α-Linolenic acid-regulated testosterone biosynthesis via activation of the JNK-SF-1 signaling pathway in primary rooster Leydig cells.

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster. METHODS: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 µmol/L) or pretreated with a p38 inhibitor (50 µmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 µmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 µmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR). RESULTS: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 µmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression significantly increased (P < 0.05) in the 40 µmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 µmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 µmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 µmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3ß-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3ß-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3ß-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05). CONCLUSION: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3ß-HSD, and P450c17 expression in primary rooster Leydig cells.

Inhibition of high-fat diet-induced inflammatory responses in adipose tissue by SF1-expressing neurons of the ventromedial hypothalamus.

Inflammation and thermogenesis in white adipose tissue (WAT) at different sites influence the overall effects of obesity on metabolic health. In mice fed a high-fat diet (HFD), inflammatory responses are less pronounced in inguinal WAT (ingWAT) than in epididymal WAT (epiWAT). Here we show that ablation and activation of steroidogenic factor 1 (SF1)-expressing neurons in the ventromedial hypothalamus (VMH) oppositely affect the expression of inflammation-related genes and the formation of crown-like structures by infiltrating macrophages in ingWAT, but not in epiWAT, of HFD-fed mice, with these effects being mediated by sympathetic nerves innervating ingWAT. In contrast, SF1 neurons of the VMH preferentially regulated the expression of thermogenesis-related genes in interscapular brown adipose tissue (BAT) of HFD-fed mice. These results suggest that SF1 neurons of the VMH differentially regulate inflammatory responses and thermogenesis among various adipose tissue depots and restrain inflammation associated with diet-induced obesity specifically in ingWAT.

Contribution of the co-chaperone FKBP51 in the ventromedial hypothalamus to metabolic homeostasis in male and female mice.

OBJECTIVE: Steroidogenic factor 1 (SF1) expressing neurons in the ventromedial hypothalamus (VMH) have been directly implicated in whole-body metabolism and in the onset of obesity. The co-chaperone FKBP51 is abundantly expressed in the VMH and was recently linked to type 2 diabetes, insulin resistance, adipogenesis, browning of white adipose tissue (WAT) and bodyweight regulation. METHODS: We investigated the role of FKBP51 in the VMH by conditional deletion and virus-mediated overexpression of FKBP51 in SF1-positive neurons. Baseline and high fat diet (HFD)-induced metabolic- and stress-related phenotypes in male and female mice were obtained. RESULTS: In contrast to previously reported robust phenotypes of FKBP51 manipulation in the entire mediobasal hypothalamus (MBH), selective deletion or overexpression of FKBP51 in the VMH resulted in only a moderate alteration of HFD-induced bodyweight gain and body composition, independent of sex. CONCLUSIONS: Overall, this study shows that animals lacking and overexpressing Fkbp5 in Sf1-expressing cells within the VMH display only a mild metabolic phenotype compared to an MBH-wide manipulation of this gene, suggesting that FKBP51 in SF1 neurons within this hypothalamic nucleus plays a subsidiary role in controlling whole-body metabolism.

Steroidogenic Factor 1 Regulation of the Hypothalamic-Pituitary-Ovarian Axis of Adult Female Mice.

The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor-driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization.

New Insights of SF1 Neurons in Hypothalamic Regulation of Obesity and Diabetes.

Despite the substantial role played by the hypothalamus in the regulation of energy balance and glucose homeostasis, the exact mechanisms and neuronal circuits underlying this regulation remain poorly understood. In the last 15 years, investigations using transgenic models, optogenetic, and chemogenetic approaches have revealed that SF1 neurons in the ventromedial hypothalamus are a specific lead in the brain's ability to sense glucose levels and conduct insulin and leptin signaling in energy expenditure and glucose homeostasis, with minor feeding control. Deletion of hormonal receptors, nutritional sensors, or synaptic receptors in SF1 neurons triggers metabolic alterations mostly appreciated under high-fat feeding, indicating that SF1 neurons are particularly important for metabolic adaptation in the early stages of obesity. Although these studies have provided exciting insight into the implications of hypothalamic SF1 neurons on whole-body energy homeostasis, new questions have arisen from these results. Particularly, the existence of neuronal sub-populations of SF1 neurons and the intricate neurocircuitry linking these neurons with other nuclei and with the periphery. In this review, we address the most relevant studies carried out in SF1 neurons to date, to provide a global view of the central role played by these neurons in the pathogenesis of obesity and diabetes.

Ventromedial Nucleus of the Hypothalamus Neurons Under the Magnifying Glass.

The ventromedial nucleus of the hypothalamus (VMH) is a complex brain structure that is integral to many neuroendocrine functions, including glucose regulation, thermogenesis, and appetitive, social, and sexual behaviors. As such, it is of little surprise that the nucleus is under intensive investigation to decipher the mechanisms which underlie these diverse roles. Developments in genetic and investigative tools, for example the targeting of steroidogenic factor-1-expressing neurons, have allowed us to take a closer look at the VMH, its connections, and how it affects competing behaviors. In the current review, we aim to integrate recent findings into the literature and contemplate the conclusions that can be drawn.

Rap1 in the VMH regulates glucose homeostasis.

The hypothalamus is a critical regulator of glucose metabolism and is capable of correcting diabetes conditions independently of an effect on energy balance. The small GTPase Rap1 in the forebrain is implicated in high-fat diet-induced (HFD-induced) obesity and glucose imbalance. Here, we report that increasing Rap1 activity selectively in the medial hypothalamus elevated blood glucose without increasing the body weight of HFD-fed mice. In contrast, decreasing hypothalamic Rap1 activity protected mice from diet-induced hyperglycemia but did not prevent weight gain. The remarkable glycemic effect of Rap1 was reproduced when Rap1 was specifically deleted in steroidogenic factor-1-positive (SF-1-positive) neurons in the ventromedial hypothalamic nucleus (VMH) known to regulate glucose metabolism. While having no effect on body weight regardless of sex, diet, and age, Rap1 deficiency in the VMH SF1 neurons markedly lowered blood glucose and insulin levels, improved glucose and insulin tolerance, and protected mice against HFD-induced neural leptin resistance and peripheral insulin resistance at the cellular and whole-body levels. Last, acute pharmacological inhibition of brain exchange protein directly activated by cAMP 2, a direct activator of Rap1, corrected glucose imbalance in obese mouse models. Our findings uncover the primary role of VMH Rap1 in glycemic control and implicate Rap1 signaling as a potential target for therapeutic intervention in diabetes.

Defective autophagy in Sf1 neurons perturbs the metabolic response to fasting and causes mitochondrial dysfunction.

OBJECTIVE: The ventromedial nucleus of the hypothalamus (VMH) is a critical component of the forebrain pathways that regulate energy homeostasis. It also plays an important role in the metabolic response to fasting. However, the mechanisms contributing to these physiological processes remain elusive. Autophagy is an evolutionarily conserved mechanism that maintains cellular homeostasis by turning over cellular components and providing nutrients to the cells during starvation. Here, we investigated the importance of the autophagy-related gene Atg7 in Sf1-expressing neurons of the VMH in control and fasted conditions. METHODS: We generated Sf1-Cre; Atg7loxP/loxP mice and examined their metabolic and cellular response to fasting. RESULTS: Fasting induces autophagy in the VMH, and mice lacking Atg7 in Sf1-expressing neurons display altered leptin sensitivity and impaired energy expenditure regulation in response to fasting. Moreover, loss of Atg7 in Sf1 neurons causes alterations in the central response to fasting. Furthermore, alterations in mitochondria morphology and activity are observed in mutant mice. CONCLUSION: Together, these data show that autophagy is nutritionally regulated in VMH neurons and that VMH autophagy participates in the control of energy homeostasis during fasting.

Deletion of growth hormone receptor in hypothalamic neurons affects the adaptation capacity to aerobic exercise.

The hypothalamus mediates important exercise-induced metabolic adaptations, possibly via hormonal signals. Hypothalamic leptin receptor (LepR)- and steroidogenic factor 1 (SF1)-expressing neurons are directly responsive to growth hormone (GH) and deletion of GH receptor (GHR) in these cells impairs neuroendocrine responses during situations of metabolic stress. In the present study, we determined whether GHR ablation in LepR- or SF1-expressing cells modifies acute and chronic metabolic adaptations to exercise. Male mice carrying deletion of GHR in LepR- or SF1-expressing cells were submitted to 8 weeks of treadmill running training. Changes in aerobic performance and exercise-induced metabolic adaptations were determined. Mice carrying GHR deletion in LepR cells showed increased aerobic performance after 8 weeks of treadmill training, whereas GHR ablation in SF1 cells prevented improvement in running capacity. Trained mice carrying GHR ablation in SF1 cells exhibited increased fat mass and reduced cross-sectional area of the gastrocnemius muscle. In contrast, deletion of GHR in LepR cells reduced fat mass and increased gastrocnemius muscle hypertrophy, energy expenditure and voluntary locomotor activity in trained mice. Although glucose tolerance was not significantly affected by targeted deletions, glycemia before and immediately after maximum running tests was altered by GHR ablation. In conclusion, GHR signaling in hypothalamic neurons regulates the adaptation capacity to aerobic exercise in a cell-specific manner. These findings suggest that GH may represent a hormonal cue that informs specific hypothalamic neurons to produce exercise-induced acute and chronic metabolic adaptations.

Preliminary study of Yulin mixture affecting the miR-320/SF-1/Cyp19a1 on mouse polycystic ovary syndrome model.

In this study, the polycystic ovary syndrome (PCOS) mice model was randomly divided into 6 groups: blank control group, Clomiphene group, PCOS group, and Yulin mixture high-/medium-/low-dose group. Rats were killed after 5 weeks of administration. The expression levels of serum E2,T,Insulin and LH were detected by ELISA. The localizations and quantities of Steroid-generating factor-1 (SF-1) and Cytochrome protein P450 a1 (Cyp19a1) were detected by immunohistochemistry and western blot. The quantities of miR-320 were detected by RT-PCR. The results showed that the mechanism of Yulin mixture inhibiting the growth of polycystic ovary on mouse PCOS model may be through the decreasing of serum T and LH levels and then reducing local estrogen content to make the polycystic ovary atrophy. Yulin mixture can decrease the level of miR-320 and increase the expression of SF-1 and Cyp19a1 in ovary, thereby regulating the ovarian granulosa cell proliferation and apoptosis.

A Layer 3→5 Circuit in Auditory Cortex That Contributes to Pre-pulse Inhibition of the Acoustic Startle Response.

While connectivity within sensory cortical circuits has been studied extensively, how these connections contribute to perception and behavior is not well understood. Here we tested the role of a circuit between layers 3 and 5 of auditory cortex in sound detection. We measured sound detection using a common variant of pre-pulse inhibition of the acoustic startle response, in which a silent gap in background noise acts as a cue that attenuates startle. We used the Nr5a-Cre driver line, which we found drove expression in the auditory cortex restricted predominantly to layer 3. Photoactivation of these cells evoked short-latency, highly reliable spiking in downstream layer 5 neurons, and attenuated startle responses similarly to gaps in noise. Photosuppression of these cells did not affect behavioral gap detection. Our data provide the first demonstration that direct activation of auditory cortical neurons is sufficient to attenuate the acoustic startle response, similar to the detection of a sound.

Growth hormone enhances the recovery of hypoglycemia via ventromedial hypothalamic neurons.

Growth hormone (GH) is secreted during hypoglycemia, and GH-responsive neurons are found in brain areas containing glucose-sensing neurons that regulate the counter-regulatory response (CRR). However, whether GH modulates the CRR to hypoglycemia via specific neuronal populations is currently unknown. Mice carrying ablation of GH receptor (GHR) either in leptin receptor (LepR)- or steroidogenic factor-1 (SF1)-expressing cells were studied. We also investigated the importance of signal transducer and activator of transcription 5 (STAT5) signaling in SF1 cells for the CRR. GHR ablation in LepR cells led to impaired capacity to recover from insulin-induced hypoglycemia and to a blunted CRR caused by 2-deoxy-d-glucose (2DG) administration. GHR inactivation in SF1 cells, which include ventromedial hypothalamic neurons, also attenuated the CRR. The reduced CRR was prevented by parasympathetic blockers. Additionally, infusion of 2DG produced an abnormal hyperactivity of parasympathetic preganglionic neurons, whereas the 2DG-induced activation of anterior bed nucleus of the stria terminalis neurons was reduced in mice without GHR in SF1 cells. Mice carrying ablation of Stat5a/b genes in SF1 cells showed no defects in the CRR. In summary, GHR expression in SF1 cells is required for a normal CRR, and these effects are largely independent of STAT5 pathway.-Furigo, I. C., de Souza, G. O., Teixeira, P. D. S., Guadagnini, D., Frazão, R., List, E. O., Kopchick, J. J., Prada, P. O., Donato, J., Jr. Growth hormone enhances the recovery of hypoglycemia via ventromedial hypothalamic neurons.

P110ß in the ventromedial hypothalamus regulates glucose and energy metabolism.

Phosphoinositide 3-kinase (PI3K) signaling in hypothalamic neurons integrates peripheral metabolic cues, including leptin and insulin, to coordinate systemic glucose and energy homeostasis. PI3K is composed of different subunits, each of which has several unique isoforms. However, the role of the PI3K subunits and isoforms in the ventromedial hypothalamus (VMH), a prominent site for the regulation of glucose and energy homeostasis, is unclear. Here we investigated the role of subunit p110ß in steroidogenic factor-1 (SF-1) neurons of the VMH in the regulation of metabolism. Our data demonstrate that the deletion of p110ß in SF-1 neurons disrupts glucose metabolism, rendering the mice insulin resistant. In addition, the deletion of p110ß in SF-1 neurons leads to the whitening of brown adipose tissues and increased susceptibility to diet-induced obesity due to blunted energy expenditure. These results highlight a critical role for p110ß in the regulation of glucose and energy homeostasis via VMH neurons.

Circular IRE-type RNAs of the NR5A1 gene are formed in adrenocortical cells.

The recently discovered circular RNAs (circRNAs) are mostly formed by back-splicing where the downstream 5' splice site splices to the upstream 3' splice site by conventional pre-mRNA splicing. These circRNAs regulate gene expression by acting as sponges for micro-RNAs or RNA-binding proteins. Here we show that the NR5A1 (previously called Ad4BP or SF-1) gene which is exclusively expressed in the adrenal cortex and steroidogenic tissue can form atypical circRNAs by unconventional splicing. Two stem loops with inositol-requiring protein-1α (IRE1α) cleavage sites are connected by an IRE1α cleavage site to form a circRNA (circIRE RNA). From total RNA of normal human adrenal cortex, we detected a circIRE RNA with connected ends by IRE1α cleavage sites in exon 6 and exon 1 (circIRE NR5A1 ex6-1 RNA). circIRE NR5A1 ex6-1 RNA was not detected in the adrenocortical cancer cell line, H295R. When IRE1α was expressed in H295R cells a different circIRE NR5A1 RNA connecting IRE1-cleavage sites in exon 7 and exon 1 was detected (circIRE NR5A1 ex7-1 RNA). The expression of this circIRE RNA was inhibited by the IRE1 inhibitor 1, STF-083010, implicating that it was formed via the ER stress pathway, where IRE1α is a major factor. This is the first report of this type of circular RNA connected by IRE1-cleavage sites found to be expressed in mammalian cells in a tissue-specific manner. To our surprise, the concomitant expression of NR5A1 was increased by IRE1α implicating that NR5A1 was not subjected to IRE1-dependent decay of mRNA (RIDD) but rather activating a transcriptional regulatory network to cope with ER stress in steroidogenic tissue reminiscent to XBP1 in other tissue. We believe this is the first report of such tissue-specific transcriptional cascade responding to ER stress as well as the novel finding of circular RNAs connected by IRE1α cleavage sites expressed in mammalian tissue.

Expression of progesterone receptor, estrogen receptors α and ß, and kisspeptin in the hypothalamus during perinatal development of gonad-lacking steroidogenic factor-1 knockout mice.

Gonadal hormones contribute to brain sexual differentiation. We analyzed expression of progesterone receptor (PR), estrogen receptor-α (ERα), ERß, and kisspeptin, in the preoptic area (POA) and/or the arcuate nucleus (ARC), in gonad-lacking steroidogenic factor-1 knockout (KO) mice during perinatal development. At postnatal-day (P) 0-P7, POA PR levels were higher in wild-type (WT) males compared with WT females, while those in KO males were lower than in WT males and similar to those in WT and KO females. At P14-P21, PR levels in all groups increased similarly. POA ERα levels were similar in all groups at embryonic-day (E) 15.5-P14. Those in WT but not KO males reduced during postnatal development to be significantly lower compared with females at P21. POA ERß levels were higher in WT males than in WT females, while those in KO males were lower than in WT males and similar to those in WT and KO females at P0-P21. POA kisspeptin expression was female-biased in WT mice, while levels in KO females were lower compared with WT females and similar to those in WT and KO males. ARC kisspeptin levels were equivalent among groups at E15.5-P0. At P7-P21, ARC levels in WT but not KO males became lower compared with WT females. Diethylstilbestrol exposure during P0-P6 and P7-P13 increased POA PR and ERß, and decreased POA ERα and ARC kisspeptin levels at P7 and/or P14 in both sexes of KO mice. These data further understanding of gonadal hormone action on neuronal marker expression during brain sexual development.

p53 in AgRP neurons is required for protection against diet-induced obesity via JNK1.

p53 is a well-known tumor suppressor that has emerged as an important player in energy balance. However, its metabolic role in the hypothalamus remains unknown. Herein, we show that mice lacking p53 in agouti-related peptide (AgRP), but not proopiomelanocortin (POMC) or steroidogenic factor-1 (SF1) neurons, are more prone to develop diet-induced obesity and show reduced brown adipose tissue (BAT) thermogenic activity. AgRP-specific ablation of p53 resulted in increased hypothalamic c-Jun N-terminal kinase (JNK) activity before the mice developed obesity, and central inhibition of JNK reversed the obese phenotype of these mice. The overexpression of p53 in the ARC or specifically in AgRP neurons of obese mice decreased body weight and stimulated BAT thermogenesis, resulting in body weight loss. Finally, p53 in AgRP neurons regulates the ghrelin-induced food intake and body weight. Overall, our findings provide evidence that p53 in AgRP neurons is required for normal adaptations against diet-induced obesity.

Continuous soy isoflavones exposure from weaning to maturity induces downregulation of ovarian steroidogenic factor 1 gene expression and corresponding changes in DNA methylation pattern.

Female Wistar rats were treated with orally administered soy isoflavones at concentrations of 0, 25, 50, or 100mg/kg body weight from weaning until sexual maturity (3 mo.), and ovarian steroidogenesis was evaluated. After soy isoflavones were administered, a significant (P<0.05) decrease (44%) in the serum estrodial levels of the high-dose (HD) group were observed. Cultured granulosa cells from the middle- (MD) and HD groups showed significantly (P<0.05) reduced (31%, 45%, respectively) in vitro estradiol secretion, and those from the HD group showed significantly (P<0.05) reduced progesterone (25%) secretion. Compared with the control group, the mRNA expression of the steroidogenic acute regulatory protein (Star), cytochromeP450 cholesterol side chain cleavage (Cyp11a1 and Cyp19a1), and hydroxysteroid dehydrogenase 3b (Hsd3b) genes also decreased. Real-time quantitative PCR and Western blotting revealed a significant (P<0.05) decrease in key transcription factor steroidogenic factor-1 (SF-1) expression in the HD group. The detection of DNA methylation using bisulfitesequencing PCR (BSP) suggested a significantly (P<0.05) increased total methylation rate in the proximal SF-1 promoter in the HD group. Further studies showed that treatment with soy isoflavones can significantly (P<0.05) increase the mRNA expression of DNA methyltransferase (DNMT) 1 and DNMT3a. This study proved that soy isoflavone administration from weaning until sexual maturity could inhibit ovarian steroidogenesis, suggesting that SF-1 might play an important role in this effect. In addition, DNA methylation might play a role in the downregulation of SF-1 gene expression induced by soy isoflavones.

Developmental changes in hypothalamic SF-1, POMC, and ERα mRNA expression and their sensitivity to fasting in male and female rats.

Estrogen plays pivotal roles in body weight regulation through its effects on central estrogen receptor-α (ERα) expression. ERα is found on neurons that express the hypothalamic anorexigenic factors steroidogenic factor-1 (SF-1) and pro-opiomelanocortin (POMC) mediate these effects of estrogen. As the gonadal hormonal milieu is drastically altered during the developmental period, the expression levels of SF-1 and POMC might also change during this period. In this study, we showed that hypothalamic SF-1 and ERα mRNA expression did not change during the neonatal to pre-pubertal period (from postnatal day 10 to 30), and there were no significant differences in the hypothalamic mRNA expression levels of these molecules between males and females at any examined age. On the other hand, hypothalamic POMC mRNA expression and the serum estradiol (E2) level increased during development in both males and females. Significant positive correlations were detected between the serum E2 level and hypothalamic POMC mRNA expression in both males and females. Hypothalamic ERα and POMC mRNA expression were decreased by fasting in male rats at all examined ages, whereas fasting had no effect on hypothalamic ERα or POMC mRNA expression in the female rats. These results indicate that the regulatory system involving E2 and hypothalamic POMC expression might already be established in the neonatal period and that the roles of POMC and ERα in body weight regulation during development might differ slightly between males and females.

Expression of Kisspeptin in Gonadotrope Precursors in the Mouse Pituitary during Embryonic and Postnatal Development and in Adulthood.

BACKGROUND: Kisspeptins are important regulators of the development and function of the hypothalamic-pituitary-gonadal axis. However, the importance of kisspeptin at the pituitary level is unclear. METHODS: We examined the expression profile of kisspeptin in the mouse pituitary during development and in adulthood using RT-PCR, quantitative PCR and immunohistochemistry. RESULTS: Kiss1 mRNA was detected in both embryonic and postnatal pituitaries. Kisspeptin-immunoreactive (+) cells were detected from embryonic day (E) 13.5 throughout adulthood, being localized to the rostroventral portion in the anterior pituitary (AP) in embryos, and also to the dorsocaudal AP postnatally. A large proportion of kisspeptin+ cells were double-labeled with gonadotrope markers including Foxl2, SF-1, and LHß, and the percentage of LHß+ cells in kisspeptin+ cells increased during development. No kisspeptin+ cells were positive for the proliferating cell marker MCM7 (minichromosome maintenance protein 7), but a few kisspeptin+ cells co-expressed the stem/progenitor cell marker Sox2. Kisspeptin expression was similar between sexes and between agonadal SF-1 knockout embryos and wild-type littermates. Kiss1 mRNA levels were not significantly different between sexes or during early postnatal development, but levels in females increased when puberty began and were significantly higher than in males at postpubertal ages. CONCLUSIONS: These results suggest that kisspeptin is expressed in gonadotrope precursors during gonadotrope differentiation, and that kisspeptin expression begins soon after the initiation of αGSU production and is extinguished soon after the initiation of LH production. Furthermore, pituitary kisspeptin expression may be regulated in a gonad-independent manner during development, but may be associated with gonadotrope function in adulthood.

Effects and mechanisms of waterborne copper exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to determine the effects and mechanism of waterborne copper (Cu) exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco. To this end, two experiments were conducted. In Exp. 1, the partial cDNA sequences of three steroidogenesis-related genes (androgen receptor (ar), steroidogenic factor 1 (sf-1) and steroidogenic acute regulatory protein (star)) were firstly characterized from P. fulvidraco. The predicted amino acid sequences for the P. fulvidraco ar, sf-1 and star contained the main structural features characteristic in other species. In Exp. 2, P. fulvidraco were exposed to three waterborne Cu concentrations (control, 30µg/l and 60µg/l, respectively) for 56days. Sampling occurred on day 28 and day 56, respectively. On day 28, the levels of serum sex-steroid hormones (FSH and LH) and the mRNA levels of steroidogenesis-related genes (3ß-hsd, cyp11a1, cyp17, cyp19a, sf-1 and star) were significantly increased in ovary of P. fulvidraco exposed to 30µg Cu/l. The immunohistochemical analysis showed the positive reaction of ER, VTG and aromatase in low dose exposure group. These indicated that in low dose and relative short-term exposure, Cu was beneficial. In contrast, 60µg Cu/l exposure significantly reduced the levels of serum FSH, LH, E2 and P, and the mRNA levels of ovarian 20ß-hsd, cyp19a and erα in P. fulvidraco. On day 56, waterborne Cu concentration exposure reduced the levels of serum gonadotropins and sex hormones, and down-regulated the mRNA levels of steroidogenesis-related genes, indicating long-term Cu exposure had toxic effect on the secretion of sex-steroid hormone in P. fulvidraco. For the first time, our study cloned cDNA sequences of ar, sf-1 and star in P. fulvidraco, and demonstrated the effects and mechanism of waterborne Cu exposure influencing hormones secretion and synthesis in dose- and time-dependent manner in P. fulvidraco, which will help to understand the Cu-induced reproductive toxicity at both protein and transcriptional levels in fish.