Expression Of Lhcgr Research Articles

Monocyte Chemoattractant Protein-1 stimulates the differentiation of rat stem and progenitor Leydig cells during regeneration

BackgroundMonocyte chemoattractant protein-1(MCP-1) is a chemokine secreted by Leydig cells and peritubular myoid cells in the rat testis. Its role in regulating the development of Leydig cells via autocrine and paracrine is still unclear. The objective of the current study was to investigate the effects of MCP-1 on Leydig cell regeneration from stem cells in vivo and on Leydig cell development in vitro.ResultsIntratesticular injection of MCP-1(10 ng/testis) into Leydig cell-depleted rat testis from post-EDS day 14 to 28 significantly increased serum testosterone and luteinizing hormone levels, up-regulated the expression of Leydig cell proteins, LHCGR, SCARB1, CYP11A1, HSD3B1, CYP17A1, and HSD17B3 without affecting progenitor Leydig cell proliferation, as well as increased ERK1/2 phosphorylation. MCP-1 (100 ng/ml) significantly increased medium testosterone levels and up-regulated LHCGR, CYP11A1, and HSD3B1 expression without affecting EdU incorporation into stem cells after in vitro culture for 7 days. RS102895, a CCR2 inhibitor, reversed MCP-1-mediated increase of testosterone level after culture in combination with MCP-1.ConclusionMCP-1 stimulates the differentiation of stem and progenitor Leydig cells without affecting their proliferation.

Global analysis of FSH-regulated gene expression and histone modification in mouse granulosa cells.

Follicle-stimulating hormone (FSH) regulates ovarian follicular development through a specific gene expression program. We analyzed FSH-regulated transcriptome and histone modification in granulosa cells during follicular development. We used super-stimulated immature mice and collected granulosa cells before and 48 h after stimulation with equine chorionic gonadotropin (eCG). We profiled the transcriptome using RNA-sequencing (N = 3/time-point) and genome-wide trimethylation of lysine 4 of histone H3 (H3K4me3; an active transcription marker) using chromatin immunoprecipitation and sequencing (ChIP-Seq; N = 2/time-point). Across the mouse genome, 14,583 genes had an associated H3K4me3 peak and 63-66% of these peaks were observed within ≤1 kb promoter region. There were 72 genes with differential H3K4me3 modification at 48 h eCG (absolute log fold change > 1; false discovery rate [FDR] < 0.05) relative to 0 h eCG. Transcriptome data analysis showed 1463 differentially expressed genes at 48 h eCG (absolute log fold change > 1; FDR < 0.05). Among the 20 genes with differential expression and altered H3K4me3 modification, Lhcgr had higher H3K4me3 abundance and expression, while Nrip2 had lower H3K4me3 abundance and expression. Using ChIP-qPCR, we showed that FSH-regulated expression of Lhcgr, Cyp19a1, Nppc, and Nrip2 through regulation of H3K4me3 at their respective promoters. Transcript isoform analysis using Kallisto-Sleuth tool revealed 875 differentially expressed transcripts at 48 h eCG (b > 1; FDR < 0.05). Pathway analysis of RNA-seq data demonstrated that TGF-βsignaling and steroidogenic pathways were regulated at 48 h eCG. Thus, FSH regulates gene expression in granulosa cells through multiple mechanisms namely altered H3K4me3 modification and inducing specific transcripts. These data form the basis for further studies investigating how these specific mechanisms regulate granulosa cell functions.

Impact of lipopolysaccharide administration on luteinizing hormone/choriogonadotropin receptor (Lhcgr) expression in mouse ovaries

Lipopolysaccharide (LPS) is isolated from the genital tract of animals suffering from uterine damage and ovarian dysfunction. This study provides direct molecular evidence about the mechanism through which endotoxins cause reproductive disorders. Granulosa cells and ovaries were collected from immature mice treated with eCG or with eCG and LPS injection intraperitoneally. Normal large antral follicles were observed in ovaries obtained from eCG and LPS coinjected mice, and the morphology of the ovaries was similar to that observed in the control group. These antral follicles were not deemed atretic because few TUNEL-positive cells were observed. However, the granulosa cells of large antral follicles did not acquire the ability to respond to hCG stimulation. The number of ovulated oocytes was significantly lower in LPS-injected mice after superovulation compared to mice that were not exposed to LPS. The low reactivity was caused by the limited expression of the Lhcgr gene, which encodes the LH receptor in granulosa cells as well as an LPS-induced increase in the level of Dnmt1 expression. The methylation rate of the Lhcgr promoter region was significantly higher in granulosa cells obtained from the LPS treatment group compared with the control group. Together, these findings demonstrated that the decrease in the expression of Lhcgr due to LPS was a result of the epigenetic regulatory action of LPS. Our studies suggest that ovarian follicular cysts that is characterized by bacterial infection in humans and animals, is closely connected to the level of methylation of the Lhcgr promoter region.

Di-n-hexyl phthalate causes Leydig cell hyperplasia in rats during puberty

Di-n-hexyl phthalate (DNHP) is commonly used as a plasticizer. However, whether DNHP influences Leydig cell development during puberty remains unexplored. In this study, DNHP (0, 10, 100, and 1000 mg/kg) was administered via gavage to 35-day-old male Sprague-Dawley rats for 21 days. Serum levels of testosterone, luteinizing hormone, follicle-stimulating hormone, Leydig cell number, the expression of Leydig and Sertoli cell genes and proteins were investigated. DNHP significantly increased serum testosterone levels at 10 mg/kg but lowered its level at 1000 mg/kg. DNHP significantly increased luteinizing hormone levels at 1000 mg/kg without affecting follicle-stimulating hormone levels. DNHP increased Leydig cell number at all doses but down-regulated the expression of Lhcgr, Hsd3b1, Hsd17b3, and Hsd11b1 in Leydig cell per se at 1000 mg/kg. DNHP elevated phosphorylation of ERK1/2 and GSK-3β at 10 mg/kg but decreased SIRT1 and PGC-1α levels at 1000 mg/kg. In conclusion, DNHP exposure causes Leydig cell hyperplasia possibly via stimulating phosphorylation of ERK1/2 and GSK-3β signaling pathways.

SAT-224 Recurrent Co-Driver Mutation in CTNNB1-Mutant Aldosterone-producing Adenomas (APA), Causing Reversible Hypertension in Puberty, Pregnancy or Menopause

Background: Three patients with a syndrome of LH/HCG-activated primary aldosteronism in pregnancy or menopause carrying somatic CTNNB1 mutations were reported four years ago (Teo et al. NEJM 2015). This proved but half the story. Diagnosis of an APA in a 12-year old boy with severe hypertension revealed an apparently essential co-driver mutation. Methods: WES of tumour and blood was performed in the pubertal boy. Candidate genes were Sanger sequenced in other APAs from GB/Ireland, and France with known or suspected CTNNB1 mutations. LHCGR, GNRHR and CYP11B2 expression were measured in all available patients’ APAs and the adjacent adrenal gland (AAG) by RT-PCR. RNA and gDNA from the zona glomerulosa (ZG) of the proband’s AAG were collected by laser capture microdissection for Sanger sequencing of GNA11 and CTNNB1. Function of mutant genes was assessed by measurement of aldosterone production and LHCGR expression by immunofluorescence (IFC) in NCI-H295R adrenocortical cells and primary human APA cells. Results: The proband’s APA contained a p.(S45F) somatic mutation in CTNNB1, and a p.(Q209P) somatic mutation of the GTPase-activating residue (Q209) in GNA11. Mutations of Q209, to P or H, were also found in six other GB/Irish patients with previously identified mutations of CTNNB1 (S33C, G34R, T41A, S45F, or S45P). All seven patients remain normotensive 2-12 years post-adrenalectomy, including some with long-standing pre-operative hypertension. Four of the 13 French patients with CTNNB1 exon 3 mutant APAs have somatic mutation of Q209 of either GNA11 (n=3) or GNAQ. In comparison with their own AAG, the GB/Irish double mutant APAs showed an increase in expression of LHCGR, CYP11B2 and GNRHR by 32-166, 158-18980, and 1174-6642 fold, respectively. All four French double-mutants had >10 fold higher LHCGR than APAs with single mutations of CTNNB1 or other genes. Hyperplasia of ZG was observed in the ZG of the boy’s AAG but no APCC was detected. Homozygous or heterozygous Q209P mutation of GNA11 was detected in multiple ZG samples in RNA and/or gDNA but WT in CTNNB1 exon 3. H295R cells (CTNNB1 S45P) were GNA11 WT. Overexpression of GNA11 Q209 mutation increased aldosterone secretion to 465% of GNA11 WT overexpressing cells (n=6, P<0.001) and CYP11B2 expression was also increased several-fold. Smaller increases were seen in primary human adrenal cells after double-transfection by GNA11 and CTNNB1 mutants (n=3, P<0.001). This also caused membrane expression of LHCGR, visualised by IFC. Conclusions: APAs with double mutation of GNA11/GNAQ Q209 and CTNNB1 have a distinct phenotype, in which hypertension is triggered by high LH or HCG, and cured in all cases by adrenalectomy. GNA11/Q mediates the aldosterone response to ANGII, and the Q209 codon is analogous to the Q227 of GNAS, mutated in McCune Albright. Mosaicism for GNA11 may cause ZG hyperplasia.

Anti-Müllerian hormone inhibits luteinizing hormone-induced androstenedione synthesis in porcine theca cells

AMH (Anti-Müllerian Hormone) is involved in the regulation of follicle growth initiation and inhibits FSH-induced aromatase expression and estrogen production in granulosa cells. However, the function of AMH in steroidogenesis by theca cells remains unclear. The aim of this study is to investigate the role of AMH as a regulator of the basal and stimulated steroid production by pig granulosa cells (pGCs) and theca cells (pTCs). PGCs and pTCs were incubated with hormones AMH, LH (luteinizing hormone), FSH (follicle stimulating hormone), individually or in combination. The expression of CYP19A1, HSD3B1, CYP11A1, LHCGR, and CYP17A1 mRNA were evaluated by quantitative reverse transcriptase PCR. In pGCs, 10 ng/mL AMH significantly decreased the FSH-stimulated effect on FSHR and CYP19A1 expression and estradiol production. In pTCs, LH treatment significantly increased the expression of HSD3B1, CYP11A1, LHCGR, and androstenedione or progesterone production (P < 0.05). Additionally, 10 ng/mL AMH also significantly decreased the LH-stimulated effects on the expression of HSD3B1, CYP11A1, CYP17A1, LHCGR and androstenedione production. Transfection with siAMHR2-I abolished the suppressive effects of AMH on LH-induced HSD3B1 expression and androstenedione production. Taken together, these results demonstrate that AMH is involved in FSH induced estradiol production in pGCs and LH induced androstenedione production in pTCs by regulating the steroidogenesis pathway.

Paraquat exposure delays late-stage Leydig cell differentiation in rats during puberty

Paraquat is a fast and non-selective herbicide that is widely used in crop cultivation and conservation tillage systems. Animal experiments have shown that paraquat decreases sperm quality and testicular organ coefficient, but its effects on the development of Leydig cells remain unclear. The objective of the current study was to investigate the effects of paraquat exposure on the Leydig cell development in rats during puberty. Twenty-eight male 35-day-old Sprague-Dawley rats were divided into 4 groups: 0, 0.5, 2.0, and 8 mg kg−1 d−1 paraquat. Paraquat was gavaged for 10 d. Adult Leydig cells were isolated and treated with paraquat for 24 h. Paraquat in vivo significantly decreased body and testis weights at 8 mg kg−1 and lowered serum testosterone levels at 2 and 8 mg kg−1 without affecting the levels of serum luteinizing hormone and follicle-stimulating hormone. Paraquat did not alter Leydig cell number and PCNA labeling index. Real-time PCR showed that paraquat down-regulated the expression of Lhcgr, Scarb1, Cyp11a1, Cyp17a1, and Hsd17b3 genes and their proteins at 2 or 8 mg kg−1, while it up-regulated the expression of Srd5a1 at 8 mg kg−1. Paraquat increased ROS and decreased testosterone production by Leydig cells at 1 and 10 μM after in vitro 24-h exposure. Vitamin E (40 μg/ml) reversed paraquat-induced ROS and suppression of testosterone synthesis in vitro. In conclusion, paraquat directly delays Leydig cell differentiation to block testosterone synthesis via down-regulating the expression of critical testosterone synthesis-related genes and up-regulating the expression of testosterone metabolic enzyme (Srd5a1) gene and possibly via increasing ROS production.

Hypoxia Limits the Growth of Bovine Follicles in Vitro by Inhibiting Estrogen Receptor α.

Simple SummaryIt is well documented that a hypoxic environment at high altitudes decreased the fertility of female domestic animals because of embryonic or fetal losses, intrauterine growth restriction, and birth weight reduction. However, little study has been performed on for the effects of hypoxia on bovine ovary function. In this study, we found that the hypoxia plays an important role in regulating follicular survival and genes expression. Hypoxia limits the growth of bovine follicles cultured in vitro through inhibition of ERα, which could provide useful information for future studies relating to reproduction of cattle.Female animals living in the Qinghai-Tibet Plateau have lower ovulation rates because of the hypoxic environment, however, the mechanism of hypoxia on animal follicles is unclear. In this study, the effects of hypoxia on bovine follicles were investigated using an in vitro follicular culture system. The results show that there was a significant decrease in follicular diameter from day 3 to day 6 in both hypoxia and hypoxia with estrogen (E2) and fulvestrant (ICI 182780) (hypoxia + E2 + ICI) groups, when compared with a normoxia group (p < 0.05). We also observed significant downregulation of ERα and FSHR, while upregulation of LHCGR expression in the hypoxia group and hypoxia + E2 + ICI groups compared to the normoxia group (p < 0.05). The expression of IGF1 gene was significantly downregulated in hypoxia + E2 + ICI group when compared to the hypoxia + E2 group (p < 0.05). The expression of HIF1A, ADAMTS1, VEGFA, and EDN2 were upregulated in both hypoxia and hypoxia + E2 + ICI groups in comparison to normoxia group (p < 0.05). Under hypoxic conditions, the addition of E2 resulted in a decrease of HIF1A protein but an increase of ERα protein in cultured bovine follicles (p < 0.05). In summary, hypoxia limits the growth of bovine follicle cultured in vitro through inhibition of ERα.

Chitosan Hydrogel Supports Integrity of Ovarian Follicles during In Vitro Culture: A Preliminary of A Novel Biomaterial for Three Dimensional Culture of Ovarian Follicles.

Objective Testing novel biomaterials for the three dimensional (3D) culture of ovarian follicles may ultimately lead to a culture model which can support the integrity of follicles during in vitro culture (IVC). The present study reports the first application of a chitosan (CS) hydrogel in culturing mouse preantral follicles. Materials and Methods In this interventional experiment study, CS hydrogels with the concentrations of 0.5, 1, and 1.5% were first tested for fourier transform infrared spectroscopy (FT-IR), Compressive Strength, viscosity, degradation, swelling ratio, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and live/dead assay. Thereafter, mouse ovarian follicles were encapsulated in optimum concentration of CS (1%) and compared with those in alginate hydrogel. The follicular morphology, quality of matured oocyte and steroid secretion in both CS and alginate were assessed by enzyme-linked immunosorbent assay (ELISA). The expression of folliculogenesis, endocrine, and apoptotic related genes was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and compared with day that in 0. Results The rates of survival, and diameter of the follicles, secretion of estradiol, normal appearance of meiotic spindle and chromosome alignment were all higher in CS group compared with those in alginate group (P≤0.05). The expression of Cyp19a1 and Lhcgr in CS group was significantly higher than that of the alginate group (P≤0.05). ConclusionThe results showed that CS is a permissive hydrogel and has a beneficial effect on encapsulation of ovarian follicle and its further development during 3D culture.

Propofol Inhibits Androgen Production in Rat Immature Leydig Cells.

Background: Propofol is a widely used anesthetic. Whether propofol inhibits androgen production by rat Leydig cells and the underlying mechanism remains unclear. The objective of the current study was to examine the effects of propofol exposure to rat primary immature Leydig cells and to define propofol-induced inhibition of steroidogenic enzymes in both rat and human testes in vitro. Methods: Immature Leydig cells were purified from 35-day-old male Sprague–Dawley rats and were exposed to propofol for 3 h. The androgen production by Leydig cells under basal, luteinizing hormone, 8bromo-cAMP, and steroid-substrate stimulated conditions and gene expression of Leydig cells after exposure to propofol were measured. Immature Leydig cells were treated with propofol for 3 h and switched to propofol-free medium for additional 3 and 9 h to test whether propofol-induced inhibition is reversible. 3H-Steroids were used to evaluate the direct action of propofol on cytochrome P450 cholesterol side chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase (HSD3B), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1), and 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) activities in rat and human testes in vitro. Results: Propofol significantly lowered luteinizing hormone and 8bromo-cAMP stimulated androgen production by Leydig cells after 3-h exposure. Further investigation showed that propofol down-regulated the expression of Cyp11a1 and Cyp17a1 and their proteins at 5 and 50 µM, although it up-regulated Lhcgr expression at 50 µM. Propofol significantly suppressed phosphorylation of ERK1/2 and induced ROS production in immature Leydig cells at 5 and 50 µM. Propofol significantly induced apoptosis of immature Leydig cells at 50 µM. Propofol specifically inhibited rat and human testis HSD3B activities in vitro. The half maximal inhibitory concentrations of propofol for rat and human HSD3B enzymes were 1.011 ± 0.065 and 3.498 ± 0.067 µM, respectively. The mode of action of propofol of inhibiting HSD3B was competitive when pregnenolone was added. At 50 µM, propofol did not directly inhibit rat and human testis CYP11A1, CYP17A1, and HSD17B3 activities in vitro. Conclusion: Propofol inhibits androgen production via both directly inhibiting HSD3B activity and down-regulating Cyp11a1 and Cyp17a1 expression in Leydig cells. Suppression of steroidogenic enzymes is presumably associated with the lower production of androgen by Leydig cells after propofol treatment. However, propofol-induced inhibition on androgen production is reversible.

Flurbiprofen Inhibits Androgen Productions in Rat Immature Leydig Cells.

Flurbiprofen is one of the nonsteroidal anti-inflammatory drugs. Whether flurbiprofen affects androgen biosynthesis in Leydig cells is still unknown. Immature Leydig cells (ILCs) isolated from 35-day-old male Sprague-Dawley rats were cultured with 0-100 μM flurbiprofen for 24 h and medium androgen levels and Leydig cell mRNA levels were measured. Immature Leydig cells were also incubated with 100 μM flurbiprofen for 3 h in combination with luteinizing hormone (LH), 8bromo-cAMP, 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively, and medium androgen levels were measured. The ROS generation and apoptosis rate were also investigated. The direct effects of flurbiprofen on androgen biosynthetic and metabolizing enzyme activities were measured. Flurbiprofen significantly inhibited basal, LH, and 8bromo-cAMP stimulated androgen production at 10 and 100 μM. Further study demonstrated that flurbiprofen competitively inhibited rat and human testis 3β-hydroxysteroid dehydrogenase (HSD3B) activity with the half maximal inhibitory concentration (IC50) values of 0.95 μM for rat enzyme and 6.31 μM for human enzyme. In addition, flurbiprofen down-regulated the expression of Srd5a1 and Akr1c14 at 1, 10, and 100 μM. Flurbiprofen also down-regulated Lhcgr expression at 100 μM. Flurbiprofen at 10 and 100 μM increased ROS production and apoptosis rate of rat Leydig cells. In conclusion, flurbiprofen directly inhibits HSD3B activity and the expression levels of Srd5a1 and Akr1c14 in rat Leydig cells, thus leading to the reduction of androgen secretion.

Effects of dexmedetomidine on the steroidogenesis of rat immature Leydig cells

Dexmedetomidine (DEX), an imidazole compound, is an anesthetic drug used perioperatively. In the current study, we investigated the effects of DEX on androgen production in rat immature Leydig cells in vitro. Leydig cells isolated from pubertal Sprague Dawley rats were treated with various concentrations of DEX (0.015–1.5 µM) for 3 h and medium 5α-androstanediol and testosterone levels and the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1 and Akr1c14 in Leydig cells were determined. At 0.015–1.5 μM, DEX concentration-dependently inhibited androgen secretion and downregulated Cyp17a1 and Srd5a1 mRNA levels. DEX equally blocked the LH- and cAMP-stimulated secretion of androgens. Using the steroid substrates, 22R-hydroxycholesterol (for cytochrome P450 cholesterol side chain cleavage), pregnenolone (for 3β-hydroxysteroid dehydrogenase 1), progesterone (for cytochrome P450 17α-hydroxylase/C17,C20-lyase), androstenedione (for 17β-hydroxysteroid dehydrogenase 3), testosterone (for steroid 5α-reductase 1), and dihydrotestosterone (for 3α-hydroxysteroid dehydrogenase), it was demonstrated that DEX inhibited 22R-hydroxycholesterol, pregnenolone, progesterone, and testosterone-mediated 5α-androstanediol formation at 1.5 μM. Further study demonstrated that DEX also directly inhibited rat testis cholesterol side chain cleavage, 3β-hydroxysteroid dehydrogenation, and 17α-hydroxylation at 1.5 μM. DEX induced ROS production and increased apoptosis rate in immature Leydig cells after 24-h treatment at ≥0.15 μM. In conclusion, DEX directly inhibits the activities of some steroidogenic enzymes and downregulates the expression of Cyp17a1 and Srd5a1, and increases ROS production, thus leading to lower production of androgens in immature Leydig cells.

MON-232 Differential Regulation of Genes Relevant to Reproductive Function in Human Granulosa Cells of Small Antral Follicles from Women with or without Polycystic Ovary Syndrome

Introduction: Polycystic Ovary Syndrome (PCOS) is the most common endocrinopathy in young women, affecting 5-10% of women of reproductive age. Despite its high prevalence, the underlying pathogenesis of PCOS remains complex and incompletely understood. Aberrant function of granulosa cells has been implicated in the aetiology of PCOS. Material and methods: Granulosa cells (GCs) were obtained from follicular fluid from (unstimulated) individual small antral follicles (SAF) isolated at the time of removal of ovarian tissue for fertility cryopreservation in young women. Granulosa luteal (GL) cells were collected from pooled follicular fluid collected at the time of oocyte retrieval in women undergoing IVF. RNA was extracted and RT-qPCR carried out to analyse expression of genes involved in granulosa cell function, including steroidogenesis and growth. 30 GC samples from individual small antral follicles from 15 women with regular cycles and 31 samples from 10 women with PCOS were included. Mean follicle size was similar between groups (mean 5.5±1mm). Luteinised GCs (GLCs) from 8 women with and without PCOS were also included. Results: 92% of GC samples from SAF expressed LH receptor (LHCGR) RNA. Expression of LHCGR was not significantly higher in PCOS GCs but there was a difference in distribution of results with a subset of PCOS samples (13%) with 4-6 times higher expression than in that of the controls. LHCGR expression was higher in GLCs compared to GCs (P<0.05). FSH receptor (FSHR) expression was significantly lower in PCOS GCs from SAFs (P<0.05), unlike PCOS GLCs, which expressed higher levels of FSHR (p<0.05). FSHR expression was 14 fold higher in GCs compared to GLCs (p<0.0001). There was no correlation between LHCGR, FSHR or follicle size. Androgen receptor (AR) expression was lower in PCOS GCs from SAF (p<0.01) unlike PCOS GLCs, which expressed more AR than GL controls (P<0.05). AR expression correlated with FSHR (p<0.0001) but not LHCGR. CYP11A1 was lower in both PCOS GCs and GLCs (p<0.05) and expression was 50 fold higher in GLCs than GCs (p<0.0001). Expression of 3BHSD2 was reduced in PCOS GCs (p=0.06). There were no significant differences in expression of STAR, CYP19, INHBA or INHBB between control and PCOS in either GCs or GLCs. Conclusions: Granulosa cells from small antral follicles from women with PCOS have a lower expression of FSHR, AR and CYP11A1 and a trend towards higher LHCGR compared to controls. This expression pattern is suggestive of early luteinisation of granulosa cells in unstimulated small antral follicles in PCOS.

SUN-213 Hsd17b3 Knockout Mice Serve As A Model To Define Mechanisms Behind The Altered Endocrine Phenotype Of Patients With Hsd17b3 Mutations

Hydroxysteroid (17β) dehydrogenase type 3 (HSD17B3) is known to be the primary HSD17B enzyme converting androstenedione (A) to testosterone (T) in human testes, and thus, to be important for sexual maturation in boys and for reproductive health later in life. In particular, inherited HSD17B3 deficiency is a known cause of 46,XY disorder of sex development in humans, where affected males are born with outwardly female genitalia, but are virilized during puberty. In the present study, Hsd17b3 knockout mice (HSD17B3KO) were established and characterized. HSD17B3KO males were born apparently phenotypically normal, but presented signs of hypogonadism at adulthood, including delayed puberty, subfertility, a shortened anogenital distance and lowered weight of several androgen-sensitive tissues. The loss of HSD17B3 also affected the androgen levels both in the testes and in circulation. Compared with the wild-type mice, androstenedione concentration was increased 10-fold in the testes and 100-fold in the serum in adult HSD17B3KO animals, accompanied with 3-fold increase in serum testosterone. The serum testosterone/androstenedione ratio in the adult HSD17B3KO mice reduced 10-fold. We also observed that circulating LH was increased 40-fold in adult KO animals, with a 200-fold increase in testicular Lhcgr expression. In addition, several genes related to steroid and cholesterol biosynthesis were upregulated in the testes, those including Star, Cyp11a1, Cyp17a1, Cyp51, Nsdhl and Hsd17b7. Our results demonstrate that, similar to humans, HSD17B3 is essential for proper androgen homeostasis in adult mice. However, like in humans, other, still unknown, enzymes can partly compensate the HSD17B3 deficiency, resulting in high amounts of serum T and DHT at adulthood. The persisting hypogonadal phenotype in the presence of high LH levels and high serum T suggest that the lack of HSD17B3 results in a defect in the pituitary-gonadal feedback system, and reduced androgen sensitivity of the extragonadal androgen target tissues. The very similar endocrine disruption present in the adult mice and men with HSD17B3 deficiency provides us the possibility to use the HSD17B3KO mice as a model to further understand the mechanisms behind the altered endocrine phenotype of the patients carrying inactivating mutations at HSD17B3 gene, and to identify potential secondary complications caused by the disease later in the life.

Fibroblast Growth Factor 1 Promotes Rat Stem Leydig Cell Development.

Fibroblast growth factor 1 (FGF1) is reported to be expressed in the testis. How FGF1 affects stem Leydig cell development remains unclear. Here, we report the effects of FGF1 on rat stem Leydig cell development in an ethane dimethane sulfonate (EDS)-treated model. FGF1 (100 ng/testis) significantly increased serum testosterone level, increased PCNA-positive Leydig cell percentage and Leydig cell number, but down-regulated the expression of Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd11b1 in Leydig cells per se, after its daily intratesticular injection from post-EDS day 14 for 14 days. Primary culture of the seminiferous tubules showed that FGF1 stimulated EdU incorporation to stem Leydig cells but blocked the differentiation into the Leydig cell lineage, possibly via FGFR1-mediated mechanism. In conclusion, FGF1 promotes stem Leydig cell proliferation but blocks its differentiation.

Effect of recombinant bovine somatotropin (rbST) treatment on follicular population and development in non-lactating dairy cows.

The aim of this study was to evaluate the long-term effects of recombinant bovine somatotropin (rbST) on follicle population and ovulatory follicle development in non-lactating dairy cows. Twenty-one Jersey cows were allocated in rbST (n=11) or control (n=10) groups. On day -60, cows in rbST group received 500 mg of somatotropin (s.c. Lactotropin®, Elanco). On day 0, control and rbST cows received an intravaginal progesterone-releasing device (1.9 g, CIDR®, Zoetis) and GnRH (100 mg, IM, Factrel®, Zoetis). On day 8, cows received PGF2α (25 mg, IM, Lutalyse®, Zoetis) and the CIDR® was removed. Twelve hours after device removal (D8), serum, follicular fluid and granulosa cells samples were collected. Serum and follicular concentration of estradiol (E2) and progesterone (P4) were analyzed. Total RNA was extracted from granulosa cells to measure gene expression of LHCGR, STAR, HSD-3B1, CYP11A1, CYP19A1, CYP17A1, IGFR and PAPPA by real-time PCR. Ultrasonography was performed on days -60, -53, -46, -14, -7, 0 and 8 for antral follicle count. Results were analyzed by repeated measures ANOVA and t-test. There was no effect of rbST treatment on the number of follicles during the 60 days period, as well as no effect on serum and follicular fluid E2 and follicular fluid P4 at the moment of follicle aspiration. There was a reduction in PAPPA (P = 0.006), CYP11A1 (P = 0.04) and CYP19A1 (P = 0.002) mRNA levels in granulosa cells of the pre-ovulatory follicle of rbST treated cows. In conclusion, a single dose of rbST did not have long-term effects on antral follicle population, serum and follicular E2/P4 concentrations in non-lactating dairy cows. Despite that, rbST injection decreased granulosa cell expression of genes related to steroidogenesis in the pre-ovulatory follicle.

Differential expression of LHCGR and its isoforms is associated to the variability in superovulation responses of Gir cattle

The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3 ± 1.2 ova/embryos per flush, n = 5) or poor responders (1.1 ± 0.3 ova/embryos per flush, n = 5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8 mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater®. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P < 0.01) as well as isoforms S10, S11 and S10 + 11, compared to good responders. In both phenotypes, superovulation down-regulated total LHCGR expression (−0.5 ± 0.2 and −0.9 ± 0.0 for good and poor responders, respectively; P < 0.05). However, in poor responders the exogenous FSH treatment up-regulated the S10 (2.4 ± 2.0; P < 0.05), S10 + 11 (3.8 ± 3.2; P < 0.01), and S1 isoforms (1.8 ± 1.3; P < 0.05), compared to good responders We conclude that down-regulation of total LHCGR, associated to up-regulation of their inactive isoforms, may have compromised follicle development and thus contributed to the low efficiency of superovulation in heifers with a poor responder phenotype.

In utero exposure to hexavalent chromium disrupts rat fetal testis development

Hexavalent chromium (Cr6+) acts as an endocrine disruptor. Herein, we investigated effects of Cr6+ on the development of rat fetal Leydig and Sertoli cells, which support differentiation of the male reproductive tract in late gestation. Female pregnant Sprague Dawley rats were gavaged with potassium dichromate (0, 3, 6, and 12 mg/kg) from gestational days (GD) 12 to GD 21. Leydig and Sertoli cell function was evaluated by investigating serum testosterone levels, cell number and distribution, and the expression levels of Leydig and Sertoli cell genes and proteins. Cr6+ increased serum testosterone level at dose of 3 mg/kg (1.170 ± 0.121 ng/ml vs. 0.720 ± 0.082 ng/ml in the control), while lowered it at dose of 12 mg/kg (0.400 ± 0.098 ng/ml). In addition, it showed that Cr6+ dose-dependently reduced Leydig cell size and cytoplasmic size and decreased the percentage of medium fetal Leydig cell cluster at dose of 12 mg/kg. Further study demonstrated that the expression of Leydig cell (Lhcgr, Scarb1, and Hsd3b1) and Sertoli cell (Fshr, Pdgfa, and Lif) genes in the testis was upregulated at dose of 3 mg/kg while the expression of Lhcgr, Hsd17b3 and Igf1 was downregulated by Cr6+ at dose of 12 mg/kg. In conclusion, Cr6+ had biphasic effects on fetal Leydig cell development with low dose to stimulate testosterone production and high dose to inhibit it, possibly via biphasically regulating growth factor gene expression in fetal Sertoli cells.

ECG stimulation in domestic cats increases the expression of gonadotrophin-induced genes improving oocyte competence during the non-breeding season.

The objective of this study was to evaluate the eCG stimulation on domestic cat oocyte competence during the non-breeding season. Four experimental groups were made: (a) untreated cycling cats (Breeding season group; BS), (b) untreated anestrous cats (Non-breeding group; NB), (c) anestrous cats treated with 200IU of eCG (eCG group) and (d) anestrous cats treated with 200IU of eCG and 100IU of hCG four days later (hCG group). In the BS, NB and eCG groups, grade I and II immature cumulus-oocyte complexes (COCs) were subjected to in vitro maturation or used for the gene expression analysis of FSHR, LHCGR, EGFR, EGR1, ESR2, PTGS2, GDF9, BMP15 and GATM. The in vitro matured oocytes from the BS, NB and eCG groups and the in vivo matured oocytes from the hCG group were subjected to parthenogenetic activation. The grade I and II COCs from the eCG group had an increased expression of FSHR, LHCGR, EGFR, EGR1 and ESR2 and a higher maturation rate than the BS and NB groups (p<0.05). After parthenogenetic activation, the blastocyst rate from the hCG, eCG and BS groups was higher than the NB group (p<0.05). However, no significant improvement was observed in the blastocyst rate in the hCG group compared to the eCG group (p>0.05). In conclusion, the eCG treatment increases the expression of specific genes improving the oocyte competence during the cat non-breeding season, which is reflected in an enhanced in vitro maturation and in vitro embryo development after parthenogenetic activation.

Gestational exposure to ziram disrupts rat fetal Leydig cell development

Ziram is an endocrine disruptor and may cause birth abnormality of the male reproductive system. However, the effects of ziram on fetal Leydig cell (FLC) development are still unknown. The objective of the present study was to determine the endocrine-disrupting effect of ziram on rat FLC development after gestational exposure. Pregnant Sprague Dawley dams were randomly divided into 5 groups and were gavaged with 0 (corn oil, the control), 1, 2, 4, or 8 mg/kg ziram from gestational day 12 (GD12) to GD21. FLC development was evaluated by measuring serum testosterone, FLC number and distribution, and the expression levels of Leydig and Sertoli cell genes. Ziram significantly increased serum testosterone level at 1 mg/kg (1.350 ± 0.099 ng/ml vs. 0.989 ± 0.106 ng/ml in the control), while it remarkably lowered it at 8 mg/kg (0.598 ± 0.086 ng/ml). Quantitative immunohistochemical staining showed that ziram increased FLC number via stimulating cell proliferation at 1 mg/kg and lowered it via inhibiting its proliferation at 8 mg/kg without affecting Sertoli cell number. Further study demonstrated that the expression of Nr5a1, Lhcgr, Scarb1, Star, Cyp11a1, and Cyp17a1 genes and proteins in the testis was upregulated at 1 mg/kg and the expression of Leydig (Nr5a1, Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, and Insl3) and Sertoli cell (Fshr, Hsd17b3, Dhh, Amh, and Sox9) genes and proteins was downregulated by ziram at 8 mg/kg. In conclusion, ziram had biphasic effects on FLC development with low dose to increase FLC number and function and high dose to decrease them.