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Effect of exogenous gonadotropin on the transcriptome of human granulosa cells and follicular fluid hormone profiles.

ABSTRACT: BACKGROUND:Superovulation treatment had some adverse effects on maturity and development of oocytes. Can superovulation dose of gonadotropins (Gns) affect the transcriptome of granulosa cells and follicular fluid (FF) hormone levels? METHODS:One leading pre-ovulatory follicle per subject was used from three natural-cycle and four Gn-stimulated patients. Granulosa cells and FF samples were collected from the same leading follicle of each patient. RNA was extracted from granulosa cells and subjected to deep sequencing and analysis. Follicle-stimulating hormone (FSH), estradiol (E2), androstenedione (AND), testosterone (T), luteinizing hormone (LH), and progesterone (P4) levels in FF were measured by immunoassays. Student's t test was used for statistical analysis. RESULTS:A total of 715 genes were up-regulated, and 287 genes were down-regulated, in the Gn-stimulated group relative to the control group. Gene Ontology analysis revealed that the down-regulated genes were enriched in cell cycle and meiosis pathways, primarily those associated with follicle or oocyte maturation and quality. On the other hand, the up-regulated genes were enriched in functions related to immunity and cytokine-cytokine receptor interactions. Compared to the follicles of natural cycle, the E2 and LH concentrations were significantly reduced (P?

SUBMITTER: Lu CL

PROVIDER: S-EPMC6591892 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Effect of exogenous gonadotropin on the transcriptome of human granulosa cells and follicular fluid hormone profiles.

Lu Cui-Ling CL Yan Zhi-Qiang ZQ Song Xue-Ling XL Xu Yang-Ying YY Zheng Xiao-Ying XY Li Rong R Liu Ping P Feng Huai-Liang HL Qiao Jie J

Reproductive biology and endocrinology : RB&E 20190624 1

<h4>Background</h4>Superovulation treatment had some adverse effects on maturity and development of oocytes. Can superovulation dose of gonadotropins (Gns) affect the transcriptome of granulosa cells and follicular fluid (FF) hormone levels?<h4>Methods</h4>One leading pre-ovulatory follicle per subject was used from three natural-cycle and four Gn-stimulated patients. Granulosa cells and FF samples were collected from the same leading follicle of each patient. RNA was extracted from granulosa ce ...[more]

PMID: 31234873

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Project description:BACKGROUND:Nerve growth factor (?-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified ?-Nerve Growth Factor (?-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas. METHODS:Experiment I: Female llamas (n?=?64) were randomly assigned to receive an intramuscular administration of: a) 50??g gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n?=?16), b) 1.0?mg of purified llama ?-NGF (n?=?16), or c) 1?ml phosphate buffered saline (PBS, negative control group, n?=?16). An additional group of llamas (n?=?16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20?h after treatment (Time 0?=?administration of treatment, n?=?8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n?=?24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20?h (n?=?4 replicates) and progesterone secretion at 48?h (n?=?4 replicates) after LH or ?-NGF treatment. RESULTS:Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified ?-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified ?-NGF with respect to the control group. All treatments except ?-NGF (20?h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20?h), or ?-NGF at 10 or 20?h after treatment. VEGF was up regulated only in those llamas submitted to mating (10?h) or treated with purified ?-NGF (10 and 20?h). Experiment II: Only ?-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20?h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20?h of in vitro culture from granulosa cells treated with ?-NGF whereas LH treatment increases VEGF mRNA abundance only at 20?h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48?h after LH or ?-NGF treatment. CONCLUSIONS:Systemic administration of purified ?-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or ?-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

Project description:ObjectiveThe follicular fluid (FF) plays an essential role in the physiology of the follicle and the oocyte. Gonadotropin stimulation affects the FF steroid hormone and anti-Mullerian hormone (AMH) concentrations, which has been suggested to be the reason for lower oocyte competence in conventional gonadotropin stimulated in vitro fertilisation (cIVF) compared to natural cycle IVF (NC-IVF). To analyse the effect of gonadotropin stimulation on a broad spectrum of signalling proteins, we ran proteomic antibody arrays on FF of women undergoing both treatments NC-IVF and cIVF.MethodTwenty women underwent one NC-IVF and one cIVF treatment cycle. Follicular fluids of the first aspirated follicle were compared between the two groups using a protein microarray which included antibodies against 224 proteins related to cell signalling and reference proteins. Each of the 40 albumin-stripped, matched-pair samples was labelled in the reverse-dye (Cy3/Cy5) procedure before undergoing array hybridisation. Signal analysis was performed using normalisation algorithms in dedicated software. Five proteins yielding a value of P < 0.05 in the array experiment (Cystatin A, Caspase-3, GAD65/67, ERK-1, and ERK-2) were then submitted to quantitative determination by ELISA in the same follicular fluids.ResultsArray analysis yielded only a small number of differentially expressed signalling markers by unadjusted P values. Adjustment as a consequence of multiple determinations resulted in the absence of any significant differential marker expression on the array. Five unadjusted differentially expressed proteins were quantified immunometrically with antibodies from different sources. Follicular fluid concentrations of Cystatin A and MAP kinase ERK-1 concentrations were significantly higher in the cIVF than in the NC-IVF follicles, while GAD-2 (GAD65/67) did not differ. The assays for Caspase-3 and MAP kinase ERK-2 did not have the required sensitivities.ConclusionIn contrast to FF steroid hormones and AMH, FF concentrations of signalling proteins are not or only marginally altered by gonadotropin stimulation.

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Effect of exogenous gonadotropin on the transcriptome of human granulosa cells and follicular fluid hormone profiles.

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Effect of exogenous gonadotropin on the transcriptome of human granulosa cells and follicular fluid hormone profiles.

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