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.

Effect Modification of LHCGR Gene Variant (rs2293275) on Clinico-Biochemical Profile, and Levels of Luteinizing Hormone in Polycystic Ovary Syndrome Patients.

 Polycystic ovary syndrome (PCOS) is a common multifaceted endocrine disorder among reproductive-aged women. Deranged luteinizing hormone levels and associated downstream signaling cascade mediated by its receptor luteinizing hormone chorionic gonadotropin receptor (LHCGR) are pivotal in the etiopathogenesis of PCOS. Genetic variations in the LHCGR have been associated with PCOS risk. However, the results are mixed and inconclusive. We evaluated the association of the LHCGR rs2293275 polymorphic variant with PCOS risk and its association with clinico-biochemical features of PCOS. 120 confirmed PCOS cases and an equal number of age-matched controls were subjected to clinical, biochemical, and hormonal investigations. Genotyping for rs2293275 was performed using polymerase chain reaction-restriction fragment length polymorphism. Logistic regression models were used to calculate odds ratios (ORs) at 95% confidence intervals (95% CIs). In the current study, PCOS cases reported a lower number of menstrual cycles per year than respective controls. A significantly higher BMI, Ferriman Galway score, levels of serum testosterone, insulin, TSH, FSH, and fasting glucose were observed in cases than in controls (p < 0.01). Compared to GG carriers, we observed a higher risk of developing PCOS in the subjects who harbored GA (OR 10.4, p < 0.0001) or AA (OR 7.73, p = 0.02) genotype. The risk persisted in the dominant model (GA + AA) as well (OR 10.29, p = 0.01). On stratification, a higher risk of developing PCOS was observed in variant genotype carriers who had a family history of either type two diabetes mellitus (OR 117; p < 0.0001) or hirsutism (OR 79; p < 0.0001). We also found significantly elevated levels of serum LH levels in the subject harboring GA and AA genotypes when compared to GG carriers. In the present study, we report a significant association of the LHCGR rs2293275 variant with the PCOS risk.

Active kisspeptin DNA vaccines oral immunization disrupt mRNA hormone receptors expression in ram lambs.

To evaluate the efficacy of oral immunization with active kisspeptin DNA vaccine on the expression of hormone receptor mRNA. For this study, ten 56-day-old Hu breed ram lambs were randomly assigned to the treatment and control groups (n = 5). Treatment Experimental group received C500/pKS-asd and the control group received C500/pVAX-asd (aspartate-ß semialdehyde dehydrogenase orally on days 0, 28, and 56, and blood samples were taken at each immunization interval (14-day) and tissues samples were collected at the end of the experimental period (day 98). The collected samples were stored in the refrigerator at -20 °C and liquid nitrogen, respectively, for laboratory examination. Total RNA was extracted from samples using TRIzol reagent and quantitative real-time polymerase chain reaction (QPCR) was used to quantify the levels of KISS1, G protein-coupled receptor-54 (Kiss1r), and gonadotrophin-releasing hormone (GnRH) mRNA in the hypothalamus. Levels of luteinizing hormone receptor (LHR) and luteinizing hormone beta (LHß) mRNA, and follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone beta (FSHß) mRNA in the testes and pituitary were analyzed, respectively. Further, gonadotropin-releasing hormone receptor (GnRHR) mRNA expression level in the pituitary was measured. Moreover, the Kiss1r concentration level in the blood was measured using an indirect ELISA. The concentration of Kiss1r in the blood was lower in the treatment group than in the control group (p < 0.05). The levels of testicular FSHR and LHR mRNA were significantly lower in the treatment group (p < 0.05) when compared to the control group. Furthermore, the treatment group's levels of hypothalamic KISS1, Kiss1r, and GnRH mRNA were significantly lower (p < 0.05) than the controls. LH, FSH, and GnRHR mRNA expression in the pituitary were also significantly lower in the treatment group (p < 0.01 and p < 0.05, respectively). These findings imply that oral immunization with active kisspeptin DNA vaccine suppresses hormone receptor mRNA expression in the ram lambs.

Luteinizing hormone signaling restricts hematopoietic stem cell expansion during puberty.

The number and self-renewal capacity of hematopoietic stem cells (HSCs) are tightly regulated at different developmental stages. Many pathways have been implicated in regulating HSC development in cell autonomous manners; however, it remains unclear how HSCs sense and integrate developmental cues. In this study, we identified an extrinsic mechanism by which HSC number and functions are regulated during mouse puberty. We found that the HSC number in postnatal bone marrow reached homeostasis at 4 weeks after birth. Luteinizing hormone, but not downstream sex hormones, was involved in regulating HSC homeostasis during this period. Expression of luteinizing hormone receptor (Lhcgr) is highly restricted in HSCs and multipotent progenitor cells in the hematopoietic hierarchy. When Lhcgr was deleted, HSCs continued to expand even after 4 weeks after birth, leading to abnormally elevated hematopoiesis and leukocytosis. In a murine acute myeloid leukemia model, leukemia development was significantly accelerated upon Lhcgr deletion. Together, our work reveals an extrinsic counting mechanism that restricts HSC expansion during development and is physiologically important for maintaining normal hematopoiesis and inhibiting leukemogenesis.

The predictive factors of successful sperm retrieval for men with Y chromosome AZFc microdeletion.

PURPOSE: To identify key predictors for successful sperm retrieval in men with AZFc microdeletion. METHODS: Totally, 71 infertile men with confirmed AZFc microdeletion were studied. For each patient, the endocrine profile including serum follicle stimulating hormone (FSH), luteinizing hormone, total testosterone, prolactin, and estradiol was recorded, along with intratesticular testosterone levels (ITT), age, and testicular size. The factors were further analyzed to determine the key predictors for successful sperm retrieval. RESULTS: Of the 71 men with AZFc microdeletion, 52 (73.2%) were classified as having non-obstructive azoospermia (NOA), 7 (9.9%) as having cryptozoospermia, and 12 (15.8%) as having severe oligoasthenoteratozoospermia. Of the 52 men with azoospermia, 47 received microdissection testicular sperm retrieval, and sperm retrieval was successful in 35 of those cases (74.5%). A significantly lower serum FSH (p = 0.03) was found in those patients from whom sperm could be successfully retrieved. The area under the receiving operating characteristic curve for FSH was determined to be 0.721. Using an FSH cutoff point of 12.95 mIU/mL, the model for predicting successful sperm retrieval was found to have 51.4% sensitivity, 83.3% specificity, 90.0% positive predictive value, and 37.0% negative predictive value. ITT levels were obtained from 7 NOA patients, the mean ITT and the mean ITT/serum testosterone ratio was 1932.8 ng/ml and 567.2 in 6 men with successful sperm retrieval, whereas, in a patient with fail sperm retrieval, the levels were 2370 ng/ml and 393.0. CONCLUSION: Men exhibiting AZFc microdeletion with discernible spermatogenesis from whom sperm was successfully retrieved by mTESE generally presented with relatively lower FSH levels.

In vitro action of leptin on gonadotropin secretion in pre-pubertal male chub mackerel.

Leptin directly influences gonadotropin (GTH) secretion from female pituitary cells in vitro and is a key signal at the onset of puberty in female chub mackerel (Scomber japonicus). Here, we investigated whether leptin also influences GTH secretion in male chub mackerel. The addition of 1 nM homologous recombinant leptin to pre-pubertal male pituitary cells stimulated follicle-stimulating hormone secretion after 1 and 2 h of culture. Therefore, leptin signaling could also directly facilitate GTH secretion in male chub mackerel.

Elevated ovarian pentraxin 3 in polycystic ovary syndrome.

PURPOSE: Pentraxin 3 (PTX3) plays a crucial role in cumulus expansion and fertilization. The ovarian PTX3 level in polycystic ovary syndrome (PCOS) remains uncertain. In the present study, we investigated the follicular PTX3 levels and found the influence of reproductive hormones on ovarian PTX3 concentration. METHODS: This study was based on 204 healthy-weight women (102 PCOS and 102 normal ovulating subjects) undergoing in vitro fertilization (IVF). Follicular fluid (FF) was collected during oocyte retrieval. The PTX3 levels and other hormone levels in FF samples were analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The PTX3 level in the follicle was significantly higher in the healthy-weight PCOS women than controls. Positive correlations were found between ovarian PTX3 level and the existence of PCOS, cycle length, basal LH to FSH ratio and TT in serum, antral follicle count, and ovarian insulin and androgen level, and inverse correlations with the basal serum PRL and ovarian SHBG. In multivariant linear regression analysis, the presence of PCOS diagnosis, participants' basal LH to FSH ratio, and ovarian androstenedione level were the main predictors of ovarian PTX3 level among the enrolled subjects. CONCLUSION: Elevated ovarian PTX3 level supports the low-grade chronic inflammatory state in the follicles of PCOS. The existence of PCOS, disturbed pituitary gland, and ovarian hyperandrogenism might also be related to this state of low-grade chronic inflammation and could be a subject of further study.

The impact of timing modified natural cycle frozen embryo transfer based on spontaneous luteinizing hormone surge.

PURPOSE: To evaluate whether adjusting timing of modified natural cycle frozen embryo transfer (mNC-FET) 1 day earlier in the setting of a spontaneous LH surge has an impact on pregnancy outcomes. METHODS: This retrospective cohort study evaluated all mNC-FET with euploid blastocysts from May 1, 2016 to March 30, 2019, at a single academic institution. Standard protocol for mNC-FET included ultrasound monitoring and hCG trigger when the dominant follicle and endometrial lining were appropriately developed. Patients had serum LH, estradiol, and progesterone checked on day of trigger. If LH was ≥ 20 mIU/mL, trigger was given that day and FET was performed 6 days after surge (LH/HCG+6), with the intent of transferring 5 days after ovulation. If LH was < 20 mIU/mL, FET was performed 7 days after trigger (hCG+7). Primary outcomes included clinical pregnancy and live birth rates. To account for correlation between cycles, a generalized estimating equation (GEE) method for multivariable logistic regression was used. RESULTS: Four hundred fifty-three mNC-FET cycles met inclusion criteria, of which 205 were in the LH/HCG+6 group and 248 were in the HCG+7 group. The overall clinical pregnancy rate was 64% and clinical miscarriage rate was 4.8%, with similar rates between the two groups. The overall live birth rate was 60.9% (61.0% in LH/HCG+6 group and 60.9% in HCG+7 group). After implementing GEE, the odds of CP (aOR 0.97, 95% CI [0.65-1.45], p = 0.88) and LB (aOR 0.98, 95% CI [0.67-1.45], p = 0.93) were similar in both groups. CONCLUSIONS: In our study cohort, mNC-FET based on LH/HCG+6 versus HCG+7 had similar pregnancy outcomes.

Genetic Regulation of Physiological Reproductive Lifespan and Female Fertility.

There is substantial genetic variation for common traits associated with reproductive lifespan and for common diseases influencing female fertility. Progress in high-throughput sequencing and genome-wide association studies (GWAS) have transformed our understanding of common genetic risk factors for complex traits and diseases influencing reproductive lifespan and fertility. The data emerging from GWAS demonstrate the utility of genetics to explain epidemiological observations, revealing shared biological pathways linking puberty timing, fertility, reproductive ageing and health outcomes. The observations also identify unique genetic risk factors specific to different reproductive diseases impacting on female fertility. Sequencing in patients with primary ovarian insufficiency (POI) have identified mutations in a large number of genes while GWAS have revealed shared genetic risk factors for POI and ovarian ageing. Studies on age at menopause implicate DNA damage/repair genes with implications for follicle health and ageing. In addition to the discovery of individual genes and pathways, the increasingly powerful studies on common genetic risk factors help interpret the underlying relationships and direction of causation in the regulation of reproductive lifespan, fertility and related traits.

Differential Regulation of Gonadotropins as Revealed by Transcriptomes of Distinct LH and FSH Cells of Fish Pituitary.

From mammals to fish, reproduction is driven by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) temporally secreted from the pituitary gland. Teleost fish are an excellent model for addressing the unique regulation and function of each gonadotropin cell since, unlike mammals, they synthesize and secrete LH and FSH from distinct cells. Only very distant vertebrate classes (such as fish and birds) demonstrate the mono-hormonal strategy, suggesting a potential convergent evolution. Cell-specific transcriptome analysis of double-labeled transgenic tilapia expressing GFP and RFP in LH or FSH cells, respectively, yielded genes specifically enriched in each cell type, revealing differences in hormone regulation, receptor expression, cell signaling, and electrical properties. Each cell type expresses a unique GPCR signature that reveals the direct regulation of metabolic and homeostatic hormones. Comparing these novel transcriptomes to that of rat gonadotrophs revealed conserved genes that might specifically contribute to each gonadotropin activity in mammals, suggesting conserved mechanisms controlling the differential regulation of gonadotropins in vertebrates.

Biphasic Roles of Clock Genes and Bone Morphogenetic Proteins in Gonadotropin Expression by Mouse Gonadotrope Cells.

Roles of Clock genes and the bone morphogenetic protein (BMP) system in the regulation of gonadotropin secretion by gonadotropin-releasing hormone (GnRH) were investigated using mouse gonadotropin LßT2 cells. It was found that luteinizing hormone (LH)ß mRNA expression level in LßT2 cells changed gradually over time, with LHß expression being suppressed in the early phase up to 12 h and then elevated in the late phase 24 h after GnRH stimulation. In addition, the mRNA expression levels of Clock genes, including Bmal1, Clock, Per2, and Cry1, also showed temporal changes mimicking the pattern of LHß expression in the presence and absence of GnRH. Notably, the expression levels of Bmal1 and Clock showed strong positive correlations with LHß mRNA expression levels. Moreover, a functional link of the ERK signaling of mitogen-activated protein kinases (MAPKs) in the suppression of LHß mRNA expression, as well as Bmal1 and Clock mRNA expression by GnRH at the early phase, was revealed. Inhibition of Bmal1 and Clock expression using siRNA was involved in the reduction in LHß mRNA levels in the late phase 24 h after GnRH stimulation. Furthermore, in the presence of BMP-6 and -7, late-phase Bmal1 and LHß mRNA expression after GnRH stimulation was significantly attenuated. Collectively, the results indicated that LH expression in gonadotrope cells exhibits Bmal1/Clock-dependent fluctuations under the influence of GnRH and that the fluctuations are regulated by ERK and BMPs in the early and late stages, respectively, in a phase-dependent manner after GnRH stimulation.

Transcriptome characterization of BPG axis and expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine.

BACKGROUND: The clupeoid fishes are ecologically and commercially important fish species worldwide that exhibit a high level of population fluctuation, accompanied by alteration of reproductive traits. However, knowledge about their reproductive physiology in order to understand mechanisms underlying such population dynamics is limited. The endocrine system along with the brain-pituitary-gonadal (BPG) axis is critical for regulating reproduction. The aims of this study were to provide transcript data and genes related to the BPG axis, and to characterize the expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine (Sardinops melanostictus, Clupeidae). RESULTS: RNA sequencing was performed using the sardine brain, pituitary, and gonad in both sexes. A total of 290,119 contigs were obtained and 115,173 non-redundant ORFs were annotated. The genes differentially expressed between ovary and testis were strongly associated with GO terms related to gamete production. The tissue-specific profile of the abundance of transcripts was characterized for the major regulators in the BPG axis, such as gonadotropin-releasing hormone, gonadotropin, and steroidogenic enzyme. By comparing between ovary and testis, out of eight different 17ß-hydroxysteroid dehydrogenase (Hsd17b) genes identified, higher hsd17b7 expression was found in testis, whereas higher expression of hsd17b8, hsd17b10, hsd17b12a, and hsd17b12b was found in ovary. The cDNAs encoding key endocrine factors in the ovarian steroidogenic pathway were cloned, sequenced, and quantitatively assayed. In the pituitary, follicle-stimulating hormone beta peaked during vitellogenesis, while luteinizing hormone beta peaked at the completion of vitellogenesis. In the ovary, follicle-stimulating hormone receptor and luteinizing hormone receptor were upregulated from mid- to late phase of vitellogenesis. Furthermore, three steroidogenic enzyme genes (cyp11a1, cyp17a1, and cyp19a1a) gradually increased their expression during ovarian development, accompanying a rise in serum estradiol-17ß, while 3ß-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein did not change significantly. CONCLUSIONS: This is the first report of deep RNA sequencing analysis of Japanese sardine, in which many key genes involved in the BPG axis were identified. Expression profiles of ovarian steroidogenesis-related genes provide a molecular basis of the physiological processes underlying ovarian development in the sardine. Our study will be a valuable resource for clarifying the molecular biology of clupeoid fishes.

Genome-wide association study of semen volume, sperm concentration, testis size, and plasma inhibin B levels.

Semen quality is affected by environmental factors, endocrine function abnormalities, and genetic factors. A GWAS recently identified ERBB4 at 2q34 as a genetic locus associated with sperm motility. However, GWASs for human semen volume and sperm concentration have not been conducted. In addition, testis size also reportedly correlates with semen quality, and it is important to identify genes that affect testis size. Reproductive hormones also play an important role in spermatogenesis. To date, genetic loci associated with plasma testosterone, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels have been identified using GWASs. However, GWASs have not identified any relevant loci for plasma inhibin B levels. We conducted a two-stage GWAS using 811 Japanese men in a discovery stage followed by a replication stage using an additional 721 Japanese men. The results of the discovery and replication stages were combined into a meta-analysis. After setting a suggestive significance threshold for P values < 5 × 10-6　in the discovery stage, we identified ten regions with SNPs (semen volume: one, sperm concentration: three, testes size: two, and inhibin B: four). We selected only the most significant SNP in each region for replication genotyping. Combined discovery and replication results in the meta-analysis showed that the locus 12q21.31 associated with plasma inhibin B levels (rs11116724) had the most significant association (P = 5.7 × 10-8). The LRRIQ1 and TSPAN19 genes are located in the 12q21.31 region. This study provides new susceptibility variants that contribute to plasma inhibin B levels.

A genome-wide association study identifies FSHR rs2300441 associated with follicle-stimulating hormone levels.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play critical roles in female reproduction, while the underlying genetic basis is poorly understood. Genome-wide association studies (GWASs) of FSH and LH levels were conducted in 2590 Chinese females including 1882 polycystic ovary syndrome (PCOS) cases and 708 controls. GWAS for FSH level identified multiple variants at FSHR showing genome-wide significance with the top variant (rs2300441) located in the intron of FSHR. The A allele of rs2300441 led to a reduced level of FSH in the PCOS group (ß = -.43, P = 6.70 × 10-14 ) as well as in the control group (ß = -.35, P = 6.52 × 10-4 ). In the combined sample, this association was enhanced after adjusting for the PCOS status (before: ß = -.38, P = 1.77 × 10-13 ; after: ß = -.42, P = 3.33 × 10-16 ), suggesting the genetic effect is independent of the PCOS status. The rs2300441 explained sevenfold higher proportion of the FSH variance than the total variance explained by the two previously reported FSHR missense variants (rs2300441 R2 = 1.40% vs rs6166 R2 = 0.17%, rs6165 R2 = 0.03%). GWAS for LH did not identify any genome-wide significant associations. In conclusion, we identified genome-wide significant association between variants in FSHR and circulating FSH first, with the top associated variant rs2300441 might be a primary contributor at the population level.

An epigenetic switch repressing Tet1 in gonadotropes activates the reproductive axis.

The TET enzymes catalyze conversion of 5-methyl cytosine (5mC) to 5-hydroxymethyl cytosine (5hmC) and play important roles during development. TET1 has been particularly well-studied in pluripotent stem cells, but Tet1-KO mice are viable, and the most marked defect is abnormal ovarian follicle development, resulting in impaired fertility. We hypothesized that TET1 might play a role in the central control of reproduction by regulating expression of the gonadotropin hormones, which are responsible for follicle development and maturation and ovarian function. We find that all three TET enzymes are expressed in gonadotrope-precursor cells, but Tet1 mRNA levels decrease markedly with completion of cell differentiation, corresponding with an increase in expression of the luteinizing hormone gene, Lhb We demonstrate that poorly differentiated gonadotropes express a TET1 isoform lacking the N-terminal CXXC-domain, which represses Lhb gene expression directly and does not catalyze 5hmC at the gene promoter. We show that this isoform is also expressed in other differentiated tissues, and that it is regulated by an alternative promoter whose activity is repressed by the liganded estrogen and androgen receptors, and by the hypothalamic gonadotropin-releasing hormone through activation of PKA. Its expression is also regulated by DNA methylation, including at an upstream enhancer that is protected by TET2, to allow Tet1 expression. The down-regulation of TET1 relieves its repression of the methylated Lhb gene promoter, which is then hydroxymethylated and activated by TET2 for full reproductive competence.

AgRP to Kiss1 neuron signaling links nutritional state and fertility.

Mammalian reproductive function depends upon a neuroendocrine circuit that evokes the pulsatile release of gonadotropin hormones (luteinizing hormone and follicle-stimulating hormone) from the pituitary. This reproductive circuit is sensitive to metabolic perturbations. When challenged with starvation, insufficient energy reserves attenuate gonadotropin release, leading to infertility. The reproductive neuroendocrine circuit is well established, composed of two populations of kisspeptin-expressing neurons (located in the anteroventral periventricular hypothalamus, Kiss1AVPV, and arcuate hypothalamus, Kiss1ARH), which drive the pulsatile activity of gonadotropin-releasing hormone (GnRH) neurons. The reproductive axis is primarily regulated by gonadal steroid and circadian cues, but the starvation-sensitive input that inhibits this circuit during negative energy balance remains controversial. Agouti-related peptide (AgRP)-expressing neurons are activated during starvation and have been implicated in leptin-associated infertility. To test whether these neurons relay information to the reproductive circuit, we used AgRP-neuron ablation and optogenetics to explore connectivity in acute slice preparations. Stimulation of AgRP fibers revealed direct, inhibitory synaptic connections with Kiss1ARH and Kiss1AVPV neurons. In agreement with this finding, Kiss1ARH neurons received less presynaptic inhibition in the absence of AgRP neurons (neonatal toxin-induced ablation). To determine whether enhancing the activity of AgRP neurons is sufficient to attenuate fertility in vivo, we artificially activated them over a sustained period and monitored fertility. Chemogenetic activation with clozapine N-oxide resulted in delayed estrous cycles and decreased fertility. These findings are consistent with the idea that, during metabolic deficiency, AgRP signaling contributes to infertility by inhibiting Kiss1 neurons.

Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis.

Prior to birth, oocytes within mammalian ovarian follicles initiate meiosis, but then arrest in prophase until puberty, when with each reproductive cycle, one or more follicles are stimulated by luteinizing hormone (LH) to resume meiosis in preparation for fertilization. Within preovulatory follicles, granulosa cells produce high levels of cGMP, which diffuses into the oocyte to maintain meiotic arrest. LH signaling restarts meiosis by rapidly lowering the levels of cGMP in the follicle and oocyte. Part of this decrease is mediated by the dephosphorylation and inactivation the NPR2 guanylyl cyclase in response to LH, but the mechanism for the remainder of the cGMP decrease is unknown. At least one cGMP phosphodiesterase, PDE5, is activated by LH signaling, which would contribute to lowering cGMP. PDE5 exhibits increased cGMP-hydrolytic activity when phosphorylated on serine 92, and we recently demonstrated that LH signaling phosphorylates PDE5 on this serine and increases its activity in rat follicles. To test the extent to which this mechanism contributes to the cGMP decrease that restarts meiosis, we generated a mouse line in which serine 92 was mutated to alanine (Pde5-S92A), such that it cannot be phosphorylated. Here we show that PDE5 phosphorylation is required for the LH-induced increase in cGMP-hydrolytic activity, but that this increase has only a modest effect on the LH-induced cGMP decrease in mouse follicles, and does not affect the timing of meiotic resumption. Though we show that the activation of PDE5 is among the mechanisms contributing to the cGMP decrease, these results suggest that another cGMP phosphodiesterase is also activated by LH signaling.

Zinc mediates the action of androgen in acting as a downstream effector of luteinizing hormone on oocyte maturation in zebrafish†.

The role of androgenic steroids on ovarian development has attracted much attention in recent years, but the molecular mechanism is still largely unknown. In this study, using zebrafish as a model, we found that the trace metal zinc mediates the action of androgen on oocyte maturation. The ovarian and serum testosterone is transiently stimulated by LH during oocyte maturation. Testosterone could mimic the action of LH on oocyte maturation, and its action appears to be independent of the classical nuclear androgen receptor. Consistent with a recent finding that a zinc transporter (Zip9) has been suggested as a novel androgen receptor, we found the labile zinc concentration could be induced by testosterone in the ovarian follicular cells, and zinc could mimic the action of testosterone on oocyte maturation and signaling. Moreover, the action of testosterone on oocyte maturation could be abolished by the chelation of zinc. Thus, the evidence supports the notion that zinc could mediate the action of androgen on oocyte maturation in zebrafish. This finding would shed light on understanding the role of androgen in ovary development and the molecular mechanism of oocyte maturation in fish.

Luteinizing hormone signaling is involved in synchronization of Leydig cell's clock and is crucial for rhythm robustness of testosterone production†.

In mammals, circadian clock regulates concentration of many reproductive hormones including testosterone. Previously, we characterized pattern of circadian transcription of core clock genes in testosterone-producing Leydig cells. Here, the potential role of luteinizing hormone receptor (LHR)-cAMP signaling in synchronization of Leydig cell's circadian clock and rhythmic testosterone production were examined. Results showed that activation of LHR-cAMP signaling in primary rat Leydig cell culture increased Star/STAR and changed expression of many clock genes (upregulated Per1/PER1, Dec1/2, and Rorb, and downregulated Bmal1 and Rev-erba/b). Inhibition of protein kinase A prevented LHR-triggered increase in transcription of Per1 and Dec1. Effect of stimulated LHR-cAMP signaling on Leydig cell's clock transcription was also confirmed in vivo, using rats treated with single hCG injection. To analyze in vivo effect of low LH-cAMP activity on rhythmical Leydig cell function, rats with experimental hypogonadotropic hypogonadism were used. Characteristics of hypogonadal rats were decreased LH and testosterone secretion without circadian fluctuation; in Leydig cells decreased arrhythmic cAMP and transcription of steroidogenic genes (Cyp11a1 and Cyp17a1) were observed, while decreased Star/STAR expression retains circadian pattern. However, expression of clock genes, despite changes in transcription levels (increased Bmal1, Per2, Cry1, Cry2, Rora, Rorb, Rev-erba/b/REV-ERBB, Dec1, Csnk1e, and decreased Npas2 and PER1) kept circadian patterns observed in control groups. Altogether, the results strengthened the hypothesis about role of LH-cAMP signaling as synchronizer of Leydig cell's clock. However, clock in Leydig cells is not sufficient to sustain rhythmicity of testosterone production in absence of rhythmic activity of LH-cAMP signaling.

Steroidogenic acute regulatory protein and luteinizing hormone are required for normal ovarian steroidogenesis and oocyte maturation in zebrafish†.

In recent studies, luteinizing hormone (LH) was reported to play important roles in oocyte maturation. However, the mechanism by which LH signaling, especially regarding the steroidogenesis process, affects oocyte maturation has not been clarified. In this study, zebrafish models with a functional deficiency in luteinizing hormone beta (Lhb) or steroidogenic acute regulatory protein (Star), an enzyme that promotes the transport of cholesterol into the inner mitochondrial membrane for maturation-induced hormone (MIH) production, were generated using transcription activator-like effector nucleases (TALENs). Similar phenotypes of the maturation-arrested oocytes in both female mutants have been observed. The levels of MIH in the oocytes of the female mutants were clearly decreased in both the lhb and star knockout zebrafish. The expression of star was dramatically down-regulated in the lhb mutant follicles and was clearly promoted by forskolin and hCG in vitro. Furthermore, treatment with the MIH precursors, pregnenolone or progesterone, as well as with MIH itself rescued the maturation-arrested oocyte phenotypes in both lhb and star mutants. The plasma levels of other steroids, including testosterone, estradiol, and cortisol, were not affected in the lhb mutants, while the levels of gonad hormones testosterone and estradiol were significantly increased in the star mutants. The cortisol levels were decreased in the star mutants. Collectively, our results confirm that LH plays important roles in the initiation of MIH synthesis from cholesterol and maintains oocyte maturation in zebrafish, as well as provide evidence that Star might act downstream of LH signaling in steroidogenesis.