Mechanisms of Male Reproductive Sterility Triggered by Dysbiosis of Intestinal Microorganisms.

The intestinal microbiota, comprised of bacteria, archaea, and phages, inhabits the gastrointestinal tract of the organism. Male reproductive sterility is currently a prominent topic in medical research. Increasing research suggests that gut microbiota dysbiosis can result in various reproductive health problems. This article specifically investigates the impact of gut microbiota dysbiosis on male reproductive infertility development. Gut microbiota imbalances can disrupt the immune system and immune cell metabolism, affecting testicular growth and sperm production. This dysfunction can compromise the levels of hormones produced and secreted by the endocrine glands, affecting male reproductive health. Furthermore, imbalance of the gut microbiota can disrupt the gut-brain-reproductive axis, resulting in male reproductive infertility. This article explores how the imbalance of the gut microbiota impacts male reproductive infertility through immune regulation, endocrine regulation, and interactions of the gut-brain-reproductive axis, concluding with recommendations for prevention and treatment.

Signaling Ligand Heterogeneities in the Peduncle Complex of the Cephalopod Mollusc Octopus bimaculoides.

INTRODUCTION: The octopus peduncle complex is an agglomeration of neural structures with remarkably diverse functional roles. The complex rests on the optic tract, between the optic lobe and the central brain, and comprises the peduncle lobe proper, the olfactory lobe, and the optic gland. The peduncle lobe regulates visuomotor behaviors, the optic glands control sexual maturation and maternal death, and the olfactory lobe is thought to receive input from the olfactory organ. Recent transcriptomic and metabolomic studies have identified candidate peptide and steroid ligands in the Octopus bimaculoides optic gland. METHODS: With gene expression for these ligands and their biosynthetic enzymes, we show that optic gland neurochemistry extends beyond the traditional optic gland secretory tissue and into lobular territories. RESULTS: A key finding is that the classically defined olfactory lobe is itself a heterogeneous territory and includes steroidogenic territories that overlap with cells expressing molluscan neuropeptides and the synthetic enzyme dopamine beta-hydroxylase. CONCLUSION: Our study reveals the neurochemical landscape of the octopus peduncle complex, highlighting the unexpected overlap between traditionally defined regions.

Reproductive axis ageing and fertility in men.

Compared to women, increasing male age is not accompanied by such marked changes in reproductive function but changes certainly do happen. These include alterations to the hypothalamo-pituitary-testicular axis, with resultant implications for testosterone production and bioavailability as well as spermatogenesis. There is a decline in sexual function as men age, with a dramatic increase in the prevalence of erectile dysfunction after the age of 40, which is a marker for both clinically evident as well as covert coronary artery disease. Despite a quantitative decline in spermatogenesis and reduced fecundability, the male potential for fertility persists throughout adult life, however there are also increasingly recognised alterations in sperm quality and function with significant implications for offspring health. These changes are relevant to both natural and medically assisted conception.

Utilizing the Gastrointestinal Microbiota to Modulate Cattle Health through the Microbiome-Gut-Organ Axes.

The microorganisms inhabiting the gastrointestinal tract (GIT) of ruminants have a mutualistic relationship with the host that influences the efficiency and health of the ruminants. The GIT microbiota interacts with the host immune system to influence not only the GIT, but other organs in the body as well. The objective of this review is to highlight the importance of the role the gastrointestinal microbiota plays in modulating the health of a host through communication with different organs in the body through the microbiome-gut-organ axes. Among other things, the GIT microbiota produces metabolites for the host and prevents the colonization of pathogens. In order to prevent dysbiosis of the GIT microbiota, gut microbial therapies can be utilized to re-introduce beneficial bacteria and regain homeostasis within the rumen environment and promote gastrointestinal health. Additionally, controlling GIT dysbiosis can aid the immune system in preventing disfunction in other organ systems in the body through the microbiome-gut-brain axis, the microbiome-gut-lung axis, the microbiome-gut-mammary axis, and the microbiome-gut-reproductive axis.

The expression of IGFBP-5 in the reproductive axis and effect on the onset of puberty in female rats.

Insulin-like growth factor-binding protein-5 (IGFBP-5) has recently been shown to alter the reproductive capacity by regulating insulin-like growth factor (IGF) bioavailability or IGF-independent effects. The present study aimed to investigate the effect and mechanism of IGFBP-5 on the onset of puberty in female rats. Immunofluorescence and real-time quantitative PCR were used to determine the expression and location of IGFBP-5 mRNA and protein distribution in the infant's hypothalamus-pituitary-ovary (HPO) axis prepuberty, peripuberty, puberty and adult female rats. Prepubertal rats with IGFBP-5 intracerebroventricular (ICV) were injected to determine the puberty-related genes expression and the concentrations of reproductive hormones. Primary hypothalamic cells were treated with IGFBP-5 to determine the expression of puberty-related genes and the Akt and mTOR proteins. Results showed that Igfbp-5 mRNA and protein were present on the HPO axis. The addition of IGFBP-5 to primary hypothalamic cells inhibited the expression of Gnrh and Igf-1 mRNAs (P < 0.05) and increased the expression of AKT and mTOR protein (P < 0.01). IGFBP-5 ICV-injection delayed the onset of puberty, reduced Gnrh, Igf-1, and Fshß mRNAs, and decreased the concentrations of E2, P4, FSH,serum LH levels and the ovaries weight (P < 0.05). More corpus luteum and fewer primary follicles were found after IGFBP-5 injection (P < 0.05).

Ground squirrels initiate sexual maturation during hibernation.

Adequate nutrition is essential for normal reproductive function, which is vital for species to survive. In humans and other mammals, starvation and undernutrition deplete fat reserves and cause weight loss, attenuating the function of the reproductive axis and causing hypogonadism.1-4 Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) spend 7 months of every year in hibernation without food and water. Hibernating squirrels alternate between periods of torpor and interbout arousal (IBA), when animals temporarily return to an active-like state.5 The physiological significance of IBA is unclear, but it is thought to be essential for hibernation in animals that drop their body temperature to 2°C-4°C during torpor. Here, we report that juvenile male ground squirrels initiate reproductive maturation during their first hibernation season, despite prolonged undernutrition and profound weight loss. We show that the hypothalamic reproductive axis undergoes activation during interbout arousals in the middle of hibernation, triggering production of luteinizing hormone and testosterone, and promoting testicular growth. Initiation of sexual maturation is circannually entrained and is independent of physiological state, ambient temperature, and food availability. Our study suggests a role for interbout arousals during hibernation and uncovers the neurophysiological mechanism of reproductive axis activation during conditions of extreme negative energy balance. VIDEO ABSTRACT.

Effects of stocking density on ovarian development and maturation during the rearing period in Shan-ma ducks.

Stocking density critically affects the growth and subsequent performance of animals in modern poultry production. This study investigated the effects of stocking density on ovarian development, ovarian maturation, and the mRNA expression of key genes in the reproductive axis during the rearing period of Shan-ma ducks. The experiments involved 180 healthy 7-wk-old Shan-ma ducks and randomly divided into low stocking density (LSD; n = 30, density = 5 birds/m2), medium stocking density (MSD; n = 60, density = 10 birds/m2) and high stocking density groups (HSD; n = 90, density = 15 birds/m2), for rearing. After examining ovarian development and measuring hormone levels in the plasma and expression levels of key regulatory genes in the reproductive axis at 19 wk of rearing, analysis of the gonad index analysis, reflecting stocking density, uncovered statistically significant differences. The gonad index of the LSD group was significantly higher than those of the MSD and HSD groups (P < 0.01), while no significant difference was observed between the MSD and HSD groups. pre-ovulatory follicles (POFs) and small yellow follicles (SYFs) development was only apparent in the LSD group, with the large white follicles (LWFs) number of this group being significantly higher than that of the MSD group (P < 0.05). The blood levels of E2 (estradiol), P4 (progesterone), and T (testosterone) were significantly higher in the LSD group than in the MSD and HSD groups (P < 0.05 or 0.01). Also, the levels of both P4 and T were significantly higher in the MSD group than in the HSD group (P < 0.01). The gene expression levels of GnRHR, FSH, AMHR, and FSHR were significantly increased in the LSD group compared to the MSD and HSD groups (P < 0.05 or 0.01), while the expression levels of GnIHR and GDF9 were significantly decreased in the LSD and MSD groups compared to the HSD group (P < 0.05 or 0.01). Steroid biosynthesis pathway genes such as StAR, CYP11A1, 3ß-HSD, CYP19A1, and BMP15 were significantly downregulated at greater stocking densities (P < 0.05 or 0.01). Likewise, the protein expression of StAR, 3ß-HSD, and CYP19A1 was also significantly decreased (P < 0.05 or 0.01). These results demonstrate that both medium and high stocking densities suppressed the expression of the key reproduction-promoting factors, while the expression level of the key reproductive inhibitory factors was enhanced. Therefore, rates of ovarian development and maturation could be reduced by a high stocking density leading to a delay in reproduction performance during the rearing period of Shan-ma ducks.

Mouse Cre drivers: tools for studying disorders of the human female neuroendocrine-reproductive axis†.

Benign disorders of the human female reproductive system, such primary ovarian insufficiency and polycystic ovary syndrome are associated with infertility and recurrent miscarriage, as well as increased risk of adverse health outcomes, including cardiovascular disease and type 2 diabetes. For many of these conditions, the contributing molecular and cellular processes are poorly understood. The overarching similarities between mice and humans have rendered mouse models irreplaceable in understanding normal physiology and elucidating pathological processes that underlie disorders of the female reproductive system. The utilization of Cre-LoxP recombination technology, which allows for spatial and temporal control of gene expression, has identified the role of numerous genes in development of the female reproductive system and in processes, such as ovulation and endometrial decidualization, that are required for the establishment and maintenance of pregnancy in mammals. In this comprehensive review, we provide a detailed overview of Cre drivers with activity in the neuroendocrine-reproductive axis that have been used to study disruptions in key intracellular signaling pathways. We first summarize normal development of the hypothalamus, pituitary, ovary, and uterus, highlighting similarities and differences between mice and humans. We then describe human conditions resulting from abnormal development and/or function of the organ. Finally, we describe loss-of-function models for each Cre driver that elegantly recapitulate some key features of the human condition and are associated with impaired fertility. The examples we provide illustrate use of each Cre driver as a tool for elucidating genetic and molecular underpinnings of reproductive dysfunction.

Melatonin is involved in the modulation of the hypothalamic and pituitary activity in the South American plains vizcacha, Lagostomus maximus.

Melatonin, the key messenger of photoperiodic information, is synthesized in the pineal gland by arylalkylamine N-acetyltransferase enzyme (AANAT). It binds to specific receptors MT1 and MT2 located in the hypothalamus and pituitary gland. Melatonin can modulate the reproductive axis affecting the secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). The South American plains vizcacha, Lagostomus maximus, shows natural poliovulation of up to 800 oocytes per estrous cycle, a 154-day long pregnancy, and reactivation of the reproductive axis at mid-gestation with pre-ovulatory follicular recruitment, presence of active corpora lutea, and variations of the endocrine status. Here we analyzed the involvement of melatonin in the modulation of the hypothalamic and pituitary gland physiology of vizcacha thorough several approaches, including histological localization of melatoninergic system components, assessment of melatoninergic components expression throughout the reproductive cycle, and evaluation of the effect of melatonin on hypothalamic and pituitary activities during the follicular and luteal phases of the estrous cycle. AANAT and melatonin receptors were localized in the pineal gland and preoptic area of the hypothalamus. Increase in pineal AANAT and serum melatonin expression was observed as pregnancy progressed, with the lowest hypothalamic MT1 and MT2 levels at mid-pregnancy. Pulsatility assays demonstrated that melatonin induces GnRH and LH secretion at luteal phase. The melatoninergic system effects on hypothalamic and pituitary gland hormones secretion during pregnancy pinpoint to melatonin as a potential key factor underlying the reactivation of the reproductive axis activity at mid-gestation.

Direct effect of RFRP-3 microinjection into the lateral ventricle on the hypothalamic kisspeptin neurons in ovariectomized estrogen-primed rats.

RFamide-related peptide-3 (RFRP-3) may be involved in the inhibition of kisspeptin, but there is no direct evidence that RFRP-3 can directly act on kisspeptin neurons. The present study aimed to investigate the role and mechanism of RFRP-3 and kisspeptin in the hypothalamic-pituitary reproductive axis. In order to detect the expression and localization of RFRP-3 and kisspeptin in dorsomedial hypothalamic nucleus, double immunofluorescence method combined with confocal microscopy were performed. RFRP-3 was injected into the lateral ventricle of ovariectomized estrogen primed rats. Blood and brain tissues were collected at 60-, 120-, 240- and 360-min. Serum levels of gonadotropin-releasing hormone, luteinizing hormone and follicle-stimulating hormone were detected by ELISA. Kisspeptin expression in hypothalamus was detected by western blotting. Finally, surface plasmon resonance was used to verify whether RFRP-3 can directly interact with kisspeptin. Confocal images indicated that RFRP-3 and kisspeptin were co-expressed in the same neurons in the hypothalamus of ovariectomized estrogen-primed rats. Serum concentrations of gonadotropin-releasing hormone, luteinizing hormone and follicle-stimulating hormone were demonstrated to be significantly reduced following microinjection of RFRP-3 into the lateral ventricle for 60, 120, 240 and 360 min compared with the corresponding saline groups. The expression levels of kisspeptin in hypothalamus were gradually decreased following microinjection of RFRP-3 into the lateral ventricle. In addition, the affinity constant (KD) of RFRP-3 binding to kisspeptin was 6.005x10-5 M, indicating that RFRP-3 bound directly to kisspeptin in the range of protein-protein binding strength (KD, 10-3-10-6 M). In conclusion, RFRP-3 may regulate the hypothalamic-pituitary reproductive axis by inhibiting the expression of hypothalamic kisspeptin and direct binding.

The Impact of Endocrine-Disrupting Chemicals in Male Fertility: Focus on the Action of Obesogens.

The current scenario of male infertility is not yet fully elucidated; however, there is increasing evidence that it is associated with the widespread exposure to endocrine-disrupting chemicals (EDCs), and in particular to obesogens. These compounds interfere with hormones involved in the regulation of metabolism and are associated with weight gain, being also able to change the functioning of the male reproductive axis and, consequently, the testicular physiology and metabolism that are pivotal for spermatogenesis. The disruption of these tightly regulated metabolic pathways leads to adverse reproductive outcomes. The permanent exposure to obesogens has raised serious health concerns. Evidence suggests that obesogens are one of the leading causes of the marked decline of male fertility and key players in shaping the future health outcomes not only for those who are directly exposed but also for upcoming generations. In addition to the changes that lead to inefficient functioning of the male gametes, obesogens induce alterations that are "imprinted" on the genes of the male gametes, establishing a link between generations and contributing to the transmission of defects. Unveiling the molecular mechanisms by which obesogens induce toxicity that may end-up in epigenetic modifications is imperative. This review describes and discusses the suggested molecular targets and potential mechanisms for obesogenic-disrupting chemicals and the subsequent effects on male reproductive health.

Pregnancy influences the selection of appropriate reference genes in mouse tissue: Determination of appropriate reference genes for quantitative reverse transcription PCR studies in tissues from the female mouse reproductive axis.

Selecting stably expressed reference genes which are not affected by physiological or pathophysiological conditions is crucial for reliable quantification in gene expression studies. This study examined the expression stability of a panel of twelve reference genes in tissues from the female mouse reproductive axis and the uterus. Gene expression studies were carried out using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). cDNA was synthesised from RNA extracted from hypothalami, pituitaries, ovaries and uteri of female mice at ages representing weaning, puberty and adulthood as well as pregnancy (13 ± 1 days post-coitus) (n = a minimum of 3 at each age and at pregnancy). The reference genes examined included 18 s, Actb, Atp5b, B2m, Canx, Cyc1, Eif4a2, Gapdh, Rpl13a, Sdha, Ubc and Ywhaz. The RT-qPCR raw data were imported into the qBASE+ software to analyse the expression stability using GeNorm. These data were also subsequently analysed using other software packages (Delta CT, Normfinder, BestKeeper). A comprehensive ranking was conducted considering all stability rankings generated from the different software analyses. B2m and Eif4a2 deviated from the acceptable range for amplification efficiency and therefore were excluded from the further analyses. The stability of the reference genes is influenced by the software used for the analysis with BestKeeper providing markedly different results than the other analyses. GeNorm analysis of tissues taken at different ages but not including pregnant animals, indicated that the expression of the reference genes is tissue specific with the most stable genes being: in the hypothalamus, Canx and Actb; in the pituitary, Sdha and Cyc1; in the ovary, 18s, Sdha and Ubc; and in the uterus, Ywhaz, Cyc1, Atp5b, 18s and Rpl13a. The optimal number of reference genes to be used was determined to be 2 in the first three tissues while in the uterus, the V-score generated by the GeNorm analysis was higher than 0.15 suggesting that 3 or more genes should be used for normalisation. Inclusion of tissues from pregnant mice changed the reference genes identified as being the most stable: Ubc and Sdha were the most stable genes in the hypothalamus, pituitary and the ovary. The addition of pregnant tissue had no effect on the stability of the genes in uterus (Ywhaz, Cyc1, Atp5b, 18s and Rpl13a). Identification of these stable reference genes will be of use to those interested in studying female fertility and researchers should be alert to the effects of pregnancy on reference gene stability. This study also signifies the importance of re-examining reference gene stability if the experimental conditions are changed, as shown with the introduction of pregnancy as a new factor in this research.

Abatement of the dysfunctional hypothalamic-pituitary-gonadal axis due to ciprofloxacin administration by selenium in male rats.

The present study examined the influence of selenium on ciprofloxacin-mediated reproductive dysfunction in rats. The research design consisted of five groups of eight animals each. The rats were administered 135 mg/kg body weight of ciprofloxacin per se or simultaneously with selenium at 0.25 and 0.5 mg/kg for 15 uninterrupted days. Antioxidant and inflammatory indices were assayed using the testes, epididymis, and hypothalamus of the animals after sacrifice. Results revealed that ciprofloxacin treatment per se interfered with the reproductive axis as demonstrated by diminished serum hormonal levels, sperm quality, and enzymatic indices of testicular function, which were, however, abrogated following selenium co-treatment. Besides this, administration of selenium attenuated the depletion of glutathione level, inhibition of catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase activities with a concomitant reduction in reactive oxygen and nitrogen species, and lipid peroxidation in ciprofloxacin-treated in rats. Selenium treatment also mitigated ciprofloxacin-mediated elevation in nitric oxide level and of myeloperoxidase activity as well as histological lesions in the animals. Overall, selenium attenuated impairment in the male reproductive axis due to ciprofloxacin treatment through abatement of inflammation and oxidative stress in rats.

Kolaviron suppresses dysfunctional reproductive axis associated with multi-walled carbon nanotubes exposure in male rats.

Reproductive toxicity associated with excessive exposure to multi-walled carbon nanotubes (MWCNTs), which are commonly used in medicine as valuable drug delivery systems, is well documented. Kolaviron, a bioflavonoid isolated from Garcinia kola seeds, elicits numerous health beneficial effects related to its anti-inflammatory, anti-genotoxic activities, anti-apoptotic, and antioxidant properties. However, information on the role of kolaviron in MWCNTs-induced reproductive toxicity is not available in the literature. Herein, we assessed the protective effects of kolaviron on MWCNTs-induced dysfunctional reproductive axis in rats following exposure to MWCNTs (1 mg/kg) and concurrent treatment with kolaviron (50 or 100 mg/kg body weight) for 15 successive days. Results showed that MWCNTs-induced dysfunctional reproductive axis as evidenced by deficits in pituitary and testicular hormones, marker enzymes of testicular function, and sperm functional characteristics were abrogated in rats co-administered with kolaviron. Moreover, co-administration of kolaviron-abated MWCNTs-induced inhibition of antioxidant enzyme activities increases in oxidative stress and inflammatory indices. This is evidenced by diminished levels of tumor necrosis factor-alpha, nitric oxide, lipid peroxidation, reactive oxygen, and nitrogen species as well as reduced activity of myeloperoxidase in testes, epididymis, and hypothalamus of the rats. Biochemical data on the chemoprotection of MWCNTs-induced reproductive toxicity were corroborated by histological findings. Taken together, kolaviron suppressed dysfunctional reproductive axis associated with MWCNTs exposure via abrogation of oxidative stress and inflammation in male rats.

Allium cepa Linn juice protect against alterations in reproductive functions induced by maternal dexamethsone treatment during lactation in male offspring of Wistar rats.

Treatment with dams with dexamethasone during lactation has been reported to induce oxidative stress in the testis of the offspring. Allium cepa L (Red Onion) is known to be a potent free radical scavenger. The protective role of Allium cepa against oxidative stress induced in testis following treatment with dexamehasone during lactation in Wistar rats was assessed. Twenty female rats were assigned into four groups (n = 5) during lactation and they were treated as follows: Group 1 serve as Control (distilled water), Group 2, 3, and four were admistered dexamethasone (60 µg/kg), Allium cepa (5 ml/kg) and dexamethasone + Allium cepa respectively. Testicular descent, pubertal age, sperm quality indices, and serum hormonal profile were assessed as indices of reproductive function. Testicular malondialdehyde (MDA) reduced glutathione (GSH) as well as superoxide dismutase (SOD) and catalase activities were assessed as measures of oxidative stress. Results obtained showed that dexamethasone caused significant (P < 0.05) reduction in testes weights, indices of sperm quality, serum testosterone, FSH, LH levels and testicular antioxidant enzyme activities. There was significant delay (P < 0.05) in days of testes descent, preputial separation and increase in testicular MDA. However, maternal treatment with Allium cepa Linn juice significantly (P < 0.05) improved both indices of reproductive function and testicular antioxidant enzymes. These findings suggest that Allium cepa Linn has a protective effect against testicular oxidative stress and reproductive dysfunction following treatment of dams with dexamethasone during lactation.

Stress-mediated convergence of splicing landscapes in male and female rock doves.

BACKGROUND: The process of alternative splicing provides a unique mechanism by which eukaryotes are able to produce numerous protein products from the same gene. Heightened variability in the proteome has been thought to potentiate increased behavioral complexity and response flexibility to environmental stimuli, thus contributing to more refined traits on which natural and sexual selection can act. While it has been long known that various forms of environmental stress can negatively affect sexual behavior and reproduction, we know little of how stress can affect the alternative splicing associated with these events, and less still about how splicing may differ between sexes. Using the model of the rock dove (Columba livia), our team previously uncovered sexual dimorphism in the basal and stress-responsive gene transcription of a biological system necessary for facilitating sexual behavior and reproduction, the hypothalamic-pituitary-gonadal (HPG) axis. In this study, we delve further into understanding the mechanistic underpinnings of how changes in the environment can affect reproduction by testing the alternative splicing response of the HPG axis to an external stressor in both sexes. RESULTS: This study reveals dramatic baseline differences in HPG alternative splicing between males and females. However, after subjecting subjects to a restraint stress paradigm, we found a significant reduction in these differences between the sexes. In both stress and control treatments, we identified a higher incidence of splicing activity in the pituitary in both sexes as compared to other tissues. Of these splicing events, the core exon event is the most abundant form of splicing and more frequently occurs in the coding regions of the gene. Overall, we observed less splicing activity in the 3'UTR (untranslated region) end of transcripts than the 5'UTR or coding regions. CONCLUSIONS: Our results provide vital new insight into sex-specific aspects of the stress response on the HPG axis at an unprecedented proximate level. Males and females uniquely respond to stress, yet exhibit splicing patterns suggesting a convergent, optimal splicing landscape for stress response. This information has the potential to inform evolutionary theory as well as the development of highly-specific drug targets for stress-induced reproductive dysfunction.

Leptin actions through the nitrergic system to modulate the hypothalamic expression of the kiss1 mRNA in the female rat.

Gonadotrophin-releasing hormone (GnRH) is the main controller of the reproductive axis and stimulates the synthesis and secretion of gonadotrophins. Estrogen is the main peripheral factor controlling GnRH secretion, and this action is mainly mediated by the transsynaptic pathway through nitric oxide, kisspeptin, leptin, among other factors. Kisspeptin is the most potent factor known to induce GnRH release. Nitric oxide and leptin also promote GnRH release; however, neurons expressing GnRH do not express the leptin receptor (OB-R). Leptin seems to modulate the expression of genes and proteins involved in the kisspeptin system. However, few kisspeptin-synthesizing cells in the arcuate nucleus (ARC) and few cells, if any, in the preoptic area (POA) express OB-R; this indicates an indirect mechanism of leptin action on kisspeptin. Nitric oxide is an important intermediate in the actions of leptin in the central nervous system. Thus, this work aimed to verify the numbers of nNOS cells were activated by leptin in different hypothalamic areas; the modulatory effects of the nitrergic system on the kisspeptin system; and the indirect regulatory effect of leptin on the kisspeptin system via nitric oxide. Ovariectomized rats were treated with estrogen or a vehicle and received an intracerebroventricular (i.c.v.) injection of a nitric oxide donor, leptin or neuronal nitric oxide synthase (nNOS) enzyme inhibitor. Thirty minutes after the injection, the animals were decapitated. Leptin acts directly on nitrergic neurons in different hypothalamic regions, and the effects on the ventral premammillary nucleus (PMV) and ventral dorsomedial hypothalamus (vDMH) are enhanced. The use of a nitric oxide donor or the administration of leptin stimulates the expression of the kisspeptin mRNA in the ARC of animals with or without estrogenic action; however, these changes are not observed in the POA. In addition, the action of leptin on the expression of the kisspeptin mRNA in the ARC is blocked by a nitric oxide synthesis inhibitor. We concluded that the effects of leptin on the central nervous system are at least partially mediated by the nitrergic system. Also, nitric oxide acts on the kisspeptin system by modulating the expression of the kisspeptin mRNA, and leptin at least partially modulates the kisspeptin system through the nitrergic system, particularly in the ARC.

Roles for Kisspeptin in proliferation and differentiation of spermatogonial cells isolated from mice offspring when the cells are cocultured with somatic cells.

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10-8 -10-6 M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP3 , TP1, TP2 , and, Prm1 markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis.

Quantitative analysis of in-vivo responses of reproductive and thyroid endpoints in male goldfish exposed to monocrotophos pesticide.

Cross-regulation occurs at many points between the hypothalamic-pituitary-gonad (HPG) and hypothalamic-pituitary-thyroid (HPT) axes. Monocrotophos (MCP) pesticide could disrupt HPG and HPT axes, but its direct target within the endocrine system is still unclear. In the present study, hormone concentrations and transcriptional profiles of HPG and HPT genes were examined in male goldfish (Carassius auratus) exposed to 0, 4, 40, and 400 µg/L MCP for 2, 4, 8, and 12 d. In vivo data were analyzed by multiple linear regression and correlation analysis, quantitatively indicating that MCP-induced plasma 17ß-estradiol (E2) levels were most associated with alteration of cyp19a transcription, which was also a potential point indirectly modulated by the MCP-altered thyroid hormones (THs) status; disturbance of THs pathways was most related with effect of MCP on regulation of the hypothalamic-pituitary hormones involved in the thyroid system, and the increased E2 levels might enhance the impact of MCP on HPT axis by modulating hepatic deiodinase expression. Our finding, based on these correlational data, gave a whole view of the regulations, especially on the cross-talk between sex hormone and thyroid hormone pathways upon exposure to chemicals with unknown direct target in vivo, and cautions should be exercised when developing adverse outcome pathway networks for reproductive and thyroidal endocrine disruption.

Neurokinin B signaling in hermaphroditic species, a study of the orange-spotted grouper (Epinephelus coioides).

Neurokinin B (NKB) plays important roles in the mammalian reproductive axis by modulating the release of gonadotropin-releasing hormone (GnRH) and gonadotropins. In the present study, the tac3 cDNA was cloned from a hermaphroditic species, the orange-spotted grouper. Sequence analysis showed that the grouper Tac3 precursor encoded two tachykinin peptides, NKB and NKB-related peptide (NKBRP). Expression analysis in different tissues revealed that tac3 mRNA was highly expressed in the brain of the orange-spotted grouper. In situ hybridization further revealed that it was localized in some hypothalamic nuclei associated with reproductive regulation. During ovarian development, an increase of tac3 expression in the hypothalamus was observed at vitellogenesis stage. Intraperitoneal administration of NKB could increase the gnrh1 and lhß mRNA levels, and enhance the serum estrogen levels, but did not significantly influence lhß expression in cultured pituitary cells, indicating that NKB does not directly exert its actions on the pituitary gland. However, it was found that NKBRP had no effect on the expression of two gnrhs and two gths in vivo and in vitro. Effects of sex steroids on tac3 expression were further investigated. During the 17-methyltestosterone-induced sex change in the orange-spotted grouper, hypothalamic tac3 expression showed no significant change. Interestingly, ovariectomy greatly stimulated tac3 expression, while the 17ß-estradiol treatment reversed this effect. In general, our data highly indicated that NKB signaling could activate the reproductive axis in the orange-spotted grouper. Our study is the first description of the NKB signaling in the hermaphroditic species.