeshwar. Serum Prolactin Level in Patients of Ischemic stroke. International Journal of Contemporary Medical Research 2016; 3(12): 3459-3460.

Role And Significance Of Kisspeptin In The Female Reproductive System

The functioning of the female reproductive system depends on the proper development and regulation of the hypothalamic-pituitary-ovarian axis. It is based on the secretion of GnRH, which stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). There have been many studies investigating the factors that regulate the secretion of gonadotropins. In recent years, the significance and role of kisspeptin and its receptor has aroused interest. This review will provide data from foreign studies and literature on the role of kisspeptin in the regulation of the female reproductive system.

Comprehensive transcriptome analysis of hypothalamus reveals genes associated with disorders of sex development in pigs

XX sex reversal, also called XX disorders of sex development (XX-DSD), is a condition affecting the development of the gonads or genitalia, and is relatively common in pigs. However, its genetic etiology and transcriptional regulation mechanism in the hypothalamic-pituitary-gonadal axis (HPGA) remain mostly unknown. XX-DSD (SRY-negative) pigs and normal sows were selected by external genitalia observation. The hypothalamus, which is the integrated center of the HPGA was sampled for whole-transcriptome RNA-seq. The role of DEmiRNA was validated by its overexpression and knockdown in vitro. A total of 1,258 lncRNAs, 1,086 mRNAs, and 61 microRNAs differentially expressed in XX-DSD pigs compared with normal female pigs. Genes in the hormone biosynthesis and secretion pathway significantly up-regulated, and the up-regulation of GNRH1, KISS1 and AVP may associate with the abnormal secretion of GnRH. We also predicted the lncRNA-miRNA-mRNA co-expression triplets and constructed three competing endogenous RNA (ceRNA) potentially associated with XX-DSD. Functional enrichment studies suggested that TCONS_00340886, TCONS_00000204 and miR-181a related to GnRH secretion. Further, miR-181a inhibitor up-regulated GNRH1, PAK6, and CAMK4 in the GT1-7 cells. Conversely, transfection of miR-181a mimics obtained the opposite trends. The expression levels of FSHR, LHR, ESR1 and ESR2 were significantly higher in XX-DSD gondas than those in normal sows. Taken together, we proposed that the balance of endocrine had broken in XX-DSD pigs. The current study is the first to examine the transcriptomic profile in the hypothalamus of XX-DSD pigs. It provides new insight into coding and non-coding RNAs that may be associated with DSD in pigs.

The Traditional Chinese Medicine Fuyou Formula Alleviates Precocious Puberty by Inhibiting GPR54/GnRH in the Hypothalamus.

The purpose of this study was to explore the effect of the traditional Chinese medicine Fuyou formula on precocious puberty (PP). The Fy formula may exert an effect in female rats with PP and GT-7 cells through the GPR54/GnRH signaling pathway. To confirm the effect of the Fy formula on PP through the GPR54/GnRH signaling pathway, we first treated GT1-7 cells with the Fy formula and observed changes in the expression of related genes and proteins and in GnRH secretion. Then, we randomly divided young female Sprague-Dawley rats into the control group, model group, leuprorelin group and the Fy formula group. A PP model was established by injection of danazol on postnatal day 5, and the Fy formula was administered on PND15. The time of vaginal opening, the wet weights of the ovary and uterus, serum hormone levels and the expression of hypothalamic-related genes were observed. We found that the Fy formula delayed vaginal opening, decreased the wet weights and coefficients of the ovary and uterus, decreased the levels of serum hormones (E2, follicle-stimulating hormone and luteinizing hormone) and the cellular GnRH level, and downregulated the gene expression of Kiss1, GPR54 and GnRH in the hypothalamus and the gene and protein expression of GPR54 and GnRH in GT1-7 cells. In conclusion, the Fy formula may alleviate PP via the GPR54/GnRH signaling pathway.

Chapter 18 - Kisspeptin and neurokinin B expression in the human hypothalamus: Relation to reproduction and gender identity

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus are at the core of reproductive functioning. GnRH released into the median eminence regulates the secretion of the gonadotropins from the anterior pituitary, which in turn activates gametogenesis and steroid synthesis by the gonads. The GnRH system displays functional sex differences: GnRH is secreted in pulses at a constant frequency in men, whereas in women, pulse frequency varies over the menstrual cycle. In both sexes, GnRH release is regulated by sex steroid hormones, acting at the level of the hypothalamus and the anterior pituitary in a classic feedback loop. Because GnRH neurons do not express sex steroid receptors, hormone effects on GnRH release are presumed to be mediated indirectly through other steroid-sensitive neuronal systems, which then converge onto GnRH cell bodies and/or terminals. Human genetic studies demonstrated that kisspeptin (KP) as well as neurokinin B (NKB) signaling are both potent regulators of GNRH secretion. In humans, postmortem studies using immunohistochemistry have shown that women have higher KP and NKB expression in the infundibular nucleus than men. Sex differences in KP expression are present throughout life, which is from the infant/prepubertal into the elderly period, whereas sex differences in NKB expression do not emerge until adulthood. KP and NKB are often coexpressed together with dynorphin by the same population of neurons, also known as KDNy neurons in other species. Indeed, significant coexpression between KP and NKB but not with Dynorphin has been observed thereby challenging the KDNy concept in humans. Female-typical expression of both KP and NKB were observed in the infundibular nucleus of trans women (male sex assigned at birth and female gender identity). Taken together, sex differences in KP and NKB expression most likely reflect organizational actions of sex steroid hormones on the developing brain but they also remain sensitive to circulating sex steroids in adulthood. The female-dominant sex difference in infundibular KP and NKB expression suggests that this brain region is most likely involved in both the negative and positive feedback actions of estrogens on GnRH secretion. Finally, the sex-reversal observed in KP and NKB expression in trans women might reflect, at least partially, an atypical sexual differentiation of the brain.

P53 Mediates GnRH Secretion via Lin28/let-7 System in GT1-7 Cells.

Study ObjectiveThe well-known tumor suppressor transcriptional factor p53 has been proposed to be one of the central hubs of a functionally related and hierarchically arranged gene network coordinating pubertal timing. Our previous studies revealed that p53 is involved in the metabolic control of puberty. The current study aimed to investigate the underlying signaling pathway, through which p53 mediated the metabolic control of puberty.Design, Setting, Participants, Interventions, and Main Outcome MeasuresWe engineered the expression of p53 and/or Lin28a in GT1-7 cells to investigate the interaction between p53 and Lin28/let-7 system, and their impact on GnRH secretion.ResultsOverexpression of p53 stimulated, while inhibition of p53 by pifithrin-α significantly suppressed the GnRH secretion and GPR54 expression levels in response to kisspeptin stimulation in GT1-7 cells. Furthermore, overexpressed p53 suppressed Lin28a and c-Myc expression levels and increased let-7 expression levels in GT1-7 cell lines. On the other hand, inhibition of p53 by pifithrin-α upregulated Lin28a and c-Myc levels and downregulated let-7 expression levels. Moreover, Lin28a overexpression counteracted the effect of p53 overexpression in p53 and Lin28a co-overexpression cells, whose GnRH secretion and GPR54 expression levels were not different from controls. Meanwhile, Lin28a suppression counteracted the effect of pifithrin-α, and the GnRH secretion and GPR54 expression levels are not different from controls in p53 and Lin28a co-suppression cells.ConclusionThese data suggest that p53 is a central mediator of GnRH secretion in hypothalamus, and this effect is at least partly through the Lin28/let-7 pathway.

Innervation of GnRH Neuron Distal Projections and Activation by Kisspeptin in a New GnRH-Cre Rat Model.

The neural mechanisms generating pulsatile GnRH release from the median eminence (ME) remain unclear. Studies undertaken in the mouse demonstrate that GnRH neurons extend projections to the ME that have properties of both dendrites and axons, termed "dendrons," and that the kisspeptin neuron pulse generator targets these distal dendrons to drive pulsatile GnRH secretion. It presently remains unknown whether the GnRH neuron dendron exists in other species. We report here the generation of a knock-in Gnrh1-Ires-Cre rat line with near-perfect targeting of Cre recombinase to the GnRH neuronal phenotype. More than 90% of adult male and female GnRH neurons express Cre with no ectopic expression. Adeno-associated viruses were used in adult female Gnrh1-Ires-Cre rats to target mCherry or GCAMP6 to rostral preoptic area GnRH neurons. The mCherry tracer revealed the known unipolar and bipolar morphology of GnRH neurons and their principal projection pathways to the external zone of the ME. Synaptophysin-labeling of presynaptic nerve terminals revealed that GnRH neuron distal projections received numerous close appositions as they passed through the arcuate nucleus and into the median eminence. Confocal GCaMP6 imaging in acute horizontal brain slices demonstrated that GnRH neuron distal projections lateral to the median eminence were activated by kisspeptin. These studies indicate the presence of a dendron-like arrangement in the rat with GnRH neuron distal projections receiving synaptic input and responding to kisspeptin.