Norepinephrine Neurons in the Nucleus of the Solitary Tract Suppress Luteinizing Hormone Secretion in Female Mice.

Stress impairs fertility, at least in part, via inhibition of gonadotropin secretion. Luteinizing hormone (LH) is an important gonadotropin that is released in a pulsatile pattern in males and in females throughout the majority of the ovarian cycle. Several models of stress, including acute metabolic stress, suppress LH pulses via inhibition of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy cells) which form the pulse generator. The mechanism for inhibition of KNDy neurons during stress, however, remains a significant outstanding question. Here, we investigated a population of catecholamine neurons in the nucleus of the solitary tract (NTS), marked by expression of the enzyme dopamine beta-hydroxylase (DBH), in female mice. First, we found that a subpopulation of DBH neurons in the NTS is activated (express c-Fos) during metabolic stress. Then, using chemogenetics, we determined that activation of these cells is sufficient to suppress LH pulses, augment corticosterone secretion, and induce sickness-like behavior. In subsequent studies, we identified evidence for suppression of KNDy cells (rather than downstream signaling pathways) and determined that the suppression of LH pulses was not dependent on the acute rise in glucocorticoids. Together these data support the hypothesis that DBH cells in the NTS are important for regulation of neuroendocrine and behavioral responses to stress.

Selective depletion of kisspeptin neurons in the hypothalamic arcuate nucleus in early juvenile life reduces pubertal LH secretion and delays puberty onset in mice.

Puberty is the critical developmental transition to reproductive capability driven by the activation of gonadotropin-releasing hormone (GnRH) neurons. The complex neural mechanisms underlying pubertal activation of GnRH secretion still remain unknown, yet likely include kisspeptin neurons. However, kisspeptin neurons reside in several hypothalamic areas and the specific kisspeptin population timing pubertal onset remains undetermined. To investigate this, we strategically capitalized on the differential ontological expression of the Kiss1 gene in different hypothalamic nuclei to selectively ablate just arcuate kisspeptin neurons (aka KNDy neurons) during the early juvenile period, well before puberty, while sparing RP3V kisspeptin neurons. Both male and female transgenic mice with a majority of their KNDy neurons ablated (KNDyABL) by diphtheria toxin treatment in juvenile life demonstrated significantly delayed puberty onset and lower peripubertal LH secretion than controls. In adulthood, KNDyABL mice demonstrated normal in vivo LH pulse frequency with lower basal and peak LH levels, suggesting that only a small subset of KNDy neurons is sufficient for normal GnRH pulse timing but more KNDy cells are needed to secrete normal LH concentrations. Unlike prior KNDy ablation studies in rats, there was no alteration in the occurrence or magnitude of estradiol-induced LH surges in KNDyABL female mice, indicating that a complete KNDy neuronal population is not essential for normal LH surge generation. This study teases apart the contributions of different kisspeptin neural populations to the control of puberty onset, demonstrating that a majority of KNDy neurons in the arcuate nucleus are necessary for the proper timing of puberty in both sexes.

An i-motif-regulated enhancer, eRNA and adjacent lncRNA affect Lhb expression through distinct mechanisms in a sex-specific context.

Genome-wide studies have demonstrated regulatory roles for diverse non-coding elements, but their precise and interrelated functions have often remained enigmatic. Addressing the need for mechanistic insight, we studied their roles in expression of Lhb which encodes the pituitary gonadotropic hormone that controls reproduction. We identified a bi-directional enhancer in gonadotrope-specific open chromatin, whose functional eRNA (eRNA2) supports permissive chromatin at the Lhb locus. The central untranscribed region of the enhancer contains an iMotif (iM), and is bound by Hmgb2 which stabilizes the iM and directs transcription specifically towards the functional eRNA2. A distinct downstream lncRNA, associated with an inducible G-quadruplex (G4) and iM, also facilitates Lhb expression, following its splicing in situ. GnRH activates Lhb transcription and increased levels of all three RNAs, eRNA2 showing the highest response, while estradiol, which inhibits Lhb, repressed levels of eRNA2 and the lncRNA. The levels of these regulatory RNAs and Lhb mRNA correlate highly in female mice, though strikingly not in males, suggesting a female-specific function. Our findings, which shed new light on the workings of non-coding elements and non-canonical DNA structures, reveal novel mechanisms regulating transcription which have implications not only in the central control of reproduction but also for other inducible genes.

In vivo imaging of the GnRH pulse generator reveals a temporal order of neuronal activation and synchronization during each pulse.

A hypothalamic pulse generator located in the arcuate nucleus controls episodic release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) and is essential for reproduction. Recent evidence suggests this generator is composed of arcuate "KNDy" cells, the abbreviation based on coexpression of kisspeptin, neurokinin B, and dynorphin. However, direct visual evidence of KNDy neuron activity at a single-cell level during a pulse is lacking. Here, we use in vivo calcium imaging in freely moving female mice to show that individual KNDy neurons are synchronously activated in an episodic manner, and these synchronized episodes always precede LH pulses. Furthermore, synchronization among KNDy cells occurs in a temporal order, with some subsets of KNDy cells serving as "leaders" and others as "followers" during each synchronized episode. These results reveal an unsuspected temporal organization of activation and synchronization within the GnRH pulse generator, suggesting that different subsets of KNDy neurons are activated at pulse onset than afterward during maintenance and eventual termination of each pulse. Further studies to distinguish KNDy "leader" from "follower" cells is likely to have important clinical significance, since regulation of pulsatile GnRH secretion is essential for normal reproduction and disrupted in pathological conditions such as polycystic ovary syndrome and hypothalamic amenorrhea.

The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep†.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase†.

In response to luteinizing hormone (LH), multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-min exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the phosphoprotein phosphatase (PPP) family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles.

Luteinizing hormone stimulates ingression of mural granulosa cells within the mouse preovulatory follicle†.

Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 min of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10 h after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4 h after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation.

Development of KISS1 knockout pigs is characterized by hypogonadotropic hypogonadism, normal growth, and reduced skatole.

Kisspeptin is a major regulator of gonadotropin secretion in pigs. Previously, CRISPR/Cas9 knockout of KISS1 was used to develop a mosaic parental line of pigs to generate offspring that would not need castration due to loss of kisspeptin. The current goal was to characterize growth and reproductive development of F1 pigs from this parental line. Body weights, gonadotropin concentrations and gonadal development were measured from birth through development (boars to 220 d of age, n = 42; gilts to 160 d of age, n = 36). Testosterone, skatole, and androstenone were also measured in boars. Blood samples were collected by jugular venipuncture for quantification of serum hormones, gonadal tissues collected for gross morphology and histology, and a fat biopsy collected (boars) for skatole and androstenone analysis. Body weight did not differ with genotype. There were no differences between KISS1+/+ and heterozygote KISS1+/- animals for most parameters measured. Gonadotropin concentrations were reduced in KISS1-/- boars and gilts compared with KISS1+/+ and KISS1+/- animals (P < 0.05). Concentrations of testosterone in serum and both androstenone and skatole in adipose were less in KISS1-/- boars than in KISS1+/+ and KISS1+/- boars (P < 0.05). Hypogonadism was in all KISS1-/- gilts and boars. These data indicate that knocking out KISS1 causes hypogonadotropic hypogonadism but does not negatively affect growth in pigs. Only one KISS1 allele is needed for normal gonadotropin secretion and gonadal development, and accumulation of compounds in adipose leading to boar taint.

The effects of short-term and long-term phthalate exposures on ovarian follicle growth dynamics and hormone levels in female mice†.

Di(2-ethylhexyl) phthalate and diisononyl phthalate are widely used as plasticizers in polyvinyl chloride products. Short-term exposures to phthalates affect hormone levels, ovarian follicle populations, and ovarian gene expression. However, limited data exist regarding the effects of long-term exposure to phthalates on reproductive functions. Thus, this study tested the hypothesis that short-term and long-term exposure to di(2-ethylhexyl) phthalate or diisononyl phthalate disrupts follicle dynamics, ovarian and pituitary gene expression, and hormone levels in female mice. Adult CD-1 female mice were exposed to vehicle, di(2-ethylhexyl) phthalate, or diisononyl phthalate (0.15 ppm, 1.5 ppm, or 1500 ppm) via the chow for 1 or 6 months. Short-term exposure to di(2-ethylhexyl) phthalate (0.15 ppm) and diisononyl phthalate (1.5 ppm) decreased serum follicle-stimulating hormone levels compared to control. Long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate (1500 ppm) increased the percentage of primordial follicles and decreased the percentages of preantral and antral follicles compared to control. Both phthalates increased follicle-stimulating hormone levels (di(2-ethylhexyl) phthalate at 1500 ppm; diisononyl phthalate at 1.5 ppm) and decreased luteinizing hormone levels (di(2-ethylhexyl) phthalate at 0.15 and 1.5 ppm; diisononyl phthalate at 1.5 ppm and 1500 ppm) compared to control. Furthermore, both phthalates altered the expression of pituitary gonadotropin subunit genes (Cga, Fshb, and Lhb) and a transcription factor (Nr5a1) that regulates gonadotropin synthesis. These data indicate that long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate alters follicle growth dynamics in the ovary and the expression of gonadotropin subunit genes in the pituitary and consequently luteinizing hormone and follicle-stimulating hormone synthesis.

Chronic estradiol exposure suppresses luteinizing hormone surge without affecting kisspeptin neurons and estrogen receptor alpha in anteroventral periventricular nucleus†.

Mammalian ovulation is induced by a luteinizing hormone surge, which is triggered by elevated plasma estrogen levels; however, chronic exposure to high levels of estradiol is known to inhibit luteinizing hormone secretion. In the present study, we hypothesized that the inhibition of the luteinizing hormone surge by chronic estradiol exposure is due to the downregulation of the estrogen receptor alpha in kisspeptin neurons at hypothalamic anteroventral periventricular nucleus, which is known as the gonadotropin-releasing hormone/luteinizing hormone surge generator. Animals exposed to estradiol for 2 days showed an luteinizing hormone surge, whereas those exposed for 14 days showed a significant suppression of luteinizing hormone. Chronic estradiol exposure did not affect the number of kisspeptin neurons and the percentage of kisspeptin neurons with estrogen receptor alpha or c-Fos in anteroventral periventricular nucleus, but it did affect the number of kisspeptin neurons in arcuate nucleus. Furthermore, chronic estradiol exposure did not affect gonadotropin-releasing hormone neurons. In the pituitary, 14-day estradiol exposure significantly reduced the expression of Lhb mRNA and LHß-immunoreactive areas. Gonadotropin-releasing hormone-induced luteinizing hormone release was also reduced significantly by 14-day estradiol exposure. We revealed that the suppression of an luteinizing hormone surge by chronic estradiol exposure was induced in association with the significant reduction in kisspeptin neurons in arcuate nucleus, luteinizing hormone expression in the pituitary, and pituitary responsiveness to gonadotropin-releasing hormone, and this was not caused by changes in the estrogen receptor alpha-expressing kisspeptin neurons in anteroventral periventricular nucleus and gonadotropin-releasing hormone neurons, which are responsible for estradiol positive feedback.

Quantification of urinary gonadotropins by specific assays may improve the evaluation of sex-specific hormonal changes in early infancy.

OBJECTIVE: The aim of this study was to investigate the feasibility of different gonadotropin assays for determining total and intact luteinizing hormone (LH), and follicle-stimulating hormone (FSH) immunoreactivity in urine (U-LH-ir and U-FSH-ir, respectively) during early infancy. DESIGN, PATIENTS AND MEASUREMENTS: Morning urine samples were obtained from 31 infants, aged between 0 and 6 months, to study the age-related course of urinary gonadotropins. Additionally, we investigated bi-hourly urine samples of a 5-day-old male neonate for 24 h to observe the course of urinary gonadotropins during a daily cycle. We employed different immunofluorometric assays for measuring total and intact U-LH-ir, and U-FSH-ir. RESULTS: In neonates up to 21 days of age, the U-LH-ir levels measured by the regular LH assay (also detecting hCG) were significantly higher than those determined by the total (specific) LH assays (p = .004). U-FSH-ir was higher in girls than boys during both the first and the next 5 months (p = .02 and p < .001, respectively), whereas total U-LH-ir was higher in boys until 6 months of age (p < .001). Total U-LH-ir/U-FSH-ir ratio was significantly higher in boys than girls across the first half-year (p < .001). CONCLUSIONS: The assessment of total U-LH-ir and U-FSH-ir, and their respective ratio constitutes a noninvasive, practical and scalable tool to investigate sex-specific changes during early infancy, with the ratio being significantly higher in boys than girls. Only highly specific LH assays detecting beta-subunit and its core fragment in addition to intact LH should be used for determining U-LH-ir in the neonatal period to avoid potential cross-reactivity with hCG of placental origin.

The relationship between male and female endogenous reproductive hormones levels and subjective cognitive decline score: A cross-sectional analysis of the Pingyin cohort study.

OBJECTIVE: Reproductive hormones might impact disease course in cognitive decline. We examined the association between male and female endogenous reproductive hormones and subjective cognitive decline (SCD) score. DESIGN, PATIENTS AND MEASUREMENTS: A cross-sectional study design was used with baseline data from the Pingyin cohort study, involving 1943 participants aged 45-70 years. Oestrogen (E2), testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured in females and E2 and testosterone were measured in males. We categorised hormones into three levels of low, intermediate and high level. The 9-item subjective cognitive decline questionnaire (SCD-Q9) scores were collected to assess the symptoms of SCD. Multivariable logistic regression models were used to estimate odds ratios (ORs) and 95% confidence interval (CI) between categorised hormone levels and SCD status. Multivariable linear regression models were also used. RESULTS: Overall, 1943 participants were involved and 1285 (66.1%) were female. The mean age at baseline was 59.1 (standard deviation 7.1) years. Women with high testosterone levels had a higher probability of having SCD compared with those with low testosterone levels (OR 1.43, 95% CI 1.01-2.05). Men with a high level of testosterone (0.59, 0.35-0.98) and high testosterone/E2 ratio (0.55, 0.33-0.90) were related to decreased chances of having SCD. Each one-unit increase of testosterone was linked to reduced SCD score in males [(ß: -.029, 95% CI (-0.052, -0.007)]. CONCLUSION: There was sex-specific relationship between hormone levels and SCD abnormal. Those with higher testosterone levels in females may increase likelihood of experiencing SCD. Males with higher testosterone levels and higher testosterone/E2 ratio may be associated with reduced likelihood of SCD. The roles of endogenous reproductive hormone levels and their dynamic changes in cognitive function need further investigation.

Regulation of corticosteroid-binding globulin release in murine leydig tumor cell line mLTC-1 by luteinizing hormone and interleukin-6.

Exogenous assaults interfere with homeostatic processes in the body by inducing stress responses. Corticosteroid-binding globulin (CBG) binds to stress hormone glucocorticoids to transport and dynamically control their availability to target tissues. In our previous study, we confirmed that CBG is locally produced by Leydig cells in the testes. Here, we explored the potential regulators of CBG using a murine Leydig tumor cell line (mLTC-1). Results indicated that luteinizing hormone (LH) and interleukin-6 (IL-6) were important factors stimulating the release of CBG from mLTC-1 cells. In addition, IL-6 stimulated mLTC-1 cells to release alpha-1 antitrypsin (AAT), a serine proteinase inhibitor (serpin) that affects CBG conformation. The results implied that any challenge that altered LH or IL-6 levels also changed the release and binding status of CBG with steroid hormones in the testicular microenvironment and modulated cellular responses to these stress hormones. In addition, secretory proteomic analysis indicated that the extracellular matrix (ECM), cytoskeleton, and proteasomes were essentially produced by the mLTC-1 cells, and LH evoked the secretion of proteins involved in binding and metabolism. These results emphasize that Leydig cells may undertake more functions than just steroidogenesis, and the regulation of Leydig cells by LH is versatile.

Effect of LH level on HCG trigger day on clinical outcomes in patients with diminished ovarian reserve undergoing GnRH-antagonist protocol.

RESEARCH QUESTION: Does luteinizing hormone (LH) levels on human chorionic gonadotropin (HCG) trigger day (LHHCG) affect the clinical outcomes of patients with diminished ovarian reserve (DOR) undergoing gonadotropin-releasing hormone antagonist (GnRH-ant) protocol? METHODS: Retrospective analysis fresh embryo transfer cycles of DOR patients who underwent GnRH-ant protocol from August 2019 to June 2023. The participants were divided into different groups according to LHHCG level and age. The clinical data and outcomes were compared between groups. RESULTS: In patients with DOR, the HCG positive rate (59.3% versus 39.8%, P = 0.005), embryo implantation rate (34.5% versus 19.7%, P = 0.002), clinical pregnancy rate (49.2% versus 28.4%, P = 0.003), live birth rate (41.5% versus 22.7%, P = 0.005) in LHHCG < 2.58 IU/L group were significantly higher than LHHCG ≥ 2.58 IU/L group. There was no significant correlation between LHHCG level and clinical pregnancy in POSEIDON group 3. In POSEIDON group 4, the HCG positive rate (52.8% versus 27.0%, P = 0.015), embryo implantation rate (29.2% versus 13.3%, P = 0.023), clinical pregnancy rate (45.3% versus 18.9%, P = 0.010) in LHHCG < 3.14 IU/L group were significantly higher than LHHCG ≥ 3.14 IU/L group. Logistic regression analysis indicated that LHHCG level was an independent influencing factor for clinical pregnancy in POSEIDON group 4 patients (OR = 3.831, 95% CI: 1.379-10.643, P < 0.05). CONCLUSIONS: LHHCG level is an independent factor affecting pregnancy outcome of fresh embryo transfer in DOR patients undergoing GnRH-ant protocol, especially for advanced-aged women. LHHCG had a high predictive value for POSEIDON group 4 patients, and LHHCG ≥ 3.14 IU/L predicts poor pregnancy outcomes.

Development of a reporter gene-based assay for the bioactivity determination of rhLH pharmaceutical products.

To establish an in vitro biological activity detection method for luteinizing hormone (LH), the hLHCGR-CREB-HEK293 cell line was constructed to stably express human luteinizing hormone/chorionic gonadotropin receptor (hLHCGR). After optimization, the rhLH starting working concentration was 800 mIU/mL with 4-fold serial dilutions, 10 concentrations and an incubation time of 5 h. The method was confirmed to be highly specific, with good accuracy, precision and linearity, meeting the needs of process research and release testing, and can be used as a routine detection method for LH biological activity. With the increasing demand for research and development of rhLH biologically similar drugs, establishing a stable and simple activity assay method to evaluate the biological activity of rhLH can provide technical support for quality control of rhLH products and powerful tools for comparability research of similar products.

PFAS Exposure is Associated with a Lower Spermatic Quality in an Arctic Seabird.

Several studies have reported an increasing occurrence of poly- and perfluorinated alkyl substances (PFASs) in Arctic wildlife tissues, raising concerns due to their resistance to degradation. While some research has explored PFAS's physiological effects on birds, their impact on reproductive functions, particularly sperm quality, remains underexplored. This study aims to assess (1) potential association between PFAS concentrations in blood and sperm quality in black-legged kittiwakes (Rissa tridactyla), focusing on the percentage of abnormal spermatozoa, sperm velocity, percentage of sperm motility, and morphology; and (2) examine the association of plasma levels of testosterone, corticosterone, and luteinizing hormone with both PFAS concentrations and sperm quality parameters to assess possible endocrine disrupting pathways. Our findings reveal a positive correlation between the concentration of longer-chain perfluoroalkyl carboxylates (PFCA; C11-C14) in blood and the percentage of abnormal sperm in kittiwakes. Additionally, we observed that two other PFAS (i.e., PFOSlin and PFNA), distinct from those associated with sperm abnormalities, were positively correlated with the stress hormone corticosterone. These findings emphasize the potentially harmful substance-specific effects of long-chain PFCAs on seabirds and the need for further research into the impact of pollutants on sperm quality as a potential additional detrimental effect on birds.

Pubertal luteinizing hormone levels in children with chronic kidney disease and association with change in glomerular filtration rate.

BACKGROUND: Children with chronic kidney disease (CKD) are at risk for abnormalities in pubertal development. We aimed to describe the timing of pubertal onset by luteinizing hormone (LH) levels and the association between hormonal onset of puberty with changes in GFR. METHODS: Data from the Chronic Kidney Disease in Children (CKiD) study were collected prospectively. GFR was estimated at annual visits and measured by iohexol clearance every other year. LH was measured from stored repository serum samples in a nested sample of 124 participants. Hormonal onset of puberty was defined as LH level greater than or equal to 0.3 IU/L. A mixed effects model with random intercepts and slopes was used to compare the slope of decline of GFR before and after hormonal onset of puberty. The model was adjusted for age, glomerular disease diagnosis, baseline proteinuria on the log scale, and BMI. RESULTS: Median age at hormonal onset of puberty was 9.9 years (IQR 8.1, 11.9) in girls and 10.2 years (IQR 9.2, 11.0) in boys. The mixed effects model showed faster decline in both estimated GFR and measured GFR in boys after hormonal onset of puberty (p < 0.001), and a similar but attenuated accelerated estimated GFR decline was observed for girls with no difference for measured GFR. CONCLUSIONS: LH levels in the post-pubertal range were observed prior to clinical manifestations of puberty in children with CKD. Hormonal onset of puberty was associated with faster decline in GFR, particularly among boys with CKD.

Functional characterization of follicle-stimulating hormone and luteinizing hormone receptors in cloudy catshark, Scyliorhinus torazame.

The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for characterization. Ventral lobe extract (VLE) from the pituitary contains homologous FSH and LH, and it stimulated the cAMP signaling of FSHR and LHR dose-dependently. Two transcript variants of LHR (LHR-L with exon 10 and LHR-S without) were identified, and LHR-S was the dominant form with higher basal cAMP activity without VLE stimulation. Among various developmental stages of follicles, FSHR expression was mainly associated with the pre-vitellogenic and early white follicles. When follicles were recruited into vitellogenesis, the expression of FSHR decreased while of LHR was upregulated reciprocally, suggesting that LHR may also be responsible for the control of vitellogenesis in chondrichthyans. The expression of LHR-L was upregulated among maturing follicles before ovulation, indicating LHR-L could have a specific role in receiving the LH surge signal for final maturation. Plasma LH-like activity was transiently increased prior to the progesterone (P4)-surge and testosterone-drop at the beginning of P4-phase, supporting a pituitary control of follicle-maturation via LH signaling in chondrichthyans. The expression of follicular LHR was downregulated during the P4-phase when LH-like activity was high, indicating that the LH-dependent downregulation of LHR is conserved in chondrichthyans as it is in other vertebrate lineages. (213 words).

Molecular characterization and stage-dependent gene expression of gonadotropin receptors in Pacific bluefin tuna, Thunnus orientalis, ovarian follicles.

To understand the physiological mechanisms by which pituitary-derived gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) regulate asynchronous oocyte development, we investigated the function and expression of Fsh and Lh receptors (Fshr and Lhr, respectively) in Pacific bluefin tuna (PBT, Thunnus orientalis). As a first, we cloned the full-length cDNAs encoding PBT Fshr and Lhr. Recombinant PBT Fsh and Lh single-chain proteins were produced in abundance using stable CHO-DG44 cell lines and were subsequently purified from the culture medium, culminating in their yields being 87.0 and 88.2%, respectively. An in vitro reporter assay using homologous recombinant Gths revealed that PBT Fshr and Lhr responded strongly to their corresponding ligands in a dose-dependent manner, with no cross-activation over a wide range of concentrations. Moreover, quantitative expression analysis of Fshr and Lhr at the follicle level showed that fshr gene expression was highly upregulated in the ovarian follicles through vitellogenesis, while lhr expression was significantly upregulated and peaked in fully vitellogenic ovarian follicles. These findings suggest that asynchronous-type oocyte development is primarily attributed to the differential function and expression of Gthrs, rather than the ligand, in PBT.

Characterization of gonadotropins and their receptors in a chondrichthyan, Scyliorhinus canicula, fills a gap in the understanding of their coevolution.

In Gnathostomes, reproduction is mainly controlled by the hypothalamic-pituitary-gonadal (HPG) axis, with the involvement of the pituitary gonadotropic hormones (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which activate their cognate receptors, FSHR and LHR, expressed in gonads. Each GTH consists of a common α subunit and of a specific FSHß or LHß subunit. Chondrichthyes (holocephalans and elasmobranchs) is a sister group of bony vertebrates. This position is highly favorable for the understanding of the evolution of endocrine regulations of reproduction among gnathostomes. Surprisingly, the characterization of gonadotropins and their receptors is still limited in chondrichthyes. In the present study, GTH and GTHR sequences have been identified from several chondrichthyan genomes, and their primary structures were analyzed relative to human orthologs. 3D models of GTH/GTHR interaction were built, highlighting the importance of the receptor hinge region for ligand recognition. Functional hormone-receptor interactions have been studied in HEK cells using the small-spotted catshark (Scyliorhinus canicula) recombinant proteins and showed that LHR was specifically activated by LH whereas FSHR was activated by both FSH and LH. Expression profiles of GTHs and their receptors were explored by real-time PCR, in situ hybridization and immunohistochemistry during spermatogenesis, along the male genital tract and other tissues, as well as in some female tissues for comparison. Tissue-expression analyses showed that the highest levels were observed for fshr transcripts in testis and ovary and for lhr in specific extragonadal tissues. The two receptors were expressed at all stages of spermatogenesis by both germ cells and somatic cells, including undifferentiated spermatogonia, spermatocytes, spermatids, somatic precursors and Sertoli cells; differentiated Leydig cells being absent in the testis of S. canicula. Receptors were also expressed by the lymphomyeloid epigonal tissue and the testicular tubules. These results, suggest a wide range of gonadotropin-regulated functions in Elasmobranchs, as well as functional redundancy during spermatogenesis. These extended functions are discussed in an evolutionary context in which the specificity of gonadotropin signaling must have contributed to the evolution of gonadal cells' morphology and function.