Differential proteomic analysis demonstrates follicle fluid participate immune reaction and protein translation in yak

Background Ovarian follicle fluid (FF) as a microenvironment surrounding oocyte plays critical roles in physio-biochemical processes of follicle development and oocyte maturation. It is hypothesized that proteins in yak FF participate in the physio-biochemical pathways. The primary aims of this study were to find differentially expressed proteins (DEPs) between mature and immature FF, and to elucidating functions of the mature and immature FF in yak. Results The mature and immature FF samples were obtained from three healthy yaks that were nonpregnant, aged from four to five years, and free from any anatomical reproductive disorders. The FF samples were subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ). The FF samples went through correlation analysis, principle component analysis, and expression pattern analysis based on quantification of the identified proteins. Four hundred sixty-three DEPs between mature and immature FF were identified. The DEPs between the mature and immature FF samples underwent gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) analysis. The DEPs highly expressed in the mature FF mainly took parts in the complement and coagulation cascades, defense response, acute-phase response, response to other organism pathways to avoid invasion of exogenous microorganisms. The complement activation pathway contains eight DEPs, namely C2, C5, C6, C7, C9, C4BPA, CFH, and MBL2. The three DEPs, CATHL4, CHGA, and PGLYRP1, take parts in defense response pathway to prevent invasion of exogenetic microorganism. The coagulation cascades pathway involves many coagulation factors, such as F7, F13A1, FGA, FGB, FGG, KLKB1, KNG1, MASP1, SERPINA1, and SERPIND1. While the DEPs highly expressed in the immature FF participated in protein translation, peptide biosynthetic process, DNA conformation change, and DNA geometric change pathways to facilitate follicle development. The translation pathway contains many ribosomal proteins, such as RPL3, RPL5, RPS3, RPS6, and other translation factors, such as EIF3J, EIF4G2, ETF1, MOV10, and NARS. The DNA conformation change and DNA geometric change involve nine DEPs, DDX1, G3BP1, HMGB1, HMGB2, HMGB3, MCM3, MCM5, MCM6, and RUVBL2. Furthermore, the expressed levels of the main DEPs, C2 and SERPIND1, were confirmed by western blot. Conclusions The differential proteomics revealed the up-regulated DEPs in mature FF take parts in immunoreaction to prevent invasion of microorganisms and the up-regulated DEPs in immature FF participate in protein synthesis, which may improve our knowledge of the follicular microenvironment and its biological roles for reproductive processes in yak. The DEPs, C2 and SERPIND1, can be considered as protein markers for mature yak follicle.

Comparative Analysis Among Different Species Reveals That the Androgen Receptor Regulates Chicken Follicle Selection Through Species-Specific Genes Related to Follicle Development

The differences in reproductive processes at the molecular level between viviparous and oviparous animals are evident, and the site in the ovary that synthesizes sex hormones (androgens and oestrogens) and the trends for enriching sex hormones during follicle development in chickens are different from those in mammals, suggesting that the effect of sex hormones on follicle development in chickens is probably different from that in viviparous animals. To explore the specific role of androgen receptors (ARs) on chicken follicular development, we matched the correspondence of follicular development stages among chickens, humans, cows and identified chicken-specific genes related to follicle development (GAL-SPGs) by comparing follicle development-related genes and their biological functions among species (chickens, humans, and cows). A comparison of the core transcription factor regulatory network of granulosa cells (or ovaries) based on super-enhancers among species (chicken, human, and mouse) revealed that AR is a core transcriptional regulator specific to chickens. In vivo experiments showed that inhibition of AR significantly reduced the number of syf (selected stage follicles) in chickens and decreased the expression of GAL-SPGs in F5 follicles, while in vitro experiments showed that inhibition of AR expression in chicken granulosa cells (GCs) significantly down-regulated the expression levels of GAL-SPGs, indicating that AR could regulate follicle selection through chicken-specific genes related to follicle development. A comparison among species (77 vertebrates) of the conserved genomic regions, where chicken super-enhancers are located, revealed that the chicken AR super-enhancer region is conserved in birds, suggesting that the role of AR in follicle selection maybe widespread in birds. In summary, we found that AR can regulate follicle selection through chicken-specific genes related to follicle development, which also emphasizes the important role of AR in follicle selection in chickens and provides a new perspective for understanding the unique process of follicle development in chickens. Our study will contribute to the application of androgens to the control of egg production in chickens and suggests that researchers can delve into the mechanisms of follicle development in birds based on androgen/androgen receptors.

Oocyte quality following in vitro follicle development

In vitro follicle development (IVFD) is an adequate model to obtain basic knowledge of folliculogenesis and provides a tool for ovarian toxicity screening. IVFD yielding competent oocytes may also offer an option for fertility and species preservation. To promote follicle growth and oocyte maturation in vitro, various culture systems are utilized for IVFD in rodents, domestic animals, wild animals, nonhuman primates, and humans. Follicle culture conditions have been improved by optimizing gonadotropin levels, regulatory factors, nutrient supplements, oxygen concentration, and culture matrices. This review summarizes quality assessment of oocytes generated from in vitro-developed antral follicles from the preantral stage, including oocyte epigenetic and genetic profile, cytoplasmic and nuclear maturation, preimplantation embryonic development following in vitro fertilization, as well as pregnancy and live offspring after embryo transfer. The limitations of oocyte quality evaluation following IVFD and the gaps in our knowledge of IVFD to support proper oocyte development are also discussed. The information may advance our understanding of the requirements for IVFD, with a goal of producing competent oocytes with genetic integrity to sustain embryonic development resulting in healthy offspring.

Curcumin in Combination with Aerobic Exercise Improves Follicular Dysfunction via Inhibition of the Hyperandrogen-Induced IRE1α/XBP1 Endoplasmic Reticulum Stress Pathway in PCOS-Like Rats

Combining diet with exercise can improve health and performance. Exercise can reduce androgen excess and insulin resistance (IR) in polycystic ovary syndrome (PCOS) patients. Curcumin is also presumed to improve the follicle development disorder. Here, we investigated the effects of a combination therapy of oral intake of curcumin and exercise on hyperandrogen-induced endoplasmic reticulum (ER) stress and ovarian granulosa cell (GC) apoptosis in rats with PCOS. We generated a PCOS model via continuous dehydroepiandrosterone subcutaneous injection into the necks of Sprague Dawley rats for 35 days. PCOS-like rats then received curcumin treatment combined with aerobic (treadmill) exercise for 8 weeks. We found that compared to control rats, the ovarian tissue and ovarian GCs of hyperandrogen-induced PCOS rats showed increased levels of ER stress-related genes and proteins. Hyperandrogen-induced ovarian GC apoptosis, which was mediated by excessive ER stress and unfolded protein response (UPR) activation, could cause follicle development disorders. Both curcumin gavage and aerobic exercise improved ovarian function via inhibiting the hyperandrogen-activated ER stress IRE1α-XBP1 pathway. Dihydrotestosterone- (DHT-) induced ER stress was mitigated by curcumin/irisin or 4μ8C (an ER stress inhibitor) in primary GC culture. In this in vitro model, the strongly expressed follicular development-related genes Ar, Cyp11α1, and Cyp19α1 were also downregulated.

Single-Cell Transcriptomics Reveals the Molecular Anatomy of Sheep Hair Follicle Heterogeneity and Wool Curvature

Wool is the critical textile raw material which is produced by the hair follicle of sheep. Therefore, it has important implications to investigate the molecular mechanism governing hair follicle development. Due to high cellular heterogeneity as well as the insufficient cellular, molecular, and spatial characterization of hair follicles on sheep, the molecular mechanisms involved in hair follicle development and wool curvature of sheep remains largely unknown. Single-cell RNA sequencing (scRNA-seq) technologies have made it possible to comprehensively dissect the cellular composition of complex skin tissues and unveil the differentiation and spatial signatures of epidermal and hair follicle development. However, such studies are lacking so far in sheep. Here, single-cell suspensions from the curly wool and straight wool lambskins were prepared for unbiased scRNA-seq. Based on UAMP dimension reduction analysis, we identified 19 distinct cell populations from 15,830 single-cell transcriptomes and characterized their cellular identity according to specific gene expression profiles. Furthermore, novel marker gene was applied in identifying dermal papilla cells isolated in vitro. By using pseudotime ordering analysis, we constructed the matrix cell lineage differentiation trajectory and revealed the dynamic gene expression profiles of matrix progenitors' commitment to the hair shaft and inner root sheath (IRS) cells. Meanwhile, intercellular communication between mesenchymal and epithelial cells was inferred based on CellChat and the prior knowledge of ligand–receptor pairs. As a result, strong intercellular communication and associated signaling pathways were revealed. Besides, to clarify the molecular mechanism of wool curvature, differentially expressed genes in specific cells between straight wool and curly wool were identified and analyzed. Our findings here provided an unbiased and systematic view of the molecular anatomy of sheep hair follicle comprising 19 clusters; revealed the differentiation, spatial signatures, and intercellular communication underlying sheep hair follicle development; and at the same time revealed the potential molecular mechanism of wool curvature, which will give important new insights into the biology of the sheep hair follicle and has implications for sheep breeding.

CPEB3 deficiency in mice affect ovarian follicle development and causes premature ovarian insufficiency

Premature ovarian insufficiency (POI) is a heterogeneous and multifactorial disorder. In recent years, there has been an increasing interest in research on the pathogenesis and treatment of POI, owing to the implementation of the second-child policy in China. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that can bind to specific RNA sequences. CPEB3 can bind to and affect the expression, cellular location, and stability of target RNAs. Cpeb3 is highly expressed in the ovary; however, its functions remain unknown. In this study, Cpeb3-mutant mice were used to characterize the physiological functions of CPEB3. Cpeb3-mutant female mice manifested signs of gradual loss of ovarian follicles, ovarian follicle development arrest, increased follicle atresia, and subfertility with a phenotype analogous to POI in women. Further analysis showed that granulosa cell proliferation was inhibited and apoptosis was markedly increased in Cpeb3-mutant ovaries. In addition, the expression of Gdf9, a potential target of CPEB3, was decreased in Cpeb3-mutant ovaries and oocytes. Altogether, these results reveal that CPEB3 is essential for ovarian follicle development and female fertility as it regulates the expression of Gdf9 in oocytes, disruption of which leads to impaired ovarian follicle development and POI.

Genetic Basis of Follicle Development in Dazu Black Goat by Whole-Transcriptome Sequencing

The follicle development (FD) is an important factor determining litter size in animals. Recent studies have found that noncoding RNAs (ncRNAs) play an important role in FD. In particular, the role of the regulatory mechanism of competing endogenous RNAs (ceRNAs) that drive FD has attracted increasing attention. Therefore, this study explored the genetic basis of goat FD by obtaining the complete follicular transcriptome of Dazu black goats at different developmental stages. Results revealed that 128 messenger RNAs (mRNAs), 4 long noncoding RNAs (lncRNAs), 49 microRNAs (miRNAs), and 290 circular RNAs (circRNAs) were significantly differentially expressed (DE) between large and small follicles. Moreover, DEmRNAs were enriched in many signaling pathways related to FD, as well as GO terms related to molecular binding and enzyme activity. Based on the analysis of the ceRNA network (CRN), 34 nodes (1 DElncRNAs, 10 DEcircRNAs, 14 DEmiRNAs, and 9 DEmRNAs) and 35 interactions (17 DEcircRNAs–DEmRNAs, 2 DElncRNAs–DEmiRNAs, and 16 DEmRNA–DEmiRNAs) implied that the CRN could be involved in the FD of goats. In conclusion, we described gene regulation by DERNAs and lncRNA/circRNA–miRNA–mRNA CRNs in the FD of goats. This study provided insights into the genetic basis of FD in precise transcriptional regulation.

FSH Promotes Rat Follicle Development Through Inhibition of the Hippo Signalling Pathway

Purpose: Whether FSH promotes follicle growth by inhibiting the Hippo signalling pathway.METHODS: Ovaries were cultured in vitro into a control group (no intervention), an FSH group (0.3 IU/mL FSH), and a VP group (10 µg/mL vetiporfin). HE staining and follicle counts were performed at each stage after 3 hours of in vitro culture. Immunohistochemistry was performed to study the expression levels of LATS2, YAP, PLATS2, and PYAP, and their expression levels in each group were also analysed by Western blot.The number of secondary follicles was significantly increased in the FSH group, the arrangement of granulosa cells was neater, the nuclear fixation was reduced, and the number of atretic follicles was decreased in the VP group. The number of secondary follicles was significantly increased, the number of atretic follicles was reduced, and granulosa cell nuclear consolidation was reduced in the VP+FSH group. Immunohistochemistry showed that LATS2 and YAP expression levels were significantly increased and PLATS2 and PYAP expression levels were relatively decreased in the FSH group, PYAP and PLATS2 expression levels were significantly increased and YAP expression was significantly decreased in the VP group, and YAP and LATS2 expression levels were significantly increased and PYAP and PLATS2 expression levels were significantly decreased in the VP+FSH group. By Western blot, LATS2 and YAP were elevated and PYAP and PLAT2 were decreased in the FSH group, LATS2 and YAP were decreased and PYAP and PLATS were significantly elevated in the VP group, and LATS2 and YAP were elevated and PYAP and PLATS2 were decreased in the VP+FSH group.CONCLUSION: FSH promotes follicle development by inhibiting the Hippo signalling pathway.