Follicles In Sheep Research Articles

7395 Developmental Programming: Adverse Effects Of Prenatal Exposure To A Real-Life Environmental Chemical Mixture Via Biosolids On Ovarian Folliculogenesis In Adult Sheep

Abstract Disclosure: Y. Zhou: None. K.M. Halloran: None. M. Bellingham: None. N.P. Evans: None. R.G. Lea: None. K.D. Sinclair: None. V. Padmanabhan: None. The ovarian capacity to provide fertilizable oocytes, regulate folliculogenesis as well as activate and progress primordial follicles into preovulatory follicles are key contributing factors to female fertility and reproductive aging. In sheep, as in humans, while genetics play a role in establishing the ovarian reserve during fetal development, developmental insults such as inappropriate exposure to native steroids or environmental chemicals (ECs) can adversely impact the establishment of the ovarian reserve and, additionally can affect folliculogenesis. Considering humans are exposed to multiple ECs simultaneously which could have additive, synergistic, or antagonistic effects, real-life EC exposure models are needed to assess risks posed by EC exposure. Offspring from sheep grazed on biosolids-treated pasture offers one such real-life exposure model. Studies with day 140 fetuses found maternal exposure to a real-life EC mixture via biosolids increased the proportion of unhealthy transitory follicles (Lea et al., Sci Rep. 2016;6:22279). The present study tested if biosolids exposure during fetal life would impact folliculogenesis, follicle activation and follicle atresia during their adult life. Ovaries were derived from adult EasyCare ewes (age 31.4 ± 0.5 months) whose mothers were grazed on biosolids-treated (BTP, n=10) or inorganic fertilizer-treated (Control, n=10) pastures from preconception through to birth. Ovarian morphometry was undertaken to assess the distribution and health status of follicles. Follicular atresia was evaluated via the expression of Caspase-3, an apoptosis marker. Data analysis utilized Student's t-test for normally distributed variables and Mann-Whitney U tests for non-parametric comparisons. There were no differences in the absolute counts or the percentages of follicle classes at all stages of folliculogenesis, the density of primordial follicles in cortical tissue, or the follicular activation rate. An increase in the percentage of unhealthy activated follicles (post-primordial) was noted in the BTP group (p = 0.014), predominantly in the transitory stage (p = 0.021). Consistent with this, a higher density of Caspase-3 positive primary follicles was observed in the BTP group (p = 0.033). These findings indicate the adverse impacts of fetal exposure to an EC mixture via maternal biosolid exposure on the survival of early-stage activated follicles in adult sheep. The lack of difference in ovarian reserve between the control and BTP groups suggests compensatory mechanisms may be in place to prevent premature depletion of the ovarian reserve. Whether EC mixture exposure via biosolids also has an adverse effect on the quality of the surviving activated follicles remains to be determined. Funding source: NIH R01 ES030374. Presentation: 6/3/2024

In vitro production of meiotically competent oocytes from early antral follicles in sheep

The potential of using long in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) from early antral follicles (EAFs) as an assisted reproductive technology in cattle has shown promising results. This study explored the feasibility of applying this technology to sheep as seasonal breeding animals. Ovaries from sheep were collected during both the breeding and non-breeding seasons. COCs were isolated from EAFs (350–450 μm) and cultured in TCM199 medium supplemented with 0.15 μg/mL Zn sulfate, 10−4IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 μM Cilostamide. After five days of LIVC, the COCs were submitted to an in vitro maturation procedure. The results indicate successful in vitro development of COCs, evidenced by a significant increase in oocyte diameter (p < 0.000) and the preservation of gap junction communication between oocyte and cumulus cells. The gradual uncoupling was accompanied by a progressive chromatin transition from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) (p < 0.000), coupled with a gradual decrease in global transcriptional activity and an increase in oocyte meiotic competence (p < 0.000). Maintenance of oocyte-cumulus investment architecture, viability, and metaphase II capability was significantly higher in COCs collected during the breeding season (p < 0.000), suggesting higher quality than those obtained during the non-breeding season. In conclusion, our study confirms LIVC feasibility in sheep, emphasizing increased effectiveness during the breeding season in isolating higher-quality COCs from EAFs. These findings can influence improving the LIVC system in mammals with seasonal reproduction.

POSTN promotes granulosa cell proliferation in sheep follicles through focal adhesion

BackgroundThe complex regulatory mechanisms involved in follicular development in sheep are not fully understood. This study aimed to understand the expression of periostin (POSTN) in granulosa cells (GCs) and its effect on follicle development. MethodsThe effect of luteinizing hormone (LH) treatment on POSTN expression in GCs was examined. POSTN was overexpressed or inhibited in GCs, and its effects on cell proliferation and apoptosis, as well as potential mechanisms of action, were evaluated. ResultsPOSTN showed higher expression in ovarian tissue than in other tissues and higher expression in large follicles than in small follicles. LH treated GCs promoted POSTN expression. Overexpression of POSTN in GCs significantly increased cell proliferation and inhibited apoptosis. Phosphorylation levels of focal adhesion kinase and autophagy were elevated in POSTN overexpressing GCs. ConclusionPOSTN affects GCs proliferation and follicle development through the focal adhesion, potentially improving fecundity in sheep.

MiR-134-3p targets HMOX1 to inhibit ferroptosis in granulosa cells of sheep follicles

BackgroundThe intricate interplay of gene expression within ovarian granulosa cells (GCs) is not fully understood. This study aimed to investigate the miRNA regulatory mechanisms of ferroptosis during the process of follicle development in lamb GCs.MethodsEmploying transcriptome sequencing, we compared differentially expressed mRNAs (DE-mRNAs) and miRNAs (DE-miRNAs) in GCs from lambs treated with follicle-stimulating hormone (FL) to untreated controls (CL). We further screened differentially expressed ferroptosis-related genes and identified potential miRNA regulatory factors. The expression patterns of HMOX1 and miRNAs in GCs were validated using qRT‒PCR and Western blotting. Additionally, we investigated the regulatory effect of oar-miR-134-3p on HMOX1 and its function in ferroptosis through cell transfection and erastin treatment.ResultsWe identified a total of 4,184 DE-mRNAs and 304 DE-miRNAs. The DE-mRNAs were mainly enriched in ferroptosis, insulin resistance, and the cell cycle. Specifically, we focused on the differential expression of ferroptosis-related genes. Notably, the ferroptosis-related genes HMOX1 and SLC3A2, modulated by DE-miRNAs, were markedly suppressed in FLs. Experimental validation revealed that HMOX1 was significantly downregulated in FL and large follicles, while oar-miR-134-3p was significantly upregulated compared to that in the CLs. HMOX1 expression was regulated by the targeting effect of oar-miR-134-3p. Functional assays further revealed that modulation of oar-miR-134-3p influenced HMOX1 expression and altered cellular responses to ferroptosis induction by erastin.ConclusionThis study suggested that oar-miR-134-3p and HMOX1 may be one of the pathways regulating ferroptosis in GCs. This finding provides new clues to understanding the development and regulatory process of follicles.

SOX18 Promotes the Proliferation of Dermal Papilla Cells via the Wnt/β-Catenin Signaling Pathway.

SRY-box transcription factor 18 (SOX18) is known to play a crucial role in the growth and development of hair follicles (HF) in both humans and mice. However, the specific effect of SOX18 on sheep hair follicles remains largely unknown. In our previous study, we observed that SOX18 was specifically expressed within dermal papilla cells (DPCs) in ovine hair follicles, leading us to investigate its potential role in the growth of hair follicles in sheep. In the present study, we aimed to examine the effect of SOX18 in DPCs and preliminarily study its regulatory mechanism through RNA-seq. We initially found that the overexpression of SOX18 promoted the proliferation of DPCs compared to the negative control group, while the interference of SOX18 had the opposite effect. To gain further insight into the regulatory mechanism of SOX18, we conducted RNA-seq analysis after knocking down SOX18 in Hu sheep DPCs. The result showed that the Wnt/β-Catenin signaling pathway was involved in the growth process of DPC after SOX18 knockdown. Subsequently, we investigated the effect of SOX18 on the Wnt/β-Catenin signaling pathway in DPCs using TOP/FOP-flash, qRT-PCR, and Western blot (WB) analysis. Our data demonstrated that SOX18 could activate the Wnt/β-Catenin signaling pathway in DPCs. Additionally, we observed that SOX18 could rescue the proliferation of DPCs after inhibiting the Wnt/β-Catenin signaling pathway. These findings underscore the essential role of SOX18 as a functional molecule governing the proliferation of DPCs. Additionally, these findings also greatly enhance our understanding of the role of SOX18 in the proliferation of DPCs and the growth of wool in Hu sheep.

LncRNAs regulate the cyclic growth and development of hair follicles in Dorper sheep.

Hair follicles in Dorper sheep are characterized by seasonal cyclic growth and development, consequently resulting in hair shedding during spring. The cyclic growth and development of hair follicles are regulated by several influencing factors such as photoperiods, hormones, age of the animal, genes, long non-coding RNAs (lncRNAs), and signaling pathways. In the present study, skin samples of five shedding sheep (S), used as experimental animals, and three non-shedding sheep (N), used as controls, were collected at three time points (September 27, 2019; January 3, 2020; and March 17, 2020) for RNA sequencing (RNA-seq) technology. Nine different groups (S1-vs-S2, S1-vs-S3, S2-vs-S3, N1- vs-N2, N1-vs-N3, N2-vs-N3, S1-vs-N1, S2-vs-N2, and S3-vs-N3) were compared using FDR < 0.05 and log 21 FC >as thresholds to assess the differences in the expression of lncRNAs. In total, 395 differentially expressed (DE) lncRNAs were screened. Cluster heatmap analysis identified two types of expression patterns, namely, high expression during the anagen phase (A pattern) and high expression during the telogen phase (T pattern). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the target genes were largely enriched in the Estrogen signaling pathway, PI3K-Akt signaling pathway, Fc gamma R-mediated phagocytosis, and cell adhesion molecules (CAMs), which are associated with hair follicle cyclic growth and development-related pathways. In addition, 17 pairs of lncRNAs-target genes related to hair follicle cyclic growth and development were screened, and a regulatory network was constructed. Altogether, candidate lncRNAs and their regulated target genes were screened that contributed to sheep hair follicle cyclic growth and development. We believe these findings will provide useful insights into the underlying regulatory mechanisms.

CRABP2 Promotes the Proliferation of Dermal Papilla Cells via the Wnt/β-Catenin Pathway

In our previous study of Hu sheep hair follicles, we found that CRABP2 was highly expressed in DPCs, which suggested that CRABP2 may influence the number of DPCs. In the present study, we aimed to understand the effect of CRABP2 in Hu sheep dermal papilla cells (DPCs). First, we explored the influence of CRABP2 on the ability of Hu sheep DPCs’ proliferation. Based on the results obtained from some experiments, such as CCK-8, EDU, qPCR, and Western blot experiment, we found that the overexpression of CRABP2 facilitated the proliferation of DPCs compared to the negative control group. Then, we also detected the effect of CRABP2 on the Wnt/β-catenin pathway based on the important function of the Wnt/β-catenin pathway in hair follicles. The results showed that CRABP2 could activate the Wnt/β-catenin pathway in DPCs, and it rescues the proliferation of DPCs when the Wnt/β-catenin pathway was inhibited. In summary, our findings indicate that CRABP2 is a vital functional gene in the proliferation of Hu sheep DPCs. Our study will be of great use for revealing the roles of CRABP2 in the hair follicles of Hu sheep.

New Technique for Human Sperm Cryopreservation Using Emptied Sheep's Ovarian Follicles.

This study aimed to investigate the efficacy of emptied ovarian follicles of sheep as a container for cryopreservation of human spermatozoa to preserve the presence of low concentrations of spermatozoa at the post-thawing stage. This research was performed on 30 semen samples from oligozoospermic patients and 10 samples from normozoospermic males. They were diagnosed according to the standard criteria of the World Health Organization 2010. Semen samples were classified into four groups of G1-G4 according to sperm concentration: 3-5 million/mL, 6-10 million/mL, 11-15 million/mL, and 16-20 million/mL, respectively. Each sample was divided into two equal parts. One part was cryopreserved without cryoprotectant, while the other was diluted 1:1 with 10% glycerol-based cryosolution. The ovarian follicles of sheep were obtained from a local slaughterhouse and prepared by slicing the ovaries and evacuating the follicular fluid and oocyte. The emptied follicles were injected with the prepared semen samples. After cryopreservation and thawing, the semen mixture aspired outside the follicles, and sperm parameters were measured, namely concentration, progressive motility, total motility, and normal morphology. Sperm concentration and progressive and total sperm motility were significantly (P<0.01) decreased in all groups at the post-thawing stage, compared to the pre-freezing stage. The sperm concentration was significantly higher (P<0.01) in samples cryopreserved without cryoprotectant, compared to that in those cryopreserved with glycerol. However, progressive and total motility were significantly (P<0.01) higher in samples cryopreserved with glycerol, compared to that in the samples cryopreserved without cryoprotectant in all groups. Moreover, no significant difference was found between the pre-freezing and post-thawing stages in terms of normal morphology. Emptied ovarian follicles are an appropriate carrier for cryopreservation of human sperms, especially for patients with oligozoospermia. The best sperm survival rate in this technique was observed when using glycerol-based cryosolution.

Seasonal Variability in Transcript Expression of Type I and Type II Keratins in Wool Follicles of Lustrous Fleece-Producing Magra Sheep

ABSTRACT Magra sheep reared around the Bikaner area of Rajasthan, India is famous for producing lustrous fleece as compared to other wool-producing native sheep breeds. Three clips of Magra wool, namely, February, July, and October months of the year are showing variations in luster and gross appearance. Present work has been conducted to unravel seasonal variations, in transcript expression patterns in major type I and type II keratin genes, that occur in wool follicles of lustrous wool-producing Magra sheep. Wool samples randomly collected were graded in high and low luster by Gloss 60° values. Wool follicles of the sheep with low (gloss 60° values; < 2.5) and high (gloss 60° values; > 2.5) luster were used for the analysis of type I and type II keratin transcripts. The results of Quantitative PCR had shown up-regulated expression of keratin genes in high-lustrous sheep as compared to low-lustrous sheep. K32, K40, and K82 transcript expression differed significantly (P ≤ .05) between high- and low-lustrous sheep. Nucleotide sequence analysis of the K40 and K82 genes revealed five and four non-synonymous mutations, respectively. From the present study, it can be speculated that the higher amount of keratin protein may play a crucial role in determining the luster property of the wool.

Comprehensive proteomic profiling of early antral follicles from sheep

The present study evaluates the proteome of early antral follicles from Ovis aries. Fifty follicles were collected from ovaries of adult ewes and extracted proteins were trypsin-digested, desalted and analyzed by LC-MS/MS. Genes were screened for potential modulation by miRNAs and protein data, subjected to functional enrichment analysis. Label-free mass spectrometry allowed the identification of 2503 follicle proteins, confirming vimentin, actin, lamin, heat shock proteins and histones as the most abundant ones. In silico analyses indicated that miRNAs modulate the expression of genes coding proteins of the sheep follicles involved in cell cycle, cell differentiation, aging, apoptosis, cell death, adipocyte differentiation, cell division. The most important biological processes associated with the follicle proteins were innate immune response, translation, adaptive immune response and protein folding, while molecular functions linked to the proteome of sheep antral follicles related to metal ion binding, ATP binding, oxygen binding, RNA binding and GTP binding, among others. Upload of 2503 Uniport accession codes through DAVID platform matched 1274 genes, associated with translation, metabolic process, proteolysis involved in cellular protein catabolic process, zona pellucida receptor complex and others. KEEG pathways analysis indicated genes correlated with ovine follicular development, with major pathways listed as carbon metabolism, biosynthesis of amino acids, glutathione metabolism, oxidative phosphorylation, fatty acid degradation and oocyte meiosis. This represents a comprehensive atlas of proteins expressed in sheep early antral follicles and will contribute to future identification of biomarkers for follicular development and oocyte maturation.

HDAC1 in the Ovarian Granulosa Cells of Tan Sheep Improves Cumulus Cell Expansion and Oocyte Maturation Independently of the EGF-like Growth Factors.

Simple SummaryThis study aimed to explore the possible role of histone deacetylase 1 (HDAC1), an epigenetic modifying enzyme, in the improvement of oocyte maturation recovered from the Tan sheep of China. We supplemented a specific inhibitor of HDAC1 to in vitro-cultured cumulus oocyte complexes (COCs). The results showed that the cumulus cell expansion and oocyte maturation ratio were impaired, while the production of the epidermal growth factor (EGF) was only slightly changed, despite the fact that the pan-acetylation levels of the histones were significantly elevated. This study implies that HDAC1 may participate in the maturation of oocytes in Tan sheep independently of the classic gonadotrophin-induction pathway.Previous studies have shown that some of the histone deacetylases (HDACs) play diverse roles in the regulation of ovarian somatic cell development, oocyte maturation and early embryonic development in different species including sheep. This study aimed to clarify whether HDAC1 also played pivotal roles in regulating oocyte maturation in Tan sheep. The results showed that HDAC1 was expressed in the nuclei of both the granulosa cells and oocytes of the growing follicles in the Tan sheep’s ovaries. However, the level of HDAC1 was unaffected by luteinizing hormone (LH) induction in cultured granulosa cells. Meanwhile, the specific inhibition of HDAC1 using pyroxamide did not induce significant changes in the expression levels of EGF-like growth factors in vitro, whereas both the cumulus expansion and oocyte maturation of the cultured cumulus oocyte complexes (COCs) were significantly inhibited by pyroxamide. Additionally, the numbers of histone acetylation sites (H4K5, H4K12, H3K14 and H3K9) in ovarian granulosa cells were significantly increased. In conclusion, a constant expression of HDAC1 in the growing follicles of Tan sheep may be pivotal for supporting oocyte growth and maturation, although its action may not be closely correlated with LH induction, nor does it directly affect the expression of the EGF-like factors. Our study implies that there may exist diverse functions of the respective HDACs in modulating female reproduction in sheep.

Expression and distribution patterns of VEGF, TGF-β1 and HIF-1α in the ovarian follicles of Tibetan sheep.

BackgroundHypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF‐β1) are multifunctional growth factors that play an important role in follicular growth and development. However, its biological function in the follicular development of Tibetan sheep at different stages has not been described.ObjectivesThe purpose of this study was to investigate the effect of VEGF, TGF‐β1 and HIF‐1α expression and distribution on the development of follicles of different sizes.MethodsImmunohistochemistry (IHC), western blot (WB) and quantification real‐time polymerase chain reaction (qRT‐PCR) were used to detect the localisation and quantitative expression of VEGF, TGF‐β1 and HIF‐1α proteins and mRNA in small‐ (< 3 mm), medium‐ (3 mm < diameter < 5 mm)‐, and large‐ (> 5 mm) sized follicles.ResultsThe results showed that the proteins VEGF, TGF‐β1 and HIF‐1α, as well as their mRNA, were expressed in follicles. However, the expression in medium‐sized follicles was significantly higher than that in large‐ and small‐sized follicles (p <0.05). IHC also showed that the proteins VEGF, TGF‐β1, and HIF‐1α were distributed in granulosa cells (GCs) in small‐, medium‐, and large‐sized follicles.ConclusionsThis study indicates that VEGF, TGF‐β1 and HIF‐1α, which operate in an autocrine or paracrine manner with the GCs, influence the follicular progressive growth, suggesting that these growth factors are closely associated with the follicular growth and development in ovarian.

Effects of testosterone on skin structure and factors related to androgen conversion and binding in Hetian sheep.

The effects of androgens on human skin mainly include the regulation of growth and differentiation of hair follicles and sebaceous glands. Androgens may have some physiological roles in sheep skin that are similar to those of humans, but further confirmation is needed. Therefore, Hetian sheep were chosen in this study as an animal model to explore the effects of testosterone on skin structure and factors related to androgen conversion and binding in Hetian sheep. The sheep were treated with different concentrations of testosterone for 42days. Skin tissue sections were prepared and then subjected to hematoxylin-eosin, Sacpic, Masson's trichrome, and Oil Red O staining to observe changes in skin morphology. Changes in the content of blood-related factors were also detected using ELISA kits. The skin tissue distribution of androgen receptor was explored by immunohistochemistry and immunofluorescence assays. The results showed that testosterone significantly increases the sebaceous gland area and stimulates the formation of new sebaceous glands. Further exploration revealed that testosterone promotes the proliferation of sebaceous gland juvenile cells. However, testosterone was found to have no significant effect on hair follicle density and hair follicle structure. Testosterone increased dihydrotestosterone levels but decreased 5α-reductase 1 and 5α-reductase 2 levels. The androgen receptors were distributed in the hair follicles, sebaceous glands, and some major skin appendages of Hetian sheep. This study suggests that androgens can be effective in regulating sebum production in sheep. This study will help advance research efforts to further explore the molecular and cellular mechanisms by which androgens modify sheep follicles and sebaceous glands.

Regulatory role of dihydrotestosterone on BMP-6 receptors in granular cells of sheep antral follicles.

Bone morphogenetic protein-6 (BMP-6) and dihydrotestosterone (DHT) affect steroid synthesis in follicles and regulate cell proliferation in the ovaries of female animals. However, little is known about granular cells (GCs) in sheep. We identified the key BMP-6 receptors, activin receptor-like kinase(ALK-6), and bone morphogenetic protein receptor type 2 (BMPRII) in sheep follicles using immunohistochemistry (IHC) and immunofluorescence (IF). Both ALK-6 and BMPRII were expressed in the GC layer, GC membranes, and cytoplasm. We evaluated ALK-6 and BMPRII expression at the follicular development stage using quantitative real-time PCR and western blotting to detect sheep GCs from large, medium, and small follicles (diameters of≥5, 2-5, and≤2mm, respectively). The mRNA abundance and protein expression of ALK-6 and BMPRII were significantly higher in GCs from large follicles compared to those in GCs from small follicles (P<0.05) and were the lowest in GCs from medium follicles. To assess whether DHT affects ALK-6 and BMPRII expression in sheep GCs, we cultured GCs from large follicles in vitro then incubated them with DHT (10-11, 10-9, 10-7 M). We found that 10-7-M DHT significantly inhibited ALK-6 and BMPRII mRNA and protein (P<0.05). We further explored whether DHT regulates ALK-6 and BMPRII through the nuclear androgen receptor (AR) pathway and found that 10-6-M flutamide, a non-selective androgen inhibitor, partially relieved the inhibitory effect of 10-7-M DHT on ALK-6 and BMPRII expression. Thus, GCs in sheep antral follicles differentially expressed ALK-6 and BMPRII at various stages, indicating that BMP-6 plays different roles to some extent during the development of antral follicles, and that high concentrations of DHT can inhibit the expression of ALK-6 and BMPRII via the androgen receptor pathway in sheep GCs. The present study aimed to determine the expression of the main BMP-6-related main receptors, namely, ALK-6 and BMPRII, during the development of GCs in sheep antral follicles and a potential mechanism of DHT regulation in sheep GCs. Our findings lay a foundation for the further exploration of the effects of ovarian BMP-6 expression on follicular development.

MiRNA expression analysis of the sheep follicle during the prerecruitment, dominant, and mature stages of development under FSH stimulation

Follicle stimulating hormone (FSH) has been widely reported to influence ovarian follicular development, and miRNAs play a significant role in mammalian follicular development by regulating their target genes. Therefore, it is of interest to explore the roles of miRNAs in sheep follicular development during FSH stimulation. In the current study, we constructed miRNA expression profiles of small follicles (SFs, prerecruitment stage), medium follicles (MFs, dominance stage), and large follicles (LFs, maturation stage). Three and 50 significant differentially expressed miRNAs (DEMs) were identified in the MF vs. SF and LF vs. SF comparisons, respectively, and none were identified in the LF vs. MF comparison. Oar-miR-10a was significantly downregulated in MFs compared with SFs. In LFs compared with SFs, miR-212-3p, miR-212-5p and miR-202-5p were significantly upregulated, and miR-27a-3p, miR-181a-5p, miR-204-5p, and miR-182-5p were significantly downregulated. Furthermore, we predicted the target genes of significant DEMs and performed functional enrichment analyses of these target genes. Analyses of KEGG pathways and GO terms showed that the putative target genes were significantly enriched in ovarian steroidogenesis, glutathione metabolism, positive regulation of cell differentiation, positive regulation of cell development, and cellular response to oxygen-containing compounds. Analyses of miRNA–gene regulatory networks suggested that miR-181a-5p–CYP11A1, (miR-27a-3p and miR-129-5p)–LDLR, (miR-212-3p and miR-212-5p)–EFNA5, (miR-181a-5p, miR-182-5p, and miR-27a-3p)–INHBA, and miR-182-5p–SOD2 might be involved in follicular development. The present study provides basic data and suggests research directions for further exploration of the roles of miRNAs in sheep follicular development under FSH stimulation.

Regulation of progesterone during follicular development by FSH and LH in sheep.

Progesterone (P4) can participate in the development of female mammalian antral follicles through nuclear receptor (PGR). In this experiment, the differences of P4 synthesis and PGR expression in different developmental stages of sheep antral follicles (large > 5mm, medium 2-5mm, small < 2mm) were detected by enzyme-linked immunosorbent assay, immunohistochemistry, qRT-PCR and Western blotting. Secondly, sheep follicular granulosa cells were cultured in vitro. The effects of different concentrations of FSH and LH on P4 synthesis and PGR expression were studied. The results showed that acute steroid regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3β Hydroxysteroid dehydrogenase (3β-HSD) and PGR were expressed in antral follicles, and with the development of antral follicles in sheep, StAR, P450scc and the expression of 3β-HSD and PGR increased significantly. In vitro experiments showed that FSH and LH alone or together treatment could regulate P4 secretion and PGR expression in sheep follicular granulosa cells to varying degrees, hint P4 and PGR by FSH and LH, and LH was the main factor. Our results supplement the effects of FSH and LH on the regulation of P4 synthesis during follicular development, which provides new data for further study of steroid synthesis and function in follicular development.

MiR-125b regulates the differentiation of hair follicles in Fine-wool Sheep and Cashmere goats by targeting MXD4 and FGFR2

With the development of miRNAs identification technology, more and more miRNAs have been discovered, and the role of miRNAs in the development of animal hair follicles has become a focus of research on hair-producing animals. In the previous experiment, compare the microRNA (miRNA) trancriptomes of goats and sheep skin using Solexa sequencing and differentially expressed miR-125b was screened. However, the mechanism of miR-125b regulating hair follicle development is not clear. Therefore, in the present study, the expression of miR-125b, MXD4 and FGFR2 in skin tissue of Fine-wool Sheep and Cashmere goats and HEK-293T cells was examined by qPCR and Western blot. Furthermore, the correlation between miR-125b and the predicted target gene (MXD4, FGFR2) was verified using the Dual-luciferase Reporter assay. We demonstrated that the expression of MXD4 and FGFR2 in Cashmere goats was significantly higher than that of Fine-wool Sheep, and the expression was opposite to that of miR-125b. miR-125b can down-regulate the levels of MXD4 and FGFR2. Dual-luciferase reporter gene assay showed that miR-125b could bind to the 3′-UTR region of target genes FGFR2 and MXD4, suggesting that MXD4 and FGFR2 were target genes of miR-125b. This study has shown that the growth and development of hair follicles in skin tissue of Fine-wool Sheep and Cashmere goats from the new regulatory levels of miRNAs, and clarified the mechanism of miR-125b and its target genes in the development of hair follicles in the skin.

Influence of TCM 199B, α-MEM, Waymouth MB 752/1 culture media, VEGF, Estradiol-17β, GDF-9 and FGF on in vitro development of preantral follicles in sheep.

A total of 2792 preantral follicles (PFs’) isolated from 750 ovaries of sheep were cultured in four different experiments. The efficacy of three commercially available culture media viz., TCM 199B, α-MEM and Waymouth MB 752/1 on the growth of sheep PFs’ was tested in experiment I. Among the three media TCM 199B supported better development of PFs’ in culture. The remaining experiments established the best concentrations of vascular endothelial growth factor (VEGF), Estradiol-17β (E2), GDF-9, Fibroblast growth factor (FGF) and their best combinations for the in-vitro development of PFs’. Inclusion of VEGF at 10 ng/mL, Estradiol-17β at 5 ng/mL, GDF-9 at 10 ng/mL or FGF at 10 ng/mL individually in a standard medium (SM) (containing FSH, IGF-I, GH and T4) supported better nuclear maturation of the oocytes to MII stage. Different combinations of VEGF, Estradiol-17β, GDF-9 and FGF supplemented in the SM promoted similar overall follicular growth. However, (a) SM + VEGF(10 ng/mL) + E2(5 ng/mL) supported higher increase in the diameter, (b) SM without any supplements induced antrum formation in greater proportion of follicles, and (c) SM + VEGF(10 ng/mL) + GDF 9(10 ng/mL) or SM + E2 (5 ng/mL) + FGF(10 ng/mL) supported high proportion of oocytes to reach MII stage. To conclude, TCM 199B appeared to be a better medium for development of sheep PFs’. VEGF, Estradiol-17 β, GDF-9 and FGF have beneficial influence on the development of sheep PFs’ when supplemented in TCM 199B

Follicle-stimulating hormone and luteinizing hormone regulate the synthesis mechanism of dihydrotestosterone in sheep granulosa cells.

Steroid hormones and receptors play important roles in female reproduction, and their expression patterns affect follicular growth and development. To examine the expression of dihydrotestosterone (DHT) synthases (5α-reductases (5α-red1 and 5α-red2)) and androgen receptor (AR) during follicular development, and the regulation of DHT signalling by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), we have used enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, immunohistochemical staining and Western blotting to examine DHT synthesis in small (≤2mm), medium (2-5mm) and large (≥5mm) sheep follicles. Expression of 5α-red1, 5α-red2 and AR was observed in ovine ovaries, and with the development of follicles, the expressions of 5α-red1 and 5α-red2 mRNA and protein increased, but the levels of AR mRNA, protein and DHT level decreased. In addition, granulosa cells were treated with FSH (0.01, 0.1 and 1 international unit (IU)/ml), LH (0.01, 0.1 and 1IU/ml) and testosterone (T, 10-7 M) to evaluate the effects of FSH and LH on DHT and oestradiol (E2) synthesis and 5α-red1, 5α-red2 and AR expression. We found that FSH and LH upregulated 5α-red1 and 5α-red2 in sheep granulosa cells, but downregulated the concentration of DHT and expression of AR. Meanwhile, FSH and LH significantly upregulated the expression of aromatase (P450arom) and secretion of E2. This result indicates that although FSH and LH promote the expression of 5α-red1 and 5α-red2, T is not transformed into DHT, but E2. This study reveals the reason why DHT concentration is downregulated in large follicles and lays a foundation for further exploring the synthesis mechanism of DHT during follicular development.

Effect of LH and Estradiol-17β Supplementation at Different Time Points on In vitro Development of Preantral Follicles in Sheep

Background: Ovarian follicular development and growth are controlled by many hormones and growth factors. Despite the fact that LH and estradiol-17β have been utilized for the in vitro culture of preantral follicles yet, the suitable time points of supplementation of LH and estradiol-17β is not known. Therefore this study aimed to investigate the influence of addition of LH and estradiol-17β at different time points on in vitro development of preantral follicles (PFs’) in sheep. Method: Preantral follicles isolated from the ovarian cortical slices using micro dissection method were cultured for six days in Bicarbonate buffered Tissue culture medium 199B (TCM 199B) or in a standard culture medium supplemented with LH (2 μg/ml) and estradiol-17β (5 ng/ml) at different points during the culture period. COCs isolated from the follicles at the end of six day culture in different treatments were subjected to in vitro maturation for additional 24h. Result: Supplementation of LH and estradiol-17β during last two days of the culture supported better proportion of PFs’ exhibiting growth whereas supplementation of LH and estradiol-17β during first two days of the culture supported better average increase in diameter and proportion of PFs’ exhibiting antrum formation at the end of six day culture. Further the oocytes in COCs isolated at the end of culture in these treatments and subsequently subjected to in vitro maturation (IVM) for 24hr developed at a higher frequency to MII (metaphase II) stage. Supplementation of LH and estradiol-17β to TCM 199B culture medium in early stages followed by standard medium alone in later stages supports better development of PFs’ in vitro. Following supplementation with LH and estradiol-17β for the first two days culture of PFs’ in standard medium appears to be advantageous for the development of preantral follicles in vitro.