ABSTRACT: Microarray profiling for differential gene expression in PSMG-hCG stimulated pre-ovulatory ovarian follicles of Chinese Erhualian and Large White sows
Project description:The Chinese Erhualian is one of the most prolific pig breeds in the world, which farrows at least five more piglets per litter than Western pig breeds partly due to a greater ovulation rate. Differences in the transcriptome of Chinese Erhualian and Large White ovaries directly result in variation of ovulation rate. To understand the molecular basis related to ovulation rate in Chinese indigenous and Western breeds, samples were collected and used to hybridized. This study reveals many potential avenues of investigation for seeking new insights into ovarian physiology and the genetic control of reproduction. Expression profiling experiments were conducted to identify differentially expressed genes in ovarian follicles at the preovulatory stage of a PMSG-hCG stimulated estrous cycle from 3 Chinese Erhualian and 3 Large White cycling sows by using the Affymetrix Porcine Genechip™.
Project description:Progesterone receptor (PGR) is an essential transcription factor for successful ovulation. Clinically, PGR inhibitors have been used as emergency contraceptives due to their ability to delay or block ovulation. While much of the current understanding of PGR’s function has been derived from animal models, the specific downstream target genes and pathways PGR regulates in human ovulatory follicles remain largely unexplored. In the present study, we utilized a human granulosa cell (hGLC) model that mimics preovulatory hCG-induced increases in PGR expression and progesterone production, combined with a PGR inhibitor (RU486), to identify hCG-induced, PGR-regulated genes in human ovulatory follicles. RNA-Seq and ChIP-Seq analyses revealed numerous genes regulated by PGR in response to human chorionic gonadotropin (hCG) treatment for 12 h, which are involved in diverse biological processes, including metabolism, steroidogenesis, signaling activation, cell cycle regulation, and transcriptional control. By comparing these findings with the existing data from PgrKO mouse models, we also identified both conserved and species-specific PGR-regulated genes. Notably, SOX9 was identified as a human-specific, hCG-induced PGR-target gene in hGLCs. A further characterization study using dominant follicle samples collected before and at different times after ovulatory hCG administration from regularly cycling women demonstrated that SOX9 expression is predominantly induced in the granulosa cells of ovulatory follicles and persists in the developing corpus luteum. Functional studies showed that SOX9 knockdown by siRNA reduced progesterone production and the expression of steroidogenesis-related genes (CYP11A1 and STAR) in hGLCs. In summary, the present finding provides the first comprehensive list of preovulatory PGR-downstream genes in human granulosa cells and unveils SOX9 as a novel human-specific ovulatory transcription factor with potential roles in steroidogenesis. This foundational information not only enhances our understanding of the PGR-regulated ovulatory pathway but also provides a critical resource for developing improved strategies to manage women’s fertility.
Project description:Progesterone receptor (PGR) is an essential transcription factor for successful ovulation. Clinically, PGR inhibitors have been used as emergency contraceptives due to their ability to delay or block ovulation. While much of the current understanding of PGR’s function has been derived from animal models, the specific downstream target genes and pathways PGR regulates in human ovulatory follicles remain largely unexplored. In the present study, we utilized a human granulosa cell (hGLC) model that mimics preovulatory hCG-induced increases in PGR expression and progesterone production, combined with a PGR inhibitor (RU486), to identify hCG-induced, PGR-regulated genes in human ovulatory follicles. RNA-Seq and ChIP-Seq analyses revealed numerous genes regulated by PGR in response to human chorionic gonadotropin (hCG) treatment for 12 h, which are involved in diverse biological processes, including metabolism, steroidogenesis, signaling activation, cell cycle regulation, and transcriptional control. By comparing these findings with the existing data from PgrKO mouse models, we also identified both conserved and species-specific PGR-regulated genes. Notably, SOX9 was identified as a human-specific, hCG-induced PGR-target gene in hGLCs. A further characterization study using dominant follicle samples collected before and at different times after ovulatory hCG administration from regularly cycling women demonstrated that SOX9 expression is predominantly induced in the granulosa cells of ovulatory follicles and persists in the developing corpus luteum. Functional studies showed that SOX9 knockdown by siRNA reduced progesterone production and the expression of steroidogenesis-related genes (CYP11A1 and STAR) in hGLCs. In summary, the present finding provides the first comprehensive list of preovulatory PGR-downstream genes in human granulosa cells and unveils SOX9 as a novel human-specific ovulatory transcription factor with potential roles in steroidogenesis. This foundational information not only enhances our understanding of the PGR-regulated ovulatory pathway but also provides a critical resource for developing improved strategies to manage women’s fertility.
Project description:The Chinese Erhualian is one of the most prolific pig breeds in the world, which farrows at least five more piglets per litter than Western pig breeds partly due to a greater ovulation rate. Differences in the transcriptome of Chinese Erhualian and Large White ovaries directly result in variation of ovulation rate. To understand the molecular basis related to ovulation rate in Chinese indigenous and Western breeds, samples were collected and used to hybridized. This study reveals many potential avenues of investigation for seeking new insights into ovarian physiology and the genetic control of reproduction.
Project description:Leukocytes, known as key effectors for ovulation, were found to specifically express oncostatin-M (OSM), while its receptors (OSMR and IL6ST) were abundant in granulosa cells, as shown in single-cell transcriptomics of follicular aspirates. OSM levels in the follicular fluid ranged 20.99 to 429.81 pg/ml. Increases in OSM-positive cells and OSMR and IL6ST expression were detected in ovulatory follicles post-hCG administration. Treating recombinant human OSM protein (rhOSM) to hCG-primed human granulosa/lutein cells activated signaling mediators, e.g., STATs. This treatment enabled hGLC cultures to mimic the in vivo ovulatory steroidogenesis dynamic, characterized by increased progesterone and reduced estradiol, unlike hCG alone. Additionally, rhOSM modulated gene expression associated with various ovulatory changes. This evidence suggests that leukocyte-derived OSM is a groundbreaking factor in human ovulation.
Project description:To investigate the difference between the pre-ovulatory F5 and pre-recruitment follicular stages in laying hens and identify master regulators that control the processes in these two follicular stages. RNA was isolated from a group of 3-5 pre-recruitment follicles and the most recently recruited pre-ovulatory follicle (F5). After RNA isolation, follicles underwent RNA sequencing and differential gene expression analysis occurred.
Project description:We previously established an alginate hydrogel-based 3D encapsulated in vitro follicle growth (eIVFG) system to culture mouse and human immature follicles in vitro. The alginate encapsulation maintained the 3D architecture of follicles and support their growth from the secondary to the antral stage to acquire maturation. Upon the stimulation of human chorionic gonadotropin (hCG), a LH analog, the grown antral follicle from eIVFG was able to rupture, undergo cumulus cell expansion, and ovulate a fertilizable metaphase II (MII) oocytes. We have also demonstrated that mouse follicles cultured using eIVFG preserved dynamic transcriptomic profile of many key genes that are essential for gonadotropin-dependent folliculogenesis, such as genes related to gonadotropin hormone receptors and ovarian steroidogenesis. However, it is unknown whether these follicles preserve molecular signatures of ovulation, which would enable this system to serve as a truly scalable and highly controllable system for nominating therapeutic and contraceptive candidates. In the present study, we treated mouse mature follicles from eIVFG with hCG to induce in vitro ovulation. Follicles were collected at 0, 1, 4, and 8 hours post-hCG for single-follicle RNA sequencing (RNA-seq) analysis. Our results showed that follicles grown from eIVFG preserve key ovulatory genes and signaling pathways.
Project description:A CNV map in pigs could facilitate the identification of chromosomal regions that segregate for important economic and disease phenotypes. The goal of this study was to identify CNV regions (CNVRs) in pigs based on a custom array comparative genome hybridization (aCGH). We carried out a custom-made array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the pig genome analysing animals of diverse pig breeds (White Duroc, Yangxin, Erhualian, Tongcheng, Large White, Pietrain, Landrace and Chinese new pig line DIV ) using a tiling oligonucleotide array with ~720,000 probes designed on the pig genome (Sus scrofa genome version 9.0). In this study, a custom-made tiling oligo-nucleotide 720k array was used with a median probe spacing of 2506 bp for screening 12 pigs with a female Duroc as the reference. WD: White Duroc (♀); YX: Yangxin (♂); EH: Erhualian (♀); TC: Tongcheng (♀); LW: Large White (♀); PT: Pietrain (♂); LD1: Landrace × DIV pig 1 (♂); LD2: Landrace × DIV pig 2 (♀); DIV1: Chinese new pig line DIV 1 (♀); DIV2: Chinese new pig line DIV 2 (♀); L1: Landrace 1 (♂); L2: Landrace 2 (♂).
Project description:Experiments were designed to evaluate changes in the transcriptome (mRNA levels) in the ovulatory, luteinizing follicle of rhesus monkeys, using a controlled ovulation (COv) model that permits analysis of the naturally selected, dominant follicle at specific intervals (0, 12, 24, 36 hours) after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle. Total RNA was prepared from individual follicles (n=4-8/timepoint), with an aliquot used for microarray analysis (AffymetrixTM Rhesus Macaque Genome Array) and the remainder applied to quantitative real-time PCR (q-PCR) assays. The microarray data from individual samples distinctly clustered according to timepoints, and ovulated follicles displayed markedly different expression patterns from unruptured follicles at 36 h. Between timepoint comparisons revealed profound changes in mRNA expression profiles. The dynamic pattern of mRNA expression for steroidogenic enzymes (CYP17A, CYP19A, HSD3B2, HSD11B1, HSD11B2), StAR, and gonadotropin receptors (LHCGR, FSHR) as determined by microarray analysis correlated precisely with those from blinded q-PCR assays. Patterns of mRNA expression for EGF-like factors (AREG, EREG) and processes (HAS2, TNFAIP6) implicated in cumulus-oocyte maturation/expansion were also comparable between assays. Thus, several mRNAs displayed the expected expression pattern for purported theca (e.g., CYP17A, AREG), granulosa (CYP19A, FSHR), cumulus (HAS2, TNFAIP6) cell, and surface epithelium (HSD11B) related genes in the rodent/primate preovulatory follicle. This database will be of great value in analyzing molecular and cellular pathways associated with periovulatory events in the primate follicle (e.g. follicle rupture, luteinization, inflammatory response, and angiogenesis), and for identifying novel gene products controlling mammalian fertility. Keywords: time course Total RNA was prepared from individual follicles (n=4-8/timepoint). Dominant follicles were selected specific intervals (0, 12, 24, 36 hours) after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle.