Hypothalamic-pituitary-gonadal Axis Research Articles

Molecular cloning and bioinformatic characterization of Gonadotropin Inhibitory Hormone (GnIH) and its receptors in the freshwater murrel, Channa punctatus (Bloch, 1793).

Gonadotropin inhibitory hormone belonging to the RFamide peptide family, a hypothalamic neuropeptide, regulates Hypothalamus-pituitary-gonadal (HPG) axis and inhibits gonadal development. GnIH polypeptide precursor has an Arg-Phe-NH2 (RFamide) motif at the C-terminal, which has LPXRF (X = Q or L) domain. The actions of GnIH are mediated through G-protein coupled receptors and upto threereceptors have been characterized in many teleosts. GnIH exerts its inhibitory effect on the HPG axis through direct interaction with GnRH and Kisspeptin neurons in the brain and acts directly on the pituitary gonadotrophs. To decipher the role of GnIH in Indian freshwater murrel, Channa punctatus, we sequenced the cDNA encoding GnIH and its two receptors. The identified GnIH mRNA encodes three RFamide peptides having -MPMRF, -MPQRF, and -LPQRFamide motifs. In silico analysis of the amino acid sequence of GnIH exhibits its molecular and functional properties and the protein-protein interaction with significant factors regulating the HPG axis. The 3-D structure of GnIH and its receptors, provides more relevant information about the active residues of these proteins which might be involved in their functioning and interaction with other proteins. Molecular dynamic simulation of GnIH protein has provided more insight into its dynamic behavior. The expression of GnIH and its receptors, shows an inverse correlation with gonadal development during the annual reproductive cycle.

Effects of copper oxide nanoparticles on reproductive system of zebrafish

Copper oxide nanoparticles (CuO NPs) were regarded as the versatile materials in daily life and the in-depth evaluation of their biological effects is of great concern. Herein the female and male zebrafishes were chosen as the model animals to analyze the reproductive toxicity caused by CuO NPs at low concentration (10, 50 and 100 μg/L) After 20-days exposure, the structure of zebrafish ovary and testis were impaired. Moreover, the contents of 17β-estradiol (E2) in both females and males were increased, while the contents of testosterone (T) were decreased, indicating the imbalanced sex hormones caused by CuO NPs. The expression of genes along the hypothalamic pituitary-gonad (HPG) axis, were examined with quantitative real-time PCR to further evaluate the toxic mechanisms. Meanwhile, the levels of erα/er2β and cyp19a in female zebrafishes and erα/er2β, lhr, hmgra/hmgrb, 3βhsd and 17βhsd in male zebrafishes were obviously up-regulated. While, the level of αr was obviously down-regulated in female and male zebrafishes. Thus, the obtained data uncovered that long-term exposure of CuO NPs with low dose could trigger the endocrine disorder, resulting in the disturbance of E2 and T level, inhibition of gonad development, and alteration of HPG axis genes. In brief, this study enriched the toxicological data of NPs on aquatic vertebrates and provided the theoretical support for assessing the environmental safety of NPs.

A Simple Mathematical Model Demonstrates the Potential for Cell-Based Hormone Therapy to Address Dysregulation of the Hypothalamus-Pituitary-Ovary Axis in Females with Loss of Ovarian Function.

Tissue-engineering and cell-based strategies provide an intriguing approach to treat complex conditions such as those of the endocrine system. We have previously developed a cell-based hormone therapy (cHT) to address hormonal insufficiency associated with the loss of ovarian function. To assess how the cHT strategy may achieve its efficacy, we developed a mathematical model to determine if known autocrine, paracrine, and endocrine effects of the native hypothalamus-pituitary-ovary (HPO) axis could explain our previously observed effects in ovariectomized rats following treatment with cHT. Our model suggests that cHT constructs participate in the complex machinery of the HPO axis. We were able to describe the in vivo behaviors of estrogen, progesterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin, and androgen with good accuracy. A sensitivity analysis indicated that some parameters impact the broader HPO system more than others, but that most changes in model parameters led to proportional changes in the system. We also conducted a predictive analysis on the effect of cHT dose on HPO axis hormones and found that, with the exception of estrogen, the other HPO hormones analyzed reach a saturation level within the physically possible number of constructs.

Current approaches in managing pregnancy in kidney transplant recipients

End-stage renal disease impacts fertility, causing functional menopause in female patients. Within 3 months of successful renal transplant, menstrual function normalizes, ovulation recommences due to improved hypothalamic–pituitary–ovarian axis function, and, thereby, fertility is restored in 80–90% of women in the childbearing age group. In such circumstances, any unplanned pregnancy poses a significant risk to the mother and the child, and the allograft. Pregnancy, in general, does not negatively impact long-term allograft function or survival if the baseline function of the allograft is excellent. Risk predictors of clinical adverse outcomes and graft loss during pregnancy include short transplant–pregnancy interval, preconception graft function, hypertension, preconception proteinuria, and preeclampsia. The recommended and safer maintenance immunosuppressive regimen during pregnancy is calcineurin inhibitors (CNI) (tacrolimus/cyclosporine), azathioprine, and steroids. Sirolimus/everolimus and mycophenolate mofetil should be withdrawn 6 weeks before planned conception. To avoid acute rejections, drug levels should be monitored closely, and the dosage should be modified to reach the recommended target level. Addressing contraception must be a crucial component of the pretransplant counseling process to prevent premature unplanned pregnancies. Mechanical contraceptives are safe for transplant recipients, convenient, and easy to use, with no concerns regarding interaction with immune suppressants; nevertheless, their efficacy depends on user compliance which is difficult to achieve in most cases. However, combined oral contraceptives and progestin-only contraceptives have an inhibitory effect on P 450 3A4, thus increasing the concentration of CNIs particularly cyclosporine. Furthermore, CNIs, in particular, tacrolimus, have an inductive effect on P 450 3A4, potentially reducing the contraceptive efficacy. Therefore, successful pregnancy depends on thorough prepregnancy counseling, careful family planning, and multidisciplinary teamwork. Breastfeeding is not contraindicated and should not be discouraged.

New perspectives in functional hypogonadotropic hypogonadism: beyond late onset hypogonadism.

Functional hypogonadotropic hypogonadism (FHH) is an increasingly frequent condition, whose pathological mechanisms are not yet fully clarified. The concept of FHH has now completely replaced that of late onset hypogonadism, that only concerned the ageing man. FHH is the result of an impairment of the hypothalamic-pituitary gonadal axis (HPG-A) function, resulting in decreased testosterone concentrations associated with low or inappropriately normal gonadotropin levels and infertility; it can be diagnosed once organic causes of hypogonadism are excluded. The growing occurrence of FHH derives from its association with widespread conditions, such as obesity and diabetes mellitus, but also to the increasing ease and frequency of use of several drugs, such as opioids, glucocorticoids, and sex steroids. Moreover, given the tendency of many subjects to excessive physical activity and drastic reduction in caloric intake, FHH may also be secondary to low energy availability. Finally, the association with HIV infection should not be overlooked. Therefore, there is an important variability in the diseases that can lead to FHH. Despite the heterogeneity of the underlying pathologies, the mechanisms leading to FHH would seem quite similar, with the initial event represented by the impairment at the HPG-A level. Nevertheless, many different biological pathways are involved in the pathogenesis of FHH, therefore the aim of the current paper is to provide an overview of the main relevant mechanisms, through a detailed analysis of the literature, focusing specifically on pathogenesis and clinical, diagnostic and therapeutic aspects.

P-429 Transcriptomic heterogeneity within cortical ovarian follicle pool in child and adult

Abstract Study question What are the transcriptomic differences between child and adult ovarian follicles in the human cortex? Summary answer Our results suggest that human ovarian cortical follicles show marked differences before and after puberty, with the greatest differences being present in the secondary stage. What is known already The ovaries and their main functional structures, follicles, are important for determining the reproductive health. Follicle growth and maturation are controlled by multiple mechanisms, including the hypothalamus pituitary ovarian axis and local paracrine signalling. Although dormant primordial follicles are activated to grow even in infancy, all growing follicles before the first menarche are bound to undergo atresia. Follicle growth activation in infancy and adulthood remains poorly understood. Additionally, child and adult follicles behave differently during in vitro culture suggesting differing biology, raising many questions e.g., about their suitability for fertility preservation. Study design, size, duration Ovarian cortical tissues from children were collected through fertility preservation program at the Helsinki Children’s hospital (Finland). Families of underage patients signed an informed written consent. Adult ovarian tissue was collected from gender reassignment patients at the Helsinki University hospital and Karolinska university hospital after informed written consent. Follicles at different maturation stages were isolated from ovarian cortex for single follicle transcriptomic analysis. Freshly-fixed cortical biopsies (3x3x1 mm3) were used for validation of transcriptomic analysis. Participants/materials, setting, methods Single viable follicles (n = 120, 60/age group) were mechanically and enzymatically isolated from frozen-thawed ovarian tissue from four adults (age >18 yrs) and five children (age 1-11 yrs) and processed for RNA-sequencing. Individual follicles were lysed, prepared for RNA libraries following Smart-Seq2 protocol and sequenced using NextSeq 500 platform. After quality assessment, 109 follicles were analysed (Adult n = 54, Child n = 55). DDX4, AMH, FOXL2 and DAZL transcripts and proteins localization were evaluated via immunodetection and RNA-FISH assay. Main results and the role of chance Principal component analysis (PCA) of all sequenced follicles demonstrated an expected timely progression of their stages based on maturation, and an overall similarity between child and adult. Interestingly, PCA displayed two distinct clusters of follicles in both adult and child samples. The first group contained only growing follicles and, after differentially expressed gene (DEG) analysis, demonstrated lower expression of traditional oocyte markers (group 1). The second group consisted of primordial and growing follicles and had lower expression of traditional granulosa marker (group 2). Group-related differential expression of main oocyte (DDX4, DAZL) and granulosa (FOXL2, AMH) marker genes were validated in tissue sections with RNA-FISH. Downstream analyses focused on group 2 follicles. When child and adult follicles were compared to each other, the highest number of DEGs were found in the secondary stage (primordial n = 32, primary n = 14, secondary n = 387). Pathway analysis of DEG displays enrichment of oestrogen-related related pathways in adult secondary follicles. Furthermore, we analysed DEGs between the follicular stages per age group, finding high number of DEGs between primordial and primary follicles (Adult n = 1663, Child n = 1914). The majority of those genes are unique to the age group (common=512). Limitations, reasons for caution Adult ovarian tissue is obtained from gender reassignment patients, who receive androgen treatments prior to surgery. Child ovarian tissue was collected from fertility preservation programme where some patients were exposed to chemotherapy prior to tissue collection. Wider implications of the findings The differences between child and adult follicles encourage further investigation of the suitability of child ovarian tissue in current fertility preservation protocols. Understanding transcriptomic changes during early folliculogenesis and reasons for follicle degeneration can help tailor protocols for in vitro growth of follicles. Trial registration number not applicable

P-684 Subclinical hypothyroidism and ovarian reserve indices in women with normogonadotropic anovulation

Abstract Study question Does subclinical hypothyroidism (SCH) affect ovarian reserve indices in women with Polycystic Ovary syndrome (PCOS) or other forms of Hypothalamic-Pituitary-Ovarian Axis Dysfunction (HPOD)? Summary answer SCH increases Anti-Müllerian hormone (AMH) concentration, but has no influence on Follicle-stimulating hormone (FSH). What is known already In PCOS, anovulation is caused by the arrest of growing follicles due to increased frequency and amplitude of luteinizing hormone (LH) pulses, resulting in increased number of pre-antral and antral follicles, and concentration of AMH. In other cases of HPOD, the causes of anovulation are more diverse and have not yet been clearly identified. Thyroid hormones can modify the function of the HPO axis, affecting the maturation of follicles. While overt hypothyroidism is treated to improve ovulation and fertility, the effect of subclinical hypothyroidism (SCH) and the presence of circulating antithyroid antibodies (ATA) on ovarian function is uncertain. Study design, size, duration A prospective cohort tertiary single-center study (consent no. 1072.6120.172.2022) included women aged 18-45, examined due to menstrual disorders and/or infertility, from July to August 2022. Only women without previously diagnosed thyroid dysfunction were included. All enrolled women gave informed written consent to participate in the study. Participants/materials, setting, methods Women underwent routine gynecological examination and pelvic ultrasound. Blood sample was tested for AMH, FSH, Luteinizing hormone (LH), Thyroid-stimulating hormone (TSH), anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-TG) antibodies concentrations. The Rotterdam-ESHRE-ASRM criteria were used to diagnose PCOS and its phenotypes. HPOD was diagnosed according to WHO classification. SCH was defined as TSH > 2.5 uIU/ml with normal thyroid function. The above parameters within subpopulations were calculated using regression analysis, Kruskal-Wallis and post-hoc tests. Main results and the role of chance The study included 51 euthyroid women aged 18-40. PCOS was diagnosed in 42/51 (82.35%) women, including PCOS-A phenotype in 31/51 (60.78%), PCOS-B in 5/51 (9.8%), PCOS-D in 6/51 (11.76%) women, and HPOD in 9/51 (17.65%) women. There was a significant positive correlation between the concentration of TSH and AMH in the studied population - with the increase of TSH value, the concentration of AMH increased (r = 0.4, p = 0.0035). The mean AMH concentration significantly differed between the groups and equaled to 55.4, 39.4, 31.1, 24.0 pmol/l in PCOS-A, PCOS-B, PCOS-D and HPOD (p = 0.05), respectively. The mean anti-TPO concentration was significantly different between the groups and was equal to 18.2, 19.0, 14.6, 10.9 U/ml in PCOS-A, PCOS-B, PCOS-D and HPOD (p = 0.05), respectively. There was no significant difference in FSH, LH, TSH, anti-TG concentrations between the groups. Limitations, reasons for caution The limitations of the study are small study group and single-center nature. Wider implications of the findings SCH increases the concentration of AMH, which in PCOS may exacerbate the symptoms of anovulation. Whether treatment of subclinical hypothyroidism affects ovarian reserve indices and improves ovarian function remain a subject of further research. Trial registration number 1072.6120.172.2022

Global Cerebral Ischemia Induces Short-Term Changes to Estrous Cycle Regularity and Alters Hypothalamic-Pituitary-Gonadal Axis Function

Global cerebral ischemia (GCI), which mimics cardiac arrest in humans, induces numerous functional alterations throughout the brain, but its impact on the hypothalamic-pituitary-gonadal (HPG) axis remains largely unknown. Importantly, the HPG axis modulates sex hormones and neuropeptides, such as estrogen and kisspeptin, as well as estrous cycle regularity. The goals of this study were to determine if estrous cycle regularity is affected by GCI and to characterize HPG axis functionality post-ischemia. Adult female Wistar rats underwent vaginal lavage for 2 weeks to confirm estrous cycle regularity before being subjected to GCI for 10 minutes or sham surgery. Estrous cycle regularity was monitored for 4 weeks following surgery, after which rats were euthanized. Immunofluorescence was used to detect kisspeptin receptors (Kiss1) and estrogen receptors alpha (ERα) and beta (ERβ) expression in the arcuate nucleus (ARC) of the hypothalamus, as well as the CA1 and dentate gyrus (DG) of the hippocampus. Following surgery, all sham subjects maintained a regular estrous cycle, whereas two thirds of ischemic rats presented a disrupted cycle following GCI (persistent diestrus, average length 13 days), before resuming regular cycling. T-test revealed a significant increase in Kiss1 receptors in the ARC of GCI rodents, as well as a trend for higher ERα expression in the CA1 of ischemic rats. Overall, our results highlight that while estrous cycle disruptions resolve within 2 weeks after the ischemic event, HPG axis dysregulation persists up to one month after GCI, hinting at possible long-term impacts on the reproductive axis.

Putative adverse outcome pathway development based on physiological responses of female fathead minnows to model estrogen versus androgen receptor agonists

Several adverse outcome pathways (AOPs) have linked molecular initiating events like aromatase inhibition, androgen receptor (AR) agonism, and estrogen receptor (ER) antagonism to reproductive impairment in adult fish. Estrogen receptor agonists can also cause adverse reproductive effects, however, the early key events (KEs) in an AOP leading to this are mostly unknown. The primary aim of this study was to develop hypotheses regarding the potential mechanisms through which exposure to ER agonists might lead to reproductive impairment in female fish. Mature fathead minnows were exposed to 1 or 10 ng 17α-ethynylestradiol (EE2)/L or 10 or 100 µg bisphenol A (BPA)/L for 14 d. The response to EE2 and BPA was contrasted with the effects of 500 ng/L of 17β-trenbolone (TRB), an AR agonist, as well as TRB combined with the low and high concentrations of EE2 or BPA tested individually. Exposure to 10 ng EE2/L, 100 µg BPA/L, TRB, or the various mixtures with TRB caused significant decreases in plasma concentrations of 17β-estradiol. Exposure to TRB alone caused a significant reduction in plasma vitellogenin (VTG), but VTG was unaffected or even increased in females exposed to EE2 or BPA alone or, in most cases, in mixtures with TRB. Over the course of the 14-d exposure, the only treatments that clearly did not affect egg production were 1 ng EE2/L and 10 µg BPA/L. Based on these results and knowledge of hypothalamic–pituitary–gonadal axis function, we hypothesize an AOP whereby decreased production of maturation-inducing steroid leading to impaired oocyte maturation and ovulation, possibly due to negative feedback or direct inhibitory effects of membrane ER activation, could be responsible for causing adverse reproductive impacts in female fish exposed to ER agonists.

The Thyroid Hormone Axis and Female Reproduction.

Thyroid function affects multiple sites of the female hypothalamic-pituitary gonadal (HPG) axis. Disruption of thyroid function has been linked to reproductive dysfunction in women and is associated with menstrual irregularity, infertility, poor pregnancy outcomes, and gynecological conditions such as premature ovarian insufficiency and polycystic ovarian syndrome. Thus, the complex molecular interplay between hormones involved in thyroid and reproductive functions is further compounded by the association of certain common autoimmune states with disorders of the thyroid and the HPG axes. Furthermore, in prepartum and intrapartum states, even relatively minor disruptions have been shown to adversely impact maternal and fetal outcomes, with some differences of opinion in the management of these conditions. In this review, we provide readers with a foundational understanding of the physiology and pathophysiology of thyroid hormone interactions with the female HPG axis. We also share clinical insights into the management of thyroid dysfunction in reproductive-aged women.

Assessing hypothalamic pituitary gonadal function in reproductive disorders.

Reproductive conditions secondary to disorders of the hypothalamic-pituitary-gonadal (HPG) axis are common and are associated with important health implications and considerable psychosocial impact. Basal and dynamic tests enable interrogation of individual components of the HPG axis, facilitating diagnosis and understanding of the pathophysiology of reproductive disorders. Onset of puberty is controlled by hypothalamic gonadotrophin-releasing hormone (GnRH) neuronal function. To date, a dynamic test of hypothalamic function is not yet available. Therefore, accurate differentiation of pubertal disorders such as constitutional delay of growth and puberty (CDGP) and congenital hypogonadotrophic hypogonadism (CHH) as causes of delayed puberty is challenging due to similar clinical presentations and hormonal profiles. Likewise, although the two commonest reproductive disorders in women, polycystic ovary syndrome (PCOS) and functional hypothalamic amenorrhoea (FHA) have disparate hypothalamic function, oligo/amenorrhoea frequently poses a diagnostic conundrum owing to the overlap in the criteria used to define both conditions. This review aims to describe pubertal and reproductive disorders secondary to pathologies affecting the HPG axis. Challenges encountered in clinical practice in differentiating pubertal and reproductive conditions are reviewed in conjunction with the utility of baseline and dynamic endocrine tests to interrogate specific components of the HPG axis. We also highlight putative hypothalamic, pituitary, and gonadal markers in development that could improve the diagnosis of patients presenting with disorders of puberty or reproduction.

Analysis of the incidence of central precocious puberty treated with gonadotropin-releasing hormone analogs. Impact of the COVID-19 pandemic

Introduction. Since the onset of the coronavirus disease 2019 (COVID-19) pandemic, consultations of girls with idiopathic central precocious puberty (ICPP) increased in several countries, but there were no data from Argentina. This increase may be related to changes in lifestyle and stress levels resulting from the lockdown, which particularly affected the child population. Objectives. 1) To describe the progression of the incidence of ICPP requiring inhibition of the hypothalamic-pituitary-gonadal (HPG) axis in girls between 2010 and 2021 in a cohort from the Metropolitan Area of Buenos Aires. 2) To compare the characteristics of girls diagnosed with ICPP during the pandemic with those of a control group. Methods. Interrupted time-series and case-control study. Results. The annual incidence remained stable between 2010 and 2017. Since 2017, it increased to an average of 59.9% (95% CI: 18.6-115.5) and appears to have accelerated during the pandemic. We found an association between ICPP and requiring inhibitory treatment between June 1st, 2020 and May 31st, 2021 and 2 variables: maternal age at menarche (OR: 0.46, 95% CI: 0.28-0.77) and family history of ICPP (OR: 4.42, 95% CI: 1.16-16.86). Conclusion. We evidenced a significant increase in the incidence of ICPP with requirement of HPG axis inhibition since 2017. Increased exposure to various environmental triggers during the COVID-19 pandemic may have had a greater influence in girls with some genetic predisposition.

Enduring sex-dependent effects of lipopolysaccharide treatment on the hypothalamic–pituitary–gonadal axis in mice

Pubertal stress causes enduring sexual behavior dysfunction in males and females, but the underlying mechanism remains unknown. These changes may arise from pubertal programming of the hypothalamic–pituitary–gonadal axis. Previous findings show that stress exposure downregulates the hypothalamic–pituitary–gonadal axis, particularly through the reduction of the neuropeptide kisspeptin (Kiss1) and its receptor (Kiss1R). Although acute changes in kiss1 and Kiss1r genes have been observed following pubertal immune stress, it is unclear whether immune stress-induced downregulation of kiss1 and kiss1r persists beyond puberty. The current study investigated the enduring sex-specific consequences of lipopolysaccharide on the expression of Kiss1 and Kiss1r in 160 pubertal or adult mice at multiple time points. Six-week and 10-week-old male and female mice were treated with either saline or with lipopolysaccharide. Mice were euthanized either 8 h or 4 weeks following treatment. Although we did not identify any sex differences, our results revealed that lipopolysaccharide treatment decreases hypothalamic Kiss1 and Kiss1r in both pubertal and adult mice within 8 h of treatment. The decreased hypothalamic Kiss1 expression persists 4 weeks later only in mice treated with lipopolysaccharide during puberty. Our findings highlight the age-dependent vulnerability of the hypothalamic–pituitary–gonadal axis to immune stress, providing a better understanding of the mechanisms implicated in allostatic shift during immune stress. Finally, our findings also show the effects of immune stress on various components of the hypothalamic–pituitary–gonadal axis, which could have implications for sexual and fertility-related dysfunctions.

Exposure to copper nanoparticles or copper sulfate dysregulated the hypothalamic-pituitary-gonadal axis, gonadal histology, and metabolites in Pelteobagrus fulvidraco

This study evaluated the effects of chronic exposure to copper nanoparticles (Cu-NPs) and waterborne copper (CuSO4) on the reproductive system of yellow catfish (Pelteobagrus fulvidraco). Juvenile yellow catfish were exposed to 100 and 200 μg Cu/L Cu-NPs and 100 μg Cu/L CuSO4 for 42 days. The results showed clear reproductive defects in both female and male yellow catfish in the 200 μg Cu/L Cu-NPs and 100 μg Cu/L CuSO4 groups. Exposure to Cu-NPs or CuSO4 inhibited folliculogenesis and vitellogenesis in the ovaries, and spermatogenesis in the testes, accompanied by elevation of the apoptotic signal. Ultrastructural observations also revealed damaged organelles of gonadal cells in both testes and ovaries. Most of the hypothalamic-pituitary-gonadal (HPG) axis genes examined and serum sex steroid hormones tended to be downregulated after Cu exposure. Metabolomic analysis suggested that gonadal estradiol level is sensitive to Cu-NPs or CuSO4. The heat map of gonadal metabolomics suggested a similar effect of 200 μg Cu/L Cu-NPs and 100 μg Cu/L CuSO4 in both the ovaries and testes. Additionally, metabolomics data showed that the reproductive toxicity due to Cu-NPs and CuSO4 may occur via different metabolic pathways. Cu-NPs tend to dysregulate the metabolic pathways of sphingolipid and linoleic acid metabolism in the ovary and the biosynthesis of amino acids and pantothenate and CoA in the testis. Overall, these findings revealed the toxicological effects of Cu-NPs and CuSO4 on the HPG axis and gonadal metabolism in yellow catfish.

Interactive metabolic signatures of testicular testosterone with bilateral adrenalectomy in mice

The hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) axes have reciprocal relationships with steroidogenesis regulation. However, the relationship between testicular steroids and defective glucocorticoid production under chronic stress remains unclear. Metabolic changes of testicular steroids in bilateral adrenalectomized (bADX) 8-week-old C57BL/6 male mice were measured using gas chromatography-mass spectrometry. Twelve weeks after surgery, testis samples were obtained from the model mice, which were divided into tap-water (n = 12) and 1 % saline (n = 24) supplementation groups, and their testicular steroid levels were compared with those of sham controls (n = 11). An increased survival rate with lower testicular levels of tetrahydro-11-deoxycorticosterone was observed in the 1 % saline group compared to both the tap-water (p = 0.029) and sham (p = 0.062) groups. Testicular corticosterone levels were significantly decreased in both tap-water (4.22 ± 2.73 ng/g, p = 0.015) and 1 % saline (3.70 ± 1.69, p = 0.002) groups compared to those in sham controls (7.41 ± 7.39). Testicular testosterone levels tended to increase in both bADX groups compared to those in the sham controls. In addition, increased metabolic ratios of testosterone to androstenedione in tap-water (2.24 ± 0.44, p < 0.05) and 1 % saline (2.18 ± 0.60, p < 0.05) mice compared to sham controls (1.87 ± 0.55) suggested increased production of testicular testosterone. No significant differences in serum steroid levels were observed. Defective adrenal corticosterone secretion and increased testicular production in bADX models revealed an interactive mechanism underlying chronic stress. The present experimental evidence suggests the crosstalk between the HPA and HPG axes in homeostatic steroidogenesis.

(083) EMT Induced Transcription Factors Direct Leptin-LEPR-DHH Signaling to Modulate Leydig Stem Cells Differentiation in Testis

Abstract Introduction The primary source of testosterone (T) in men are Leydig cells (LCs) and their dysfunction can lead to T deficiency. Testicular microenvironment (TME) which is comparised of cells such as Sertoli and Peritubular myoid cells releases the paracrine factors to influence the growth and differentiation of LCs. Leptin is a 16-kDa protein produced primarily by adipose tissue and exerts a significant influence on hormonal regulation. In our recent study, we demonstrated that Leptin, a paracrine factor secreted by TME, is critical for Leydig stem cell (LSCs) differentiation and subsequent T production under the regulation of the desert hedgehog signalling pathway. We also showed that this regulation is inverse concentration dependent such as Leptin increases LSC differentiation at low doses and decrease it at higher doses. However, an in-depth understanding of mechanisms that derive the Leptin secretion and decide the fate of LCs are still unclear and is the focus of the present study. Objective To study the mechanisms that derive the Leptin secretion and decide the fate of LCs. Methods A total of 18 men with testicular failure underwent testis biopsies. The testicular cells were characterized using Flow cytometry, Immunostaining and RNA sequencing then the cells were also treated with Leptin in a dose dependent (0, 1, 10 ng/mL respectively) and time dependent (24, 48, 72, and 96 hours) manner. For the functional studies, UCSC genome browser was used to study the transcription factor genes (TFs) binding the leptin receptor (LEPR) promoter. The list of TFs was subjected to enrichment analysis. To evaluate the effects of shortlisted TFs on leptin-LEPR-DHH to induce EMT in LSCs, qPCR and western blotting were performed. For statistical analysis GraphPad Prism was used. For statistical analysis GraphPad Prism was used. All data were presented as the means ± SEM. All data were presented as the means ± SEM Results Over 60 TF’s were identified by UCSC Genome browser, which were subjected to enrichment analysis using ENRICHR and Ingenuity pathway analysis. Output revealed TFs; SOX2, SOX8 and SOX9 that were enriched for mechanisms influencing hormonal regulation via hypothalamic pituitary gonadal axis. The role of these TFs has been well established in epithelial to mesenchymal transition in different cancer types, however their impact on LSC differentiation remained underexplored. RNA sequencing data from cells exposed to different concentrations of Leptin showed an inducing effect of Leptin on SOX2, SOX8 Sox9 respectively. This was confirmed at RNA as well as at protein levels. Consequently, upon inhibition of LEPR, the expression of SOX2, SOX8 and SOX9 was reduced as well. Furthermore we used SOX8, SOX9 and SOX2 antagonists in the presence or absence of Leptin to validate the effects on EMT of LCs. Conclusions Our results demonstrate that Leptin-LEPR-DHH induced Leydig stem cells differentiation in testis is modulated by EMT inducing transcription factors which binds to LEPR. Further studies are ongoing to explore the potential therapeutic effects of leptin treatment in improving fertility. Disclosure No

Hair Cortisol: A Biomarker of Chronic Stress in Animals and its Association with Reproduction

Hair cortisol concentration (HCC) is used to assess the long-term retrospective measurement of the hypothalamic-pituitary-adrenal axis (HPA axis). HCC reflects HPA axis activity over a period of months and years depending on hair growth rate and length of hair measured. HCC is being used as a biomarker of chronic stress in order to check the well-being of domestic animals (cattle, horses, dogs, pigs), wild animals, captive animals, human trauma victims, and post-traumatic stress disorders (PTSD) as it provides an innovative approach to measure chronic HPA activity retrospectively over months without being affected by short term acute stressors and diurnal patterns. Stress plays a significant role in affecting the fertility of animals. Activation of the HPA axis further activates the hypothalamic–pituitary–gonadal axis (HPG) i.e., glucocorticoids are produced causing apoptosis of Leydig cells and decreased testosterone levels in males. In females, increased cortisol levels can suppress the tonic secretion of Leutinizing hormone (LH) which might hamper ovulation. Thus, hair cortisol analysis for assessment of the HPA-HPG axis could be a very important tool for animal welfare.

The Role of G3BP1 Gene Mediates P38 MAPK/JNK Pathway in Testicular Spermatogenic Dysfunction Caused by Cyfluthrin.

In recent years, male infertility has received global attention and seriously affected the quality of human fertility, and pyrethroids (type II pyrethroids), as recognized environmental endocrine disruptors, may threaten male reproductive health. Therefore, in this study, we established an in vivo model for the development of testicular and germ cell toxicity induced by cyfluthrin and explored the role and mechanism of the G3BP1 gene-mediated P38 MAPK/JNK pathway in testicular and germ cell damage caused by cyfluthrin to find early and sensitive indicators and new therapeutic targets for the development of testicular damage. Firstly, 40 male Wistar rats (about 260 g) were divided into a control group (corn oil), low dose group (6.25 mg/kg), middle dose group (12.5 mg/kg) and high dose group (25 mg/kg). The rats were anesthetized and executed after 28 days of poisoning on alternate days. Then, HE staining, transmission electron microscopy, ELISA, q-PCR, Western blot, immunohistochemistry, double-immunofluorescence and TUNEL were used to observe the pathology, androgen levels, oxidative damage and altered expression of the key factors of the G3BP1 and MAPK pathways in rat testes. The results showed that, compared with the control group, the testicular tissue and spermatocytes were superficially damaged with an increasing dose of cyfluthrin; furthermore, it could interfere with the normal secretion of the hypothalamic-pituitary-gonadal axis (serum GnRH, FSH, T and LH levels) and cause hypergonadal dysfunction. A dose-dependent increase in MDA and a dose-dependent decrease in T-AOC indicated that the oxidative-antioxidative homeostatic balance was disrupted. The Western blot and qPCR analysis revealed that G3BP1, p-JNK1/2/3, P38 MAPK, p-ERK, COX1 and COX4 proteins and mRNA expression were decreased, and p-JNK1/2/3, p-P38MAPK, caspase 3/8/9 proteins and mRNA expression were significantly increased. The double-immunofluorescence and immunohistochemistry results showed that the protein expression of G3BP1 decreased with an increasing dose of staining, while the expression of JNK1/2/3 and P38 MAPK were increased significantly. The positive expressions of G3BP1 were mainly located in the testicular germinal epithelium and germ cell layer, and the positive expressions of JNK1/2/3 were mainly located in the testicular germinal epithelium and sperm cells, while the positive expressions of P38 MAPK were located in all levels of the germ cells and spermatozoa. Our results demonstrated that exposure to cyfluthrin caused testicular and spermatocyte damage in rats, which could cause pathomorphology, altered androgen levels and a decreased antioxidant capacity. When the intracellular antioxidant capacity was impaired, G3BP1 expression and activity were inhibited, causing activation of the P38 MAPK/JNK pathway and activation of the intracellular apoptotic pathway, which, in turn, led to germ cell apoptosis.

Effects of different photoperiods on growth and ovarian development and maturation of silver pomfret Pampus argenteus.

Photoperiod has a great influence on the growth and ovarian development and maturation of fishes. To analyse the effects of photoperiod on growth and ovarian development of an important marine economic fish, silver pomfret Pampus argenteus, short photoperiod group (L:D = 8:16), control group (L:D = 12:12) and long photoperiod group (L:D = 18:6) were set up for 60 days. The growth performance, ovarian development, changes in hormones and key enzyme activities in the hypothalamic-pituitary-gonadal (HPG) axis and expressions of key regulatory genes in the HPG axis were studied under different photoperiod conditions. The results showed that the final weight gain, body weight index, specific growth rate for weight, specific growth rate for length and average daily growth were the highest in the long photoperiod group, and the feed conversion rate was the lowest. Under long photoperiod condition, gonado-somatic index and hepato-somatic index were higher, ovarian maturity was better and expressions of HPG axis-related regulatory genes foxl2a, foxl2b, cyp19a1a, cyp19a1b, kiss, gpr54-2, gnrh2, fsh and lh were higher. When compared with the other two groups, in the long photoperiod group, the change trend of estradiol (E2) was consistent with those of luteinizing hormone, melatonin (MT) and kisspeptin, and the levels were higher on the 20th and 50th days. These results indicate that prolongation of the photoperiod can improve the growth performance of P. argenteus and promote ovary development and maturation. The authors speculate that photoperiod may regulate the ovarian activity of P. argenteus through MT and kisspeptin/gpr54 signalling pathways. The results show that photoperiod can regulate the ovarian development of P. argenteus, which would help in breaking the seasonal restrictions of animals and regulating an animal's reproductive cycle.

Effect made by the growth of multiple primary malignant tumors on the hypothalamicpituitary-gonadal axis in BALB/c Nude mice of both sexes

The aim was to study changes in the functioning of the hypothalamic-pituitary-gonadal (HPG) axis in BALB/c Nude mice of both sexes with multiple primary malignant tumors (MPMTs). The BALB/c Nude mice (n=84) of both sexes were divided into groups as follows: intact males and females (n=14), a reference group of mice of both sexes with standard subcutaneous inoculation of B16/F10 melanoma (n=14); the main group of mice with the reproduction of the MPMT model (n=14); B16/ F10 melanoma and Lewis carcinoma were inoculated into mice sequentially subcutaneously on two sides: one on the left side and the other on the right side. After 15 days of the growth of the malignant tumors, GnRH, LH and FSH contents were determined by ELISA using standard kits in 1% of the homogenates of the hypothalamic and pituitary tissues, and estradiol content (E2) was determined in 10% homogenates of ovaries and testes, as well concentrations of testosterone (T) and progesterone (P4) were identified in blood serum with the standard RIA kits (Immunotech, Czech Republic). Results. In the hypothalamus in females of the reference and main groups, the level of GnRH increased by 2.5-4 times, and in males it decreased by an average of 3.1 times; in response to those changes in animals of both sexes in the reference group and in the main group only in females in the pituitary gland the level of LH increased by 1.3-1.6 times, however a decrease in FSH in all groups was more pronounced in females: by 7.8-13.7 times and less pronounced in males: by 1.4-1.7 times (р˂0, 05). In the gonads of females, the level of E2 increased, but the levels of T and P4 decreased, while P4 increased in males. Blood parameters did not reflect hormonal changes in the organs. Conclusion. Abnormalities in the HHG axis performance, along with primary immunodeficiency, play a decisive role in overcoming antitumor protection for uncharacteristic malignant tumors.