FSH Receptor mRNA Research Articles

P-564 Decoding ovarian response: the predictive power of FSH receptor gene expression in controlled ovarian stimulation

Abstract Study question Can profiling of variation in follicle-stimulating hormone receptor (FSHR) gene expression forecast response to Controlled Ovarian Stimulation (COS) in Assisted Reproductive Techniques? Summary answer Full-length FSHR transcript was the most common mRNA species. Nine distinct transcripts were characterised, but only one (deletion Exon 6) was associated with altered response. What is known already AMH levels and age are considered useful predictors of ovarian response to COS (high AMH and young age typically associated with high yields of oocytes; older ages and low AMH associated with fewer oocytes). However, it is not uncommon to see patients who do not fit this pattern, highlighting the limitations of these parameters in predicting response. Age-independent biomarkers capable of revealing patients at elevated risk of hyperstimulation, or those who might produce fewer than the optimal number of oocytes, are desirable. Alternative transcripts of the FSHR gene have been proposed as an explanation for variation in response to COS. Study design, size, duration FSHR splice variants in mRNA in granulosa cells from 126 patients were examined utilising long-read nanopore sequencing. This strategy permitted the sequencing of mRNA variants without fragmentation, allowing the detection of all splicing events in each mRNA strand. In addition to being categorised based on age and AMH levels, patients were also classified by the expression of FSHR transcript variants and their response to stimulation (high, low, or normal response). Participants/materials, setting, methods Patterns of FSHR mRNA splice variants were analysed in each of 126 patients. This involved extraction of RNA from the granulosa cells, followed by long-read nanopore sequencing, permitting analysis of whole transcripts in a single, contiguous sequence ‘read’. Patients were classified based upon their level of response to COS (low, normal, high), taking into account their age, AMH level and the number of oocytes retrieved. Patterns of FSHR transcript variation were compared to COS response. Main results and the role of chance Full-length FSHR transcript was detected in 97.6% of patients and skipping of Exon 6 was observed in 68%. Five novel transcripts were discovered, including partial insertion of ∼100bp segments of introns 1 or 2, seen in 6.55% of samples. In total, nine distinct FSHR mRNA variants were detected. As expected, increasing age was associated with higher FSH requirement per oocyte collected (p = 0.018), and AMH levels declined with age (2.8± 1.42 in women <38 versus 1.58 ±1.29 in women ≥38; p = 0.011). Consistent with previous analyses, AMH levels and amount of FSH per oocyte retrieved were negatively correlated (p < 0.001). One-third of the patients (33%) in this cohort displayed responses to COS that were not considered to be consistent with expectations based upon age and AMH levels. Expression of the novel FSHR transcript variants was not associated with FSH needed per oocyte retrieved (p = 0.78), nor the proportion of mature oocytes (p = 0.95).The only transcript potentially linked to an altered response to COS was the previously reported mRNA species, lacking exon 6, associated with significantly greater numbers of oocytes produced (p = 0.0087). Interestingly, women ≥38 expressed novel variants more frequently than their younger counterparts (p = 0.018), potentially indicative of an age-related decline in transcriptional fidelity. Limitations, reasons for caution It is possible that the use of controlled ovarian stimulation might alter the dynamics of transcription in cumulus cells. Therefore, it is possible that the spectrum of transcripts seen in this study might differ from those present in unstimulated cycles. Wider implications of the findings Expected associations between age, AMH levels and response to COS were observed. Skipping exon 6 may be associated with a superior response to COS, suggesting FSHR mRNA profiling might have a role in tailoring controlled ovarian stimulation protocols. Novel transcript variants were detected and seen most often in older women. Trial registration number not applicable

Dynamic Expression of Follicle-Stimulating Hormone and Estrogen mRNA Receptors Associated with microRNAs 34a and -let-7c in Canine Follicles during the Estrous Cycle.

The genes encoding for estrogen receptor (ESR2) and follicle-stimulating hormone receptor (FSHR) play crucial roles in ovarian follicular development. This study aimed to determine the expression levels of miRNAs predicted against FSHR and ESR2 mRNAs in follicular cells related to their target genes during the estrous cycle in canines. Antral follicles were dissected from 72 ovaries following ovariohysterectomies. MiRNAs regulating FSHR and ESR2 genes were selected from miRNA databases, and mature miRNA and mRNA expression profiling was performed using real-time polymerase chain reaction (PCR). The best miRNA for each target gene was selected considering the quantitative PCR (qPCR) performance and target prediction probability, selecting only miRNAs with a binding p-value of 1.0, and choosing cfa-miR-34a and cfa-let-7c for FSHR and ESR2, respectively. The expression levels comparing the different phases of the estrous cycle were evaluated using ANOVA. Pearson correlations between the expression pattern of each miRNA and their target genes were performed. Each miRNA and its target genes were expressed in the granulosa cells in all estrous phases. FSHR remained low in anestrus and proestrus, increased (p < 0.05) to the highest level in estrus, and decreased (p < 0.05) in diestrus. ESR2 showed the same trend as FSHR, with the highest (p < 0.05) expression in estrus and the lowest (p < 0.05) in anestrus and proestrus. A tendency for an inverse relationship was observed between the expression of miR-34a and FSHR only in the anestrus phase, while an inverse correlation (r = -0.8) was found between miRNA-7c and ESR2 (p < 0.01). The expression profile of miR-34a and miR-let-7c and their predicted target genes of dog ovarian follicles throughout the estrous cycle observed in this study suggest a role in the transcriptional regulation of FSHR and ESR2, which is the first evidence of the involvement of these miRNAs in the canine follicular function.

Zuogui Pills improve testicular development of offspring rats exposed to prenatal stress via Cx43

This study aims to reveal the mechanism of prenatal stress in affecting the testicular development of offspring rats and the intervention effects of Zuogui Pills via connexin 43(Cx43). Forty pregnant SD rats were randomized into a blank control group, a mo-del group, a high-dose(18.9 g·kg~(-1)) Zuogui Pills group, a low-dose(9.45 g·kg~(-1)) Zuogui Pills group, and a vitamin E(1.44 mg·kg~(-1)) group. The other groups except the blank control group was subjected to chronic unpredictable mild stress for the modeling of prenatal stress. The model was evaluated by sucrose preference test, open field test, and enzyme-linked immunosorbent assay(ELISA) of the glucocorticoid level. ELISA was employed to measure the thyroxine 4(T4), testosterone(T), and follicle-stimulating hormone(FSH) levels to assess kidney deficiency. Hematoxylin-eosin(HE) staining was employed to evaluate the status of testicular germ cells. An automatic sperm analyzer was used to measure the sperm quality. Immunofluorescence double staining was employed to detect the expression of Cx43 and follicle-stimulating hormone receptor(FSHR) in the testes of offspring rats. The mRNA and protein levels of Cx43, FSHR, phosphatidylinositol 3-kinase(PI3K), and protein kinase B(Akt) were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot, respectively. Prenatal stress induced testicular development disorders in offspring rats. The HE staining results showed that on the day of birth, the model group had reduced seminiferous tubules in the testes, elevated FSH level in the serum, and lowered Cx43 level in the testicular tissue. Male offspring rats of 60 days old had reduced testicular spermatogenic function, decreased sperm quality, elevated FSH level and lowered T level in the serum, and down-regulated protein and mRNA levels of Cx43, FSHR, PI3K, and Akt in the testicular tissue. Zuogui Pills alleviated the abnormal development and dysfunction of testicles in the offspring rats caused by prenatal stress. In summary, Zuogui Pills may weaken the effects of prenatal stress on testicular development and spermatogenic function of offspring rats by activating the PI3K/Akt pathway to regulate Cx43 expression in the testicular tissue.

Significantly shortened telomere length and altered androgen receptor level in cumulus cells from women with polycystic ovary syndrome

ObjectiveThe aim of this study was to investigate the correlation between hormone receptor levels and telomere length (TL) in infertile women with and without polycystic ovary syndrome (PCOS). Materials and methodsThis prospective cohort study recruited a total of 431 cumulus oocyte complex (COC) from 88 infertile women between July 2012 and June 2014. The participants were divided into three groups: young age (<38 years, n = 42 and 227 COC), advanced age (≥38 years, n = 33 and 107 COC) and PCOS patients (n = 13 and 97 COC). Cumulus cells were collected from individual follicle during oocyte pick-up, and the mRNA levels of hormone receptors and TL were measured using real-time PCR. ResultsThe cumulus cells of PCOS patients demonstrated lower mRNA levels of LH receptor (75.57 ± 138.10 vs. 171.07 ± 317.68; p < 0.01) and androgen receptor (1.13 ± 1.52 vs. 4.08 ± 9.57; p < 0.01), as well as a shorter TL (2.39 ± 2.58 vs. 3.96 ± 4.72; p < 0.01) compared to those of the young age group. In the young age group, only androgen receptor mRNA level showed a significant association with TL (rho = 0.148, p = 0.026), while FSH receptor mRNA level was the only factor associated with TL (rho = 0.247, p = 0.015) in PCOS patients. For advanced-aged patients, no significant relationship was observed between hormone receptor mRNA levels and TL. Alternative splicing of androgen receptors was identified in some PCOS patients but not in young age controls. ConclusionThe findings suggest that the androgen receptor level and function may be altered in the cumulus cells of PCOS patients, leading to a shorter TL in cumulus cells in PCOS patients.

Effects of Dietary Betaine on the Laying Performance, Antioxidant Capacity, and Uterus and Ovary Function of Laying Hens at the Late Stage of Production.

Betaine has been found to alleviate oxidative stress, inflammation, and apoptosis. However, whether dietary betaine can protect late-laying hens against these adverse effects is unknown. Here, 270 65-week-old Jinghong-1 laying hens were randomly divided into the Control, 0.1% Betaine, and 0.5% Betaine groups and fed a basal diet, 0.1%, and 0.5% betaine supplemented diet, respectively. The trial lasted for seven weeks. Birds that consumed 0.5% betaine laid more eggs with thicker eggshells. Accordingly, uterine reduced glutathione (GSH), glutathione peroxidase (GSH-PX), and ovarian superoxide dismutase (SOD) contents were increased. The uterine calcium ion content and the mRNA expression of ovalbumin, ovotransferrin, and carbonic anhydrase two were increased. Moreover, ovarian IL-1β, Caspase-1, Caspase-8, and Caspase-9 mRNA expressions were decreased; luteinising hormone receptor (LHR) and follicle-stimulating hormone receptor mRNA expressions were increased. Furthermore, dietary betaine decreased the ovaries' mRNA expression of DNA methyltransferase 1 (DNMT)1, DNMT3a, and DNMT3b. The methylation level at the promoter region of ovarian LHR decreased. These results indicated that dietary betaine consumption with a concentration of 0.5% could increase the laying rate and the eggshell thickness during the late-laying period. The underlying mechanism may include antioxidative, anti-apoptosis, and hormone-sensitivity-enhancing properties.

Dendrobium officinale polysaccharides improved reproductive oxidative stress injury in male mice treated with cyclophosphamide.

Polysaccharides from Dendrobium officinale polysaccharides (DOPs) are the main bioactive components of Dendrobium officinale, which have the functions of antioxidation and immune regulation. However, it is not clear whether DOPs have any effect on the prevention of reproductive disorders induced by oxidative stress. The purpose of this study was to explore the protective effect of DOPs on reproductive oxidative stress injury in male mice and its possible mechanism. In this study, the mouse model of reproductive injury was established by intraperitoneal injection of cyclophosphamide (CTX). The reproductive function was evaluated by relative testicular mass, sperm parameters, and sex hormone levels. The oxidative stress level of male mice with reproductive injury treated with DOPs was analyzed by the levels of 8-hydroxydeoxyguanosine (8-OHdG), malondialdehyde (MDA), and nitric oxide (NO) in sperm. The expression of follicle-stimulating hormone receptor (FSHR) mRNA, androgen-binding (ABP) mRNA, and c-kit mRNA was detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to explore its mechanism. After CTX administration, the sperm density, sperm motility, normal sperm morphology, and sex hormone levels in mice were significantly lower than those in the control group (P < 0.05). At the same time, the expression of p53 protein was upregulated, and the expression of Bcl-2 protein was downregulated (P < 0.05). In addition, the expression of FSHR and ABP mRNA on Sertoli cells was also significantly inhibited (P < 0.05). DOPs can effectively reduce the oxidative stress injury of testicular tissue. After DOP treatment, the sperm quality and sex-related hormone levels of mice were significantly improved and positively correlated with the dose of DOPs (P < 0.05). Administration of DOPs can reduce the damage caused by oxidative stress by reducing the level of oxidative stress, improving the hormone environment in testes, and regulating the expression of specific genes in Sertoli cells and spermatogenic cells.

Endocrine changes induced by GnRH immunization and re-stimulation of testicular function with a GnRH agonist in stallions

Resumption of testicular function after GnRH immunization varies among animals and some stallions regain fertility only after a prolonged time (Janett et al., 2009, Anim Reprod Sci 115:88e102). This study evaluated endocrine effects of GnRH immunization and subsequent re-stimulation with a GnRH agonist. We hypothesized that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. Shetland stallions were assigned to a GnRH vaccination group (Improvac; 400 µg/animal in weeks 0 and 4; n=6) or a saline control group (n=6). Blood was collected weekly for hormone analyses. Each vaccinated stallion was hemi-castrated together with an age-matched control when testosterone concentration was <0.3 ng/ml for two consecutive weeks (C1). Treatment of vaccinated stallions with the GnRH agonist buserelin (4 µg/day for 4 weeks and 8 µg/day for 6 weeks) started three weeks thereafter. Irrespective of treatment, the remaining testicle was removed when testosterone concentration increased to >0.5 ng/ml for two consecutive weeks in vaccinated stallions (C2; 9-10 weeks after start of buserelin treatment). Testicular tissues were stored at – 80°C and fixed in formalin for RT-qPCR and histology. Statistical comparisons were made by repeated measures ANOVA with group as between and time as within subject factor. Data on semen characteristics in these stallions have been published recently (Gautier et al., 2022, Theriogenology 194:27-34). Plasma testosteroneconcentration decreased after GnRH-vaccination and returned to control stallion concentrations during buserelin treatment (time p<0.001, time x group p<0.001, group p=0.001). Serum LH was undetectable after the second vaccination, increased during buserelin treatment but remained lower than in control stallions (time p=0.003, time x group p<0.001). Serum FSH concentration decreased after vaccination and increased with buserelin treatment in vaccinated but even more in control stallions after both hemi-castrations (time p<0.001, time x group p<0.001, group p=0.008). Relative mRNA abundance for LH receptor (LH-R) and aromatase (CYP19A1) were lower in testicular tissue of vaccinated than control stallions at C1 (p<0.001). At C2, LH-R mRNA abundance no longer differed between groups, whereas CYP19A1 mRNA remained lower in vaccinated than in control stallions (p<0.001). Relative FSH receptor (FSH-R) mRNA abundance was higher in vaccinated than in control stallions at C1 (p<0.001) and similar in both groups at C2. Immunolabelling for LH-R was lower in testes of GnRH-vaccinated compared to control stallions at C1 and C2 (p=0.007), whereas FSH-R protein expression at no time differed between groups. Aromatase immunostaining was reduced at C1 in vaccinated stallions and increased until C2 (time p=0.035, time x group p<0.0031, group p<0.001). In conclusion, endocrine testicular function can be advanced in GnRH-vaccinated stallions by daily low dose buserelin treatment.

A Longitudinal Study of the Relationship of Adiponectin with Reproduction in Infertile Women Undergoing IVF/ICSI Treatment, and an Experimental Study in Human Granulosa Cells.

This study investigated the roles of adiponectin in IVF treatment during Phase I (the basal stage before gonadotropin administration), Phase II (approximately 8 days after gonadotropin administration), and Phase III (on the ovum pick-up day), as well as the effects of adiponectin on CYP19A1 and the FSH receptor (FSHR) mRNA expression in a human granulosa-like tumor cell line (KGN). In human subjects (a longitudinal study, n = 30), blood samples were collected in all phases, while follicular fluid (FF) was only collected in Phase III. The participants were classified into successful and unsuccessful groups based on the determination of fetal heartbeats. KGN cells were treated with adiponectin/FSH/IGF-1 (an experimental study, n = 3). There was no difference in the adiponectin levels between successful and unsuccessful pregnancies in the FF (Phase III) and in serum (all phases), as well as among the three phases in both groups. Serum FSH (Phase I) was positively associated with serum adiponectin in the unsuccessful group, but it had a negative association in the successful group (all phases). Serum adiponectin and serum FSH (Phase I) were positively correlated in the unsuccessful group, whereas they were negatively correlated (all phases) in the successful group. The serum adiponectin levels (Phase III) were significantly higher than in the FF in unsuccessful pregnancies, but there was no difference in successful pregnancies. FF adiponectin concentrations were negatively correlated with serum LH in successful subjects. In KGN cells, adiponectin had no influence on CYP19A1 and FSHR mRNA expression. High adiponectin levels in serum compared to FF (Phase III) in unsuccessful subjects might negatively impact IVF treatment.

Transcriptomic analysis of ovarian follicles uncovers the crucial genes relevant to follicle selection and preovulatory hierarchy in hens.

Follicle selection and preovulatory hierarchy of hen ovaries were important stages of follicle development and crucially determining egg-laying performance. The selected follicles with a higher expression level of follicle-stimulating hormone receptor (FSHR) mRNA that facilitates response to FSH, and rapidly develops into preovulatory follicles with distinctive characteristics of granulosa cells (GCs) proliferation and differentiation. Identification of the key genes involved in these developmental events is helpful for elucidation of the molecular mechanism underlying egg-laying traits in chicken and other domestic fowl. Herein, the comparative transcriptomic analysis of ovarian prehierarchical follicles before selection (BSF), follicles at selection stage (ASF), and hierarchical follicles (HF) were implemented in the Jilin Black chicken (JB) and Lohmann Brown layer (LB) with the divergences in their egg-laying performance by RNA-sequencing. The results showed that nine deferentially expressed genes (DEGs), including STMN4, FABP3, ROBO2, RSPO4, and DMRT1 were revealed between follicles BSF and ASF; and seventeen DEGs, such as SLC6A15, SLITRK3, PRKG2 and TMC3 were mined between ASF and HF. These two group DEGs being co-expressed between BSF and ASF, and between ASF and HF were compared and substantiated in the JB and LB layers, respectively. Furthermore, 10 signaling pathways, such as cAMP signaling, PPAR signaling pathway, AMPK(Adenosine 5'-monophosphate (AMP)-activated protein kinase) pathway, and estrogen signaling pathway were also identified. Moreover, the roles of two representative candidates ROBO2 and PRKG2 genes presented as downregulated mRNA expression pattern in the transcriptomic profiles were further verified in vitro. The results demonstrated that downregulation of ROBO2 or PRKG2 significantly increased the expression levels of FSHR mRNA and protein with the boosted expression of CCND1, STAR, and BCL-2, whereas remarkably inhibited the expression of Caspase-3, consequently, brought about the decrease of GC apoptosis in the ovarian follicles, but increase of GC proliferation and differentiation serving as the hallmarks for follicle selection. It indicated that ROBO2 and PRKG2 may play indispensable roles in follicle selection and preovulatory hierarchy of hen ovaries separately. Our findings provided a comparative transcriptomic evidence for clarifying the molecular mechanism of the follicle development underlying egg-laying traits in chicken.

OR25-3 Sperm-carried IGF2 Downregulates Mitogens Released by Sertoli Cells: A Paracrine Mechanism of Spermatogenetic Regulation?

Abstract The presence of insulin-like growth factor 2 (IGF2) mRNA has been demonstrated in human and mouse spermatozoa. However, the expression of the IGF2 protein in spermatozoa and its function is not known. To accomplish this, we searched for IGF2 protein in spermatozoa of four healthy Caucasian men by Western blot (WB) and immunofluorescence (IM). Semen samples were collected by masturbation into a sterile container after 3-4 days of sexual abstinence. The spermatozoa were separated from leucocytes and epithelial cells by swim-up. The WB demonstrated the presence of the IGF2 protein in each sample examined. IM showed that IGF2 is a cytoplasmic protein with a variable degree of expression in each spermatozoon and localized in the equatorial and post-acrosomal segments. To evaluate the effects of IGF2, porcine Sertoli cells (SCs) were isolated from neonatal Large White pigs (7-15 days) using established methods. SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. At the end of incubation, the following endpoints were evaluated: a) expression of the GDNF, FGF2, and SCF genes, known to be mitogens that promote gonocyte proliferation and differentiation towards their spermatogonial fate, by real-time PCR (RT-PCR); b) gene (RT-PCR) and protein (WB and IM) expression of the follicle-stimulating hormone receptor (FSHR); and c) proliferation of SCs by flow cytometry. Subsequently, the effects of IGF2 were re-evaluated by pre-treating SCs with NVP-AEW541, a not competitive inhibitor of the insulin-like growth factor 1 receptor (IGF1R), known to be activated by IGF2. For this purpose, the NVP-AEW541 was added to the culture medium at a concentration of 1 µg/mL one hour before the IGF2 and was left for the entire duration of the incubation. The results of this study showed that IGF2 significantly downregulates GDNF gene expression in a concentration-dependent manner (-40.3% at 0.33 ng/mL, -48.2% at 3.33 ng/mL, -55.5%, respectively, p&amp;lt;0.01). Gene expression of FGF2 and SCF was significantly downregulated only by IGF2 at the highest concentration used (-23.6% and -39.1%, respectively, p&amp;lt;0.01). Similarly, IGF2 incubation downregulated FSHR mRNA (-27.1% at 0.33 ng/mL, -45.4.2% at 3.33 ng/mL, -21.1%, respectively, p&amp;lt;0.01) and protein (for both WB and IM) expression. Finally, SC proliferation significantly decreased after incubation with IGF2 (2.82±0.1% in untreated controls, -1.87±0.1% at 0.33 ng/mL, -2.19±0.2% at 3.33 ng/mL, and -2.36±0.1% at 10 ng/mL; p&amp;lt;0.01). In the presence of NVP-AEW541, all these effects were counteracted, thus suggesting that they are mediated by the IGF1R. In conclusion, these results suggest the presence of a paracrine regulatory mechanism between germ cells and SCs by which the former can influence the proliferation of SCs, their sensitivity to FSH, and their function by reducing the secretion of germ cell mitogens. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Association between cumulus cells—mRNA levels of AMHR2 and FSHR with oocyte maturity

BackgroundThe gene expression of anti-Mullerian hormone receptor type 2 (AMHR2) and follicle stimulating hormone receptor (FSHR) in cumulus cells (CCs) isolated from mature and immature oocytes was studied as a possible non-invasive approach for determining oocyte maturity and quality. The CCs of 100 infertile women with different etiologic factors were subdivided into control (CCs from MII) and case (CCs from GV) group. Q-PCR was used to evaluate FSHR and AMHR2 mRNA expression levels in CCs from mature and immature oocytes.ResultsAMHR2 and FSHR genes are significantly overexpressed (4–6 fold) in CCs from immature relative to mature oocyte. The expression level of AMHR2 gene in terms of etiologic subgroups is significantly different (P value 0.000). FSHR mRNA expression levels in CCs show no significant difference regarding etiologic subgroups (P value 0.575).ConclusionIt seems that determining the expression level of AMHR2 and FSHR genes in CCs could help to understanding molecular mechanism of oocyte maturation process.

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-β 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 (LHβ) mRNA, and follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone beta (FSHβ) 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.

Differential expression of FSHR and LHR genes and proteins during development of rabbit ovarian follicles.

The development of an ovarian follicle is a complex process at the cellular and molecular level that is mainly regulated by follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). To elucidate the contribution of these receptors to ovarian follicle development, it is necessary to determine their expression profiles during this biological process. Therefore, this study aimed to investigate the relationship between ovarian development pattern and the differential ovarian expression pattern of FSHR and LHR genes as well as proteins at different developmental stages. Ovaries were collected from 30 New Zealand rabbits at day 0 (birth), week 2 (neonate), week 4 (cub), week 16 (maturity), and day 18 pregnancy. Ovarian histology, and gene as well as protein expression were determined using light microscopy, real-time PCR and western blotting, respectively. The results showed that the expression levels of FSHR mRNA and protein increased coincidently with age and the growth of ovarian follicles. The levels of LHR mRNA and protein remained low from the day of birth until week 4 and became significantly higher by week 16 coinciding with appearance of growing and antral follicles, which have a defined thecal layer. FSHR gene and protein expression decreased with pregnancy, whereas LHR increased, reaching a peak level during pregnancy. It can be concluded that changes in FSHR and LHR gene and protein expression could be related to the growth and development of follicles, indicating the regulatory role for these receptors in rabbit folliculogenesis.

WT1 suppresses follicle-stimulating hormone-induced progesterone secretion by regulating ERK1/2 pathway in chicken preovulatory granulosa cells.

Multiple Wilms tumor gene 1 (WT1) splicing variants are expressed in mammals, and these variants regulate tumorigenesis and mediate the development of multiple tissues and organs, including gonads. However, WT1 splicing variants (+KTS or -KTS) are expressed in only two nonmammalian vertebrates, and unexpectedly, their functions in chicken ovaries remain elusive. Progesterone (P4) secreted by chicken granulosa cells (GCs) participates in various physiological processes and plays an important role in maintaining reproductive performance. The purpose of this study was to investigate the effect of WT1(+KTS) and WT1(-KTS) on chicken P4 secretion in preovulatory GCs. First, we detected WT1 mRNA expression in GCs from follicles of different developmental stages by Quantitative real-time PCR (RT-qPCR) and found that WT1 mRNA expression was considerably increased in preovulatory GCs compared with prehierarchical GCs. Primary cells collected from preovulatory follicles were treated with WT1(+KTS) or WT1(-KTS) overexpression vectors and subsequently cultured in the absence or presence of follicle-stimulating hormone (FSH). The mRNA levels of FSH-receptor (FSHR) and steroidogenesis genes were determined by RT-qPCR, and the P4 levels in the cell supernatants were measured by radioimmunoassay (RIA). Both WT1(+KTS) and WT1(-KTS) significantly decreased P4 secretion due to a reduction in FSHR, STAR and CYP11A1 mRNA levels. Western blotting revealed that ERK1/2 and BRAF phosphorylation levels were suppressed after overexpression of WT1(+KTS) or WT1(-KTS), whereas total protein and mRNA levels were not significantly changed. In addition, CREB protein and phosphorylation levels were inhibited after overexpression of WT1(+KTS) or WT1(-KTS). In conclusion, WT1(+KTS) and WT1(-KTS) inhibited CREB protein activity and significantly reduced FSHR, STAR and CYP11A1 mRNA levels, which subsequently suppressed FSH-induced P4 secretion in preovulatory GCs by modulating ERK1/2 signaling.

Effects of different energy diets on DNA methylation and mRNA expression in follicle stimulating hormone receptor gene promoter region of Duolang sheep during estrus.

The aim of this study was to study the relationship between the methylation level of the promoter region of follicle-stimulating hormone receptor (FSHR) gene and the mRNA expression of Duolang sheep fed different energy diets. In this study, polyembryo estrus Duolang sheep under different energy levels were selected as the experimental subjects. Dietary nutrient level reference (NY/T 816-2004), medium energy level was 10.88MJ/day, high and low energy groups were increased and decreased by 15% on the basis of medium energy level, respectively 12.51MJ/day, 9.25MJ/day. Through RNA and DNA extraction, qPCR, bisulfitegenomicse-quencing PCR (BSP), sequence matching and other analysis of ovarian tissue of Duolang sheep. The difference of DNA methylation level and mRNA expression of FSHR gene during estrus in Duolang sheep fed with different energy diets was detected. The results showed the expression level of FSHR in high energy group was significantly higher than that in low energy group (P < 0.01), the expression level of FSHR in high energy group was significantly higher than that in medium energy group (P < 0.05), and the expression level of FSHR in medium energy group was significantly higher than that in low energy group (P < 0.05). In the target fragment of the promoter region of the FSHR gene, the methylation rate was 25% in the high energy group, 50% in the normal group, and 75% in the low energy group. This study revealed that different dietary energy levels had certain effects on the FSHR gene DNA methylation level and mRNA expression, and the expression level was negatively correlated with methylation level.

The Effects of Zearalenone on the Localization and Expression of Reproductive Hormones in the Ovaries of Weaned Gilts.

This study aims to investigate the effects of zearalenone (ZEA) on the localizations and expressions of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), gonadotropin releasing hormone (GnRH) and gonadotropin releasing hormone receptor (GnRHR) in the ovaries of weaned gilts. Twenty 42-day-old weaned gilts were randomly allocated into two groups, and treated with a control diet and a ZEA-contaminated diet (ZEA 1.04 mg/kg), respectively. After 7-day adjustment, gilts were fed individually for 35 days and euthanized for blood and ovarian samples collection before morning feeding on the 36th day. Serum hormones of E2, PRG, FSH, LH and GnRH were determined using radioimmunoassay kits. The ovaries were collected for relative mRNA and protein expression, and immunohistochemical analysis of FSHR, LHR, GnRH and GnRHR. The results revealed that ZEA exposure significantly increased the final vulva area (p < 0.05), significantly elevated the serum concentrations of estradiol, follicle stimulating hormone and GnRH (p < 0.05), and markedly up-regulated the mRNA and protein expressions of FSHR, LHR, GnRH and GnRHR (p < 0.05). Besides, the results of immunohistochemistry showed that the immunoreactive substances of ovarian FSHR, LHR, GnRH and GnRHR in the gilts fed the ZEA-contaminated diet were stronger than the gilts fed the control diet. Our findings indicated that dietary ZEA (1.04 mg/kg) could cause follicular proliferation by interfering with the localization and expression of FSHR, LHR, GnRH and GnRHR, and then affect the follicular development of weaned gilts.

Moxibustion improves ovarian function based on the regulation of the androgen balance.

The effect of androgens on follicular development and female reproduction has become an active research topic. Moxibustion is a Traditional Chinese Medicine therapy that has been reported to be able to prevent and treat numerous ovary-related problems. However, studies on the effect of moxibustion for diminished ovarian reserve (DOR) on androgen balance are still lacking. The present study aimed to assess the efficacy of moxibustion intervention prior to disease onset and at the early stage of disease in a rat model of DOR and explore the mechanisms of its effect on ovarian function. A total of 32 rats were randomly divided into four groups: Blank group, Model group (a drug-induced model of DOR), Moxibustion group 1 and Moxibustion group 2. Moxibustion was performed on the BL23 and RN4 acupoints of female rats daily for a total of 20 days (once a day, five times a week for a total of 4 weeks). The two moxibustion groups were established with different intervention times: One group was subjected to pre-disease intervention and the other group to early-disease intervention. The ovarian function was evaluated by detecting anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), estradiol (E2), testosterone (T), dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT) and androgen receptor (AR) levels in the serum or the ovary samples. To further investigate the downstream regulatory factors for AR after moxibustion treatment for pre-disease or early-disease intervention, FSH receptor (FSHR) and microRNA (miR)-125b expression in ovaries were also analyzed. The results indicated that AMH and DHT levels were reduced in the model group compared with those in the blank group, while FSH, T and DHEA levels were increased. AMH and DHT levels were increased in Moxibustion group 1 compared with those in the model group, while FSH, T and DHEA levels were reduced. There was no difference in E2 levels between Moxibustion group 1 and the model group. Compared with that in the model group, the AR content in the ovary was increased in Moxibustion group 1. There was no difference in FSHR mRNA in the ovaries between Moxibustion group 1 and the model group. miR-125b levels were significantly increased in Moxibustion group 1 as compared with those in the model group. Furthermore, AMH and DHT levels were increased in Moxibustion group 2 compared with those in the model group, while FSH, T and DHEA levels were reduced. E2 levels were significantly decreased in Moxibustion group 2 compared with those in the model group. The relative mRNA expression of AR, FSHR and miR-125b was decreased following establishment of the model. Compared with that in the model group, the AR content in the ovary was increased in Moxibustion group 2. In comparison with the blank and model groups, the FSHR content in the ovary of Moxibustion group 2 was significantly increased. miR-125b levels were not obviously altered in Moxibustion group 2 as compared with those in the model group. In addition, there was no significant difference in AMH, FSH, T and DHEA levels between the two moxibustion groups. E2 and DHT levels were higher in Moxibustion group 1 than in Moxibustion group 2. There was no difference in AR mRNA expression between the two moxibustion groups. FSHR mRNA levels were lower in Moxibustion group 1 than in Moxibustion group 2, while miR-125b mRNA levels were higher in Moxibustion group 1 than in Moxibustion group 2. In conclusion, the present study suggested that moxibustion intervention prior to disease onset and at the early disease stage was able to improve ovarian function via modulation of the AR-mediated stable equilibrium of androgens. However, the effects and mechanisms of moxibustion intervention for pre-disease and early-disease intervention of DOR appear to be different. The appropriate duration of treatment and the time-effect relationship require to be further studied.

The Effects of Adiponectin on Infertile Women Undergoing IVF/ICSI Treatment and on Human Granulosa Cells

Adiponectin, one of the most abundant adipocyte-secreted protein, has been involved in female reproductive regulation. This study aimed to 1) compare serum adiponectin levels in various phases of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment including Phase I (the beginning of gonadotropin stimulation), Phase II (around 8 days after gonadotropin stimulation), and Phase III (on the day of ovum pick-up) between success and unsuccess subjects; 2) compare follicular fluid (FF) adiponectin levels between success and unsuccess groups; 3) compare serum adiponectin levels among different phases of IVF/ICSI treatment in success and unsuccess groups; 4) compare the levels of adiponectin between serum and FF; and 5) investigate the effects of adiponectin on mRNA expressions of follicle stimulating hormone receptor (FSHR) and CYP19A1 (aromatase) in human granulosa-like tumor cell line (KGN) (n=3). In the human study, recruited participants (n=30) with age of 26-40 years were enrolled between April 2018 - May 2019. Blood samples were collected at Phases I, II, and III while FF samples were collected at Phase III. Adiponectin levels were comparable between success and unsuccess subjects in both serum (all phases) and FF (Phase III). Furthermore, serum adiponectin levels were comparable among Phase I, II, and III in success and unsuccess groups. In Phase III, serum adiponectin showed positive correlations with serum adiponectin in Phase I and II and serum FSH in Phase I. Interestingly, adiponectin levels in FF were significantly lower than serum at Phase III in unsuccessful pregnancies but were comparable in successful pregnancies. Moreover, FF adiponectin had a negative correlation with serum LH at Phase III in success subjects. In the KGN cell study, adiponectin had no effects on FSHR and CYP19A1 (aromatase) mRNA expression compared with control. In conclusion, high adiponectin levels in serum compared to FF might contribute to unsuccessful IVF/ICSI treatment.

Differential molecular and hormonal changes in oocytes, granulosa cells and follicular fluid of pregnant and non-pregnant camels.

This study aimed to compare the expression of genes regulating follicles development, survival and steroid hormones secretion in oocytes and granulosa cells (GCs) and study the correlation between their expression and follicular fluid (FF) levels of progesterone (P4) in pregnant and non-pregnant camels. In total, 138 ovarian pairs from slaughtered camels were used. Gene expression and hormonal assay were determined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The obtained results revealed that the number of follicles (3-8 mm) was significantly (P < 0.05) lower in pregnant, compared with non-pregnant, camels. P4 level in the FF was significantly (P < 0.05) higher in pregnant, compared with non-pregnant, camels. However, no significant (P > 0.05) difference was noticed in the oestradiol (E2) level. STAR, PTEN, IGF1 and BCL2 mRNA levels were significantly higher in GCs and significantly lower in oocytes of pregnant, compared with non-pregnant, camels. However, follicle-stimulating hormone receptor (FSHR) mRNA level was significantly lower in GCs and oocytes, and the BMP15 mRNA level was significantly lower in oocytes of pregnant, compared with non-pregnant, camels. P4 level in FF was positively correlated with STAR, PTEN, IGF1 and BCL2 mRNA levels in GCs and negatively correlated with BMP15 mRNA levels in oocytes and FSHR mRNA levels in GCs and oocytes of pregnant camels. It could be concluded that pregnancy-induced variations in oocytes and GC expression of BMP15, IGF1, FSHR, STAR, BCL2, and PTEN genes might be associated with a decrease in the number of follicles and an increase in the FF level of P4.

Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte-cumulus cell complexes in vitro.

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2–1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte–cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0–100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.