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Transfer RNA-derived small RNAs (tsRNAs) are novel regulatory small non-coding RNAs that have been found to modulate many life activities in recent years. However, the exact functions of tsRNAs in follicle development remain unclear. Follicle development is a remarkably complex process that follows a strict hierarchy and is strongly associated with reproductive performance in ducks. The process of converting small yellow follicles into hierarchal follicles is known as follicle selection, which directly determines the number of mature follicles. We performed small RNA sequencing during follicle selection in ducks and identified tsRNA-00764 as the target of interest based on tsRNA expression profiles in this study. Bioinformatics analyses and luciferase reporter assays further revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) was the target gene of tsRNA-00764. Moreover, tsRNA-00764 knockdown promoted estrogen and progesterone synthesis and lipid deposition in duck granulosa cells, while a PPAR-γ inhibitor reversed the above phenomenon. Taken together, these results demonstrate that tsRNA-00764, differentially expressed in pre-hierarchal and hierarchy follicles, modulates estrogen and progesterone synthesis and lipid deposition by targeting PPAR-γ in duck granulosa cells, serving as a potential novel mechanism of follicle selection. Overall, our findings provide a theoretical foundation for further exploration of the molecular mechanisms underlying follicle development and production performance in ducks. [ABSTRACT FROM AUTHOR]

Simple Summary: It has been observed that red light enhances egg production in pigeons. However, the underlying histological characteristics and molecular mechanisms remain unclear. In this study, fifty-four White King pigeons were selected to confirm the effect of red light on egg production in pigeons and to assess the histological characteristics of follicles, reproductive hormone levels and ovarian transcriptomics on the third day of the laying interval under both red light and white light. This study revealed a molecular basis associated with red light promoting hierarchical follicle selection, enabling a better understanding of the underlying mechanisms of red light improving egg production in pigeons. Red light (RL) has been observed to enhance egg production in pigeons, yet the underlying histological characteristics and molecular mechanisms remain less understood. This study included fifty-four pigeons to assess follicular histology, reproductive hormones, and ovarian transcriptomics on the third day of the laying interval under RL and white light (WL). The results showed that the granulosa cell layer was significantly thicker under RL (p < 0.05), whereas the theca cell and connective tissue layers showed no significant differences (p > 0.05). Higher plasma estradiol (E2) levels were recorded in the RL group (p < 0.05), as well as follicle stimulating hormone (FSH), although progesterone (P4) levels were higher under WL (p < 0.05). Moreover, P4 concentrations in follicle yolk significantly decreased under RL (p < 0.01), with higher FSH and E2 levels in F1 yolk and similar increases in SF1 yolk (p < 0.01). Transcriptomic analysis revealed 4991 differentially expressed genes in the pigeon ovary. The protein–protein interaction network highlighted genes like HSD11B1, VEGFD, WNT6, SMAD6, and LGR5 as potential contributors to hierarchical follicle selection under RL. This research provides new insights into the molecular basis by which RL may promote hierarchical follicle selection and improve egg production in pigeons. [ABSTRACT FROM AUTHOR]

The decline in reproductive efficiency during post-peak period of production in poultry species holds significant economic implications. This study aimed to investigate the productive and reproductive performance of Japanese quails across distinct production stages and the association between these parameters and some genes expression and histometric alterations within the reproductive system. A total of 180 quails from a commercial flock were selected at varying egg production stages, including young, mature, and old, with 45 female and 15 male quails allocated to each group. The quails were maintained for six weeks. During recording period, daily records of egg production and egg weight were recorded. Additionally, oviduct histometric and Follicle biometric measurements, along with mRNA transcript abundance assessments related to follicular selection and yolk accumulation, were conducted on the oviduct, ovary, and small yellow follicles at the end of the experimental period. The results revealed a decrease in egg production in the old group compared to the young and mature groups (P < 0.05); meanwhile, the old group had the highest egg weight, and F1 follicle weight (P < 0.05). Additionally, the number of prehierarchical follicles was lower in the mature and old groups compared to the young group (P < 0.05). The lowest oviduct length, primary and secondary fold height, and thickness of the isthmus and magnum were noted in the old group (P < 0.05). Fertility and hatchability were lower in the old group compared to the other groups (P < 0.05). The mRNA transcript abundance of anti-Mullerian hormone (AMH), was highest in the old group and lowest in the young group (P < 0.05), while the mRNA transcript abundance of bone morphogenetic protein 15 (BMP15) was higher in the mature group compared to the other groups (P < 0.05). Additionally, the young quails had the highest occludin (OCLN) mRNA transcript abundance compared to other groups (P < 0.05). Overall, the study findings indicate decreased production and reproductive performance, as well as reduced hatchling quality over the production period, attributed to a declining number of follicles, noncooperative gene expression related to follicle selection and yolk accumulation, and diminishing oviduct fold size.

ABSTRACT: In avian, follicular selection is a key molecular event that can determine avian egg production. Theca cells (TC) are the main components of follicles, the molecular mechanisms about TCs physiological differences during follicle selection stage are still unclear. This study revealed significant differences in proliferation, apoptosis, lipid synthesis, and steroid secretion levels between prehierarchical theca cells (phTC) and hierarchical theca cells (hTC) of Tianfu meat-type geese. A total of 1,559 differentially expressed genes (DEG) and 71 differentially expressed miRNAs (DEM) were identified between phTCs and hTCs, respectively. Functional enrichment analysis results showed that 143 DEGs were enriched in the pathways related to cell proliferation/apoptosis and lipid/steroid metabolism. Protein-protein interaction (PPI) network results indicated the 143 DEGs have functional interactions. Additionally, the predicted target genes of 71 DEMs were jointly analyzed with the above 143 DEGs, and the results showed that 15 DEMs and 17 DEGs with targeted relationships were found. Among them, miR-202-5p was significantly down-regulated both in hTCs and hierarchical theca layers, and target prediction results showed that miR-202-5p may affect TCs proliferation/apoptosis by targeting CHPT1 to regulate the expression levels of CCN1/FOXO3; meanwhile, may affect TCs lipid/steroid metabolism and proliferation/apoptosis by targeting CHPT1 to regulate the expression levels of p53/ABCA1/SREBP-2. This study provides new insights into the regulatory mechanisms of TCs physiological differences during goose follicle selection.

Follicle selection, a crucial step in maintaining continuous egg production in chickens, is a process that relies on granulosa cells (GCs). In this study, we aimed to identify the key genes that are involved in follicle selection from our previous single-cell transcriptomic data. We used a combination of techniques and assays, including quantitative real-time PCR, immunofluorescence, Oil Red O staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), monodansylcadaverine (MDC) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, cell counting Kit-8 (CCK-8) assay, and 5-ethynyl-2-deoxyuridine (EdU) assay. Multiple indices, such as cell proliferation, cell differentiation, progesterone synthesis, lipid droplet production, total and free cholesterol content, apoptosis, and autophagy, were measured to determine the states of GCs in vitro. The results demonstrated that overexpression of genes related to insulin-like growth factor 2 (IGF2) or IGF-binding protein 4 (IGFBP4) increases intracellular free cholesterol (progesterone precursors) and lipid droplet production, inhibits apoptosis through increased autophagy, and inhibits cell proliferation. This indicates that IGF2 or IGFBP4 can maintain the survival state and improve differentiation tendency of chicken granulosa cells in vitro. Therefore, this study provides new evidence on the functions of IGFs and IGFBPs in chickens, establishing a crucial experimental foundation for understanding the regulatory mechanisms of follicle selection. In addition, our study contributes to understanding follicular development and improves the egg-laying performance of chickens.

Low egg-producing turkeys reduce the profitability of a flock by limiting the number of poults that can be hatched. Understanding the biological mechanics behind egg-production rates will greatly benefit the industry. Two lines with vastly different egg production rates are the Ohio State University E line, and its unselected counterpart, the random-bred control one (RBC1). Differences between E Line and RBC1 hens (n = 4 per line) were investigated by measuring egg production traits, ovarian and follicle anatomical characteristics, and gene expression for reproductively important genes within different follicle types. Data were analyzed by an ANOVA mixed model procedure in SAS. The E line hens produced 20% more eggs than the RBC1 hens, even though they had similar numbers of preovulatory follicles in their ovaries. This was accomplished by increasing clutch length and keeping the pause length the same. On the gene expression side small white follicles (SWF) within E line hens had less LHCGR expression which coincided with downregulation of CYP11A1 and CYP17A1. Along with an upregulation of PRLR in small yellow follicles (SYF) which also coincided with downregulation of CYP17A1. In both cases changes in pituitary hormone receptor transcription levels appeared to affect the steroid hormone synthesis pathway. In SWF from E line hens ESR2 was downregulated, however in the large white follicles and selected follicles ESR1 was the estradiol receptor which was downregulated. The similarity in preovulatory numbers suggests that E line hens aren't selecting more follicles to grow, but instead, follicles are growing faster. Based on the gene expression patterns, the reduction of steroid hormone synthesis might hint at the follicles putting more energy into growth and differentiation. At the same time, the decrease in estradiol receptor might limit the negative effects of estradiol on granulosa cells and allow for more rapid growth, suggesting a possible mechanism for the higher egg production trait of the E line.

ABSTRACT: Follicle selection in chicken refers to the process of selecting a follicle to enter hierarchy from a cohort of small yellow follicles (SY) with a diameter of 6 to 8 mm. The follicle being selected will develop rapidly and ovulate. Follicle selection is a key stage affecting chicken egg-laying performance. Our previous study showed that the phosphorylation level of lysine (K)-specific demethylase 1A (LSD1) at serine 54 (LSD1Ser54p) was significantly increased in F6 follicles compared to prehierarchal SY follicles, but its function was unclear. Here, the mechanism of this modification, the effect of LSD1Ser54p dephosphorylation on gene expression profile of chicken hierarchal granulosa cells and the function of fibroblast growth factor 9 (FGF9) that is regulated by LSD1Ser54p were further investigated. The modification of LSD1Ser54p was predicted to be mediated by cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). Treatment of chicken hierarchal granulosa cells with CDK5 inhibitor significantly decreased LSD1Ser54p level (P < 0.05) and LSD1Ser54p interacted with CDK5, suggesting that, in the granulosa cells of chicken hierarchal follicles, LSD1Ser54p modification was carried out by CDK5. When the LSD1Ser54p level decreased in the granulosa cells of chicken hierarchal follicles, both the mRNA expression of FGF9 and α-actinin 2 (ACTN2) and the H3K4me2 level in their promoter regions significantly increased (P < 0.05), indicating that this phosphorylation modification enhanced the demethylation activity of LSD1. Moreover, in chicken hierarchal granulosa cells, overexpression of chicken FGF9 stimulated their proliferation and increased the mRNA expression of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b) and steroidogenic acute regulatory protein (StAR). This study collectively revealed that phosphorylation of LSD1 at serine 54 by CDK5 enhanced its demethylation activity in chicken ovarian granulosa cells and regulated genes including FGF9 that is engaged in chicken follicle selection.

Follicle selection in hens refers to a biological process that only one small yellow follicle (SYF) is selected daily or near‐daily for following hierarchical development (from F5/F6 to F1) until ovulation. MFN2 is a kind of GTPases located on the mitochondrial outer membrane, which plays a crucial role in mitochondrial fusion. This study aimed to elucidate the role of MFN2 in proliferation and progesterone biosynthesis of granulosa cells (GCs) during follicle selection in hens. The results showed that GCs began to produce progesterone (P4) after follicle selection, accompanied with changes from multi‐layer with flat cells to single layer with cubic cells. MFN2 was detected in GCs of follicles from SYF to F1. After follicle selection, the expression level of MFN2 in GCs upregulated significantly, accompanied with increases in P4 biosynthesis, ATP production, mitochondrial DNA (mtDNA) copy numbers of granulosa cells. FSH (80 ng/mL) facilitated the effects of P4 biosynthesis and secretion, ATP production, mtDNA copy numbers, cell proliferation and the MFN2 transcription of granulosa cells from F5 (F5G) in vitro. However, FSH treatment did not promote P4 secretion in granulosa cells from SYF (SYFG) in vitro. Meanwhile, we observed that change fold of MFN2 transcription, ATP production, mtDNA copy numbers and cell proliferation rate in F5G after treatment with FSH were greater than those in SYFG. Furthermore, expression levels of MFN2 protein and messenger RNA in F5G were significantly higher than those in SYFG after treatment with FSH. P4 biosynthesis, ATP production, mtDNA copy numbers as well as cell proliferation reduced significantly in F5G with MFN2 knockdown. Oppositely, P4 biosynthesis, ATP production, mtDNA copy numbers and cell proliferation increased significantly in SYFG after the overexpression of MFN2. Our results suggest that the upregulation of MFN2 may be involved in the initiation of P4 biosynthesis, and promotion of GCs proliferation during follicle selection. [ABSTRACT FROM AUTHOR]

Red light (RL) has been observed to enhance egg production in pigeons, yet the underlying histological characteristics and molecular mechanisms remain less understood. This study included fifty-four pigeons to assess follicular histology, reproductive hormones, and ovarian transcriptomics on the third day of the laying interval under RL and white light (WL). The results showed that the granulosa cell layer was significantly thicker under RL (p < 0.05), whereas the theca cell and connective tissue layers showed no significant differences (p > 0.05). Higher plasma estradiol (E2) levels were recorded in the RL group (p < 0.05), as well as follicle stimulating hormone (FSH), although progesterone (P4) levels were higher under WL (p < 0.05). Moreover, P4 concentrations in follicle yolk significantly decreased under RL (p < 0.01), with higher FSH and E2 levels in F1 yolk and similar increases in SF1 yolk (p < 0.01). Transcriptomic analysis revealed 4991 differentially expressed genes in the pigeon ovary. The protein–protein interaction network highlighted genes like HSD11B1, VEGFD, WNT6, SMAD6, and LGR5 as potential contributors to hierarchical follicle selection under RL. This research provides new insights into the molecular basis by which RL may promote hierarchical follicle selection and improve egg production in pigeons.

Simple Summary: The follicular selection stage during the development of goose follicles is a decisive step in egg production, but current research is still incomplete. With the help of high-throughput sequencing technology, we analyzed the mRNAs, miRNAs, and lncRNAs that play a critical role only in the follicular selection stage and ultimately constructed a ceRNA regulatory network. We confirmed the significant changes in the extracellular matrix, cell junction, and glucose and lipid metabolism during follicular selection. More importantly, using a new analytical approach, we identified miRNAs that play an important role in follicle selection, including miR-222-3p, miR-2954-3p, miR-126-5p, miR-2478, and miR-425-5p. This study provides potential regulatory targets for precise regulation of goose production performance. The selection of follicles determines the reproductive performance of birds, but the process of follicle selection in geese is still elusive. This study focuses on Yangzhou geese during the egg-laying period and divides the follicular development process into three stages: small follicle development, follicle selection, and follicle maturation. Transcriptome sequencing was performed on granulosa cells from large white follicles, small yellow follicles, and F5 and F4 follicles. In addition, we selected the transcripts that remained unchanged during the development and maturation of small follicles but significantly changed during the follicular selection stage as the transcript collection that plays an important role in the follicular selection process. Then, we performed functional analysis on these transcripts and constructed a ceRNA network. The results showed that during the follicular selection stage, the number of differentially expressed mRNAs, miRNAs, and lncRNAs was the highest. In addition, miR-222-3p, miR-2954-3p, miR-126-5p, miR-2478, and miR-425-5p are potential key core regulatory molecules in the selection stage of goose follicles. These results can provide a reference for a better understanding of the basic mechanisms of the goose follicle selection process and potential targets for the precise regulation of goose egg production performance. [ABSTRACT FROM AUTHOR]

Abstract Background Follicle selection in chickens refers to the process of selecting one follicle from a group of small yellow follicles (SY, 6–8 mm in diameter) for development into 12–15 mm hierarchal follicles (usually F6 follicles), which is controlled by sex hormones including follicle-stimulating factor (FSH), estrogen and progesterone. Follicle selection is a critical process impacting egg production in chicken, therefore, is the focus of many studies. Phosphorylation is important for the proper function of proteins, thus, needs to be analyzed by proteomic level. Result In this study, we compared the phosphoproteomes of SY and F6 follicles in laying hens and identified 2,386 phosphoproteins and 5,940 phosphosites, of which 4,235 sites of 1,963 phosphoproteins were quantified. From SY to F6 follicles, 190 phosphorylation sites of 149 proteins changed significantly, among which the phosphorylation level of lysine demethylase 1 A (LSD1) at the conserved 54th serine (LSD1Ser54p) was significantly upregulated in F6 follicles compared to SY follicles (p

ABSTRACT: Quercetin (Que), widely found in a huge variety of plants, plays important roles in ovarian function. However, to data, there have been no reports about Que regulating granulosa cells (GCs) in prehierarchical follicles in chicken. Herein, GCs from follicles diameter from 4 to 8 mm in chicken were treated by Que in vitro culture to investigate how Que exerts its effect on follicular development. GCs treated by Que in concentrations of 10, 100, and 1,000 ng/mL were tested for cell proliferation and progesterone secretion. Eight cDNA libraries were constructed from GCs (4 samples per group) to explore transcriptome expression changes. The role of the MAPK/ERK signaling pathway was validated in this process. Treatment with 100 and 1,000 ng/mL levels of Que significantly promoted cell proliferation and progesterone secretion (P < 0.05). RNA-seq analysis data showed that 402 and 263 differentially expressed genes (DEGs) were up- and down-regulated, respectively. Functional enrichment analysis that the pathways related to follicular development included biosynthesis of amino acids, MAPK signaling pathway, and calcium signaling pathway. Notably, the function exerted in GCs of the different levels of Que was associated with the suppression of the MAPK pathway. In conclusion, our results proved that low levels of Que could promote MAPK signaling pathway, but high levels of Que inhibit MAPK signaling pathway in GCs from the prehierarchical follicles, promote cell proliferation and progesterone secretion, and benefit follicle selection.

ABSTRACT: Follicle selection is an important step in the laying process of chicken, which is closely related to the laying performance and fecundity of hens. Follicle selection mainly depends on the regulation of follicle-stimulating hormone (FSH) secreted by pituitary gland and the expression of follicle stimulation hormone receptor. To uncover the role of FSH in chicken follicle selection, in this study, we analyzed the changes in the mRNA transcriptome profiles of FSH-treated chicken granulosa cells from prehierarchical follicles by long-read sequencing Oxford Nanopore Technologies (ONT) approach. Among the 10,764 genes detected, 31 differentially expressed (DE) transcripts of 28 DE genes were significantly upregulated by FSH treatment. These DE transcripts (DETs) were mainly related to the steroid biosynthetic process by GO analysis and enriched in pathways of ovarian steroidogenesis and aldosterone synthesis and secretion by KEGG analysis. Among these genes, the mRNA and protein expression of TNF receptor associated factor 7 (TRAF7) was upregulated after FSH treatment. Further study revealed that TRAF7 stimulated the mRNA expression of steroidogenic enzymes steroidogenic acute regulatory protein (StAR) and cytochrome P450 family 11 subfamily A member 1 (CYP11A1) genes and the proliferation of granulosa cells. This is the first study to investigate differences in chicken prehierarchical follicular granulosa cells before and after FSH treatment by using ONT transcriptome sequencing, which provides a reference for a more comprehensive understanding of the molecular mechanism of follicle selection in chicken.

Background: Follicle selection in chickens refers to the process of selecting one follicle from a group of small yellow follicles (SY, 6–8 mm in diameter) for development into 12–15 mm hierarchal follicles (usually F6 follicles), which is controlled by sex hormones including follicle-stimulating factor (FSH), estrogen and progesterone. Follicle selection is a critical process impacting egg production in chicken, therefore, is the focus of many studies. Phosphorylation is important for the proper function of proteins, thus, needs to be analyzed by proteomic level. Result: In this study, we compared the phosphoproteomes of SY and F6 follicles in laying hens and identified 2,386 phosphoproteins and 5,940 phosphosites, of which 4,235 sites of 1,963 phosphoproteins were quantified. From SY to F6 follicles, 190 phosphorylation sites of 149 proteins changed significantly, among which the phosphorylation level of lysine demethylase 1 A (LSD1) at the conserved 54th serine (LSD1Ser54p) was significantly upregulated in F6 follicles compared to SY follicles (p < 0.05); however, the expression of chicken LSD1 were not significantly different on both mRNA and protein levels. LSD1Ser54p is mainly located in the nucleus of both SY and F6 follicles, and was higher in F6 follicles than that of SY follicles revealed by both immunofluorescence and Western blotting. LSD1Ser54p level increased after treatment with 5 ng/mL and 10 ng/mL of FSH in the theca cells and the granulosa cells of pre-hierarchal follicles, and with 50 ng/mL in granulosa cells of hierarchal follicles. In the theca cells of hierarchal follicles, estrogen stimulated the level of LSD1Ser54p in a dosage-dependent manner, and in granulosa cells of pre-hierarchal follicles, 10 ng/mL of estrogen increased LSD1Ser54p expression. Treatment with 50 ng/mL of progesterone increased LSD1Ser54p expression in theca cells of pre-hierarchal follicles, and with 10 to 100 ng/ml enhanced LSD1Ser54p expression in the granulosa cells of hierarchal follicles. Conclusion: The expression dynamics of LSD1Ser54p in follicles from SY to F6 and its regulation by sex hormones suggest that it is involved in chicken follicle selection. [ABSTRACT FROM AUTHOR]

Taihang chickens are a domestic breed distributed throughout Hebei province in the Taihang Mountains of China and are characterized by their high meat and egg quality. However, the relatively limited egg production by this breed constrains their more widespread commercial utilization. The follicle selection process is closely linked to oocyte development and ovulation, making it a key determinant of laying performance and fecundity in hens. To understand the biological basis for such follicle selection and to identify the associated regulatory pathways, we conducted a genome-wide analysis of long noncoding RNAs (lncRNAs) and mRNAs from the pre-hierarchical follicles and hierarchical follicles of Taihang laying hens. We identified 81 lncRNAs and 528 mRNAs that were differentially expressed during follicle selection, and integrated network analyses suggested that these RNAs were associated with the cell cycle, focal adhesion, oocyte meiosis, peroxisome proliferator-activated receptor signaling, and steroid hormone biosynthesis pathways. The identified lncRNAs were also predicted to influence a series of target genes in cis and trans, suggesting that they may be important regulators of ovarian follicular development. Overall, the present analysis of lncRNA and mRNA expression patterns associated with ovarian follicle development offers a new foundation for future studies of reproductive physiology in Taihang chickens, highlighting new opportunities to improve the laying performance of this important domestic breed. • Long noncoding RNAs (lncRNAs) and mRNAs were widely expressed in follicle of domestic Taihang Chicken. • In chicken follicle selection, 81 lncRNAs and 528 mRNAs were differentially expressed. • Follicle selection highlighted 4 cell pathway and hormone biosynthesis pathway. • Non-coding RNAs are important regulators of ovarian follicular development. [ABSTRACT FROM AUTHOR]

The decline in reproductive efficiency during post-peak period of production in poultry species holds significant economic implications. This study aimed to investigate the productive and reproductive performance of Japanese quails across distinct production stages and the association between these parameters and some genes expression and histometric alterations within the reproductive system. A total of 180 quails from a commercial flock were selected at varying egg production stages, including young, mature, and old, with 45 female and 15 male quails allocated to each group. The quails were maintained for six weeks. During recording period, daily records of egg production and egg weight were recorded. Additionally, oviduct histometric and Follicle biometric measurements, along with mRNA transcript abundance assessments related to follicular selection and yolk accumulation, were conducted on the oviduct, ovary, and small yellow follicles at the end of the experimental period. The results revealed a decrease in egg production in the old group compared to the young and mature groups (P < 0.05); meanwhile, the old group had the highest egg weight, and F1 follicle weight (P < 0.05). Additionally, the number of prehierarchical follicles was lower in the mature and old groups compared to the young group (P < 0.05). The lowest oviduct length, primary and secondary fold height, and thickness of the isthmus and magnum were noted in the old group (P < 0.05). Fertility and hatchability were lower in the old group compared to the other groups (P < 0.05). The mRNA transcript abundance of anti-Mullerian hormone (AMH), was highest in the old group and lowest in the young group (P < 0.05), while the mRNA transcript abundance of bone morphogenetic protein 15 (BMP15) was higher in the mature group compared to the other groups (P < 0.05). Additionally, the young quails had the highest occludin (OCLN) mRNA transcript abundance compared to other groups (P < 0.05). Overall, the study findings indicate decreased production and reproductive performance, as well as reduced hatchling quality over the production period, attributed to a declining number of follicles, noncooperative gene expression related to follicle selection and yolk accumulation, and diminishing oviduct fold size. [ABSTRACT FROM AUTHOR]

Follicle selection, a crucial step in maintaining continuous egg production in chickens, is a process that relies on granulosa cells (GCs). In this study, we aimed to identify the key genes that are involved in follicle selection from our previous single-cell transcriptomic data. We used a combination of techniques and assays, including quantitative real-time PCR, immunofluorescence, Oil Red O staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), monodansylcadaverine (MDC) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, cell counting Kit-8 (CCK-8) assay, and 5-ethynyl-2-deoxyuridine (EdU) assay. Multiple indices, such as cell proliferation, cell differentiation, progesterone synthesis, lipid droplet production, total and free cholesterol content, apoptosis, and autophagy, were measured to determine the states of GCs in vitro. The results demonstrated that overexpression of genes related to insulin-like growth factor 2 (IGF2) or IGF-binding protein 4 (IGFBP4) increases intracellular free cholesterol (progesterone precursors) and lipid droplet production, inhibits apoptosis through increased autophagy, and inhibits cell proliferation. This indicates that IGF2 or IGFBP4 can maintain the survival state and improve differentiation tendency of chicken granulosa cells in vitro. Therefore, this study provides new evidence on the functions of IGFs and IGFBPs in chickens, establishing a crucial experimental foundation for understanding the regulatory mechanisms of follicle selection. In addition, our study contributes to understanding follicular development and improves the egg-laying performance of chickens. [ABSTRACT FROM AUTHOR]

In avian, follicular selection is a key molecular event that can determine avian egg production. Theca cells (TC) are the main components of follicles, the molecular mechanisms about TCs physiological differences during follicle selection stage are still unclear. This study revealed significant differences in proliferation, apoptosis, lipid synthesis, and steroid secretion levels between prehierarchical theca cells (phTC) and hierarchical theca cells (hTC) of Tianfu meat-type geese. A total of 1,559 differentially expressed genes (DEG) and 71 differentially expressed miRNAs (DEM) were identified between phTCs and hTCs, respectively. Functional enrichment analysis results showed that 143 DEGs were enriched in the pathways related to cell proliferation/apoptosis and lipid/steroid metabolism. Protein-protein interaction (PPI) network results indicated the 143 DEGs have functional interactions. Additionally, the predicted target genes of 71 DEMs were jointly analyzed with the above 143 DEGs, and the results showed that 15 DEMs and 17 DEGs with targeted relationships were found. Among them, miR-202-5p was significantly down-regulated both in hTCs and hierarchical theca layers, and target prediction results showed that miR-202-5p may affect TCs proliferation/apoptosis by targeting CHPT1 to regulate the expression levels of CCN1 / FOXO3 ; meanwhile, may affect TCs lipid/steroid metabolism and proliferation/apoptosis by targeting CHPT1 to regulate the expression levels of p53 / ABCA1 / SREBP -2. This study provides new insights into the regulatory mechanisms of TCs physiological differences during goose follicle selection. [ABSTRACT FROM AUTHOR]

In chickens, follicle selection is an important process affecting laying traits, which is characterized by the differentiation of granulosa cells and the synthesis of progesterone by granulosa cells from hierarchical follicles. By using Oxford Nanopore Technologies (ONT) approach, we compared the transcriptomes of granulosa cells between pre-hierarchical (Pre-GCs) and hierarchical follicles (Post-GCs) to identify genes underlying chicken follicle selection. A total of 2,436 differentially expressed genes (DEGs), 3,852 differentially expressed transcripts (DETs) and 925 differentially expressed lncRNA transcripts were identified between chicken Pre-GCs and Post-GCs. For all of the significant DETs, the alternative 3′splice sites (A3) accounted for a maximum of 23.74% of all alternative splicing events. Three DETs of the 7-dehydrocholesterol reductase gene (DHCR7) named as T1, T3, and T4, differing in 5′untranslated regions (UTRs), increased in Post-GCs with different folds (T1: 1.83, T3: 2.42, T4: 5.06). The expression of the three DHCR7 transcripts was upregulated by estrogen in a dose-dependent manner, while was downregulated by bone morphogenetic protein 15 (BMP15) and transforming growth factor-beta 1 (TGF-β1). Follicle-stimulating hormone (FSH) and bone morphogenetic protein 4 (BMP4) promoted the expression of the three DHCR7 transcripts in Pre-GCs at lower concentrations, while repressed their expression at higher concentrations. The data from this study may provide a reference for better understanding of the genetic mechanisms underlying follicle selection in chicken and other poultry species.

Low egg-producing turkeys reduce the profitability of a flock by limiting the number of poults that can be hatched. Understanding the biological mechanics behind egg-production rates will greatly benefit the industry. Two lines with vastly different egg production rates are the Ohio State University E line, and its unselected counterpart, the random-bred control one (RBC1). Differences between E Line and RBC1 hens (n = 4 per line) were investigated by measuring egg production traits, ovarian and follicle anatomical characteristics, and gene expression for reproductively important genes within different follicle types. Data were analyzed by an ANOVA mixed model procedure in SAS. The E line hens produced 20% more eggs than the RBC1 hens, even though they had similar numbers of preovulatory follicles in their ovaries. This was accomplished by increasing clutch length and keeping the pause length the same. On the gene expression side small white follicles (SWF) within E line hens had less LHCGR expression which coincided with downregulation of CYP11A1 and CYP17A1. Along with an upregulation of PRLR in small yellow follicles (SYF) which also coincided with downregulation of CYP17A1. In both cases changes in pituitary hormone receptor transcription levels appeared to affect the steroid hormone synthesis pathway. In SWF from E line hens ESR2 was downregulated, however in the large white follicles and selected follicles ESR1 was the estradiol receptor which was downregulated. The similarity in preovulatory numbers suggests that E line hens aren't selecting more follicles to grow, but instead, follicles are growing faster. Based on the gene expression patterns, the reduction of steroid hormone synthesis might hint at the follicles putting more energy into growth and differentiation. At the same time, the decrease in estradiol receptor might limit the negative effects of estradiol on granulosa cells and allow for more rapid growth, suggesting a possible mechanism for the higher egg production trait of the E line., Competing Interests: Declaration of competing interest The authors listed do not have any conflicts of interest to report., (Copyright © 2024. Published by Elsevier Inc.)

Objective Follicle selection is an important process in chicken egg laying. Among several small yellow (SY) follicles, the one exhibiting the highest expression of follicle stimulation hormone receptor (FSHR) will be selected to become a hierarchal follicle. The role of lncRNA, miRNA and other non-coding RNA in chicken follicle selection is unclear. Methods In this study, the whole transcriptome sequencing of SY follicles with different expression levels of FSHR in Jining Bairi hens was performed, and the expression of 30 randomly selected mRNAs, lncRNAs and miRNAs was validated by quantitative real-time polymerase chain reaction. Preliminary studies and bioinformatics analysis were performed on the selected mRNA, lncRNA, miRNA and their target genes. The effect of identified gene was examined in the granulosa cells of chicken follicles. Results Integrated transcriptomic analysis on chicken SY follicles differing in FSHR expression revealed 467 differentially expressed mRNA genes, 134 differentially expressed lncRNA genes and 34 differentially expressed miRNA genes, and sosondowah ankyrin repeat domain family member A (SOWAHA) was the common target gene of three miRNAs and one lncRNA. SOWAHA was mainly expressed in small white (SW) and SY follicles and was affected by follicle stimulation hormone (FSH) treatment in the granulosa cells. Knockdown of SOWAHA inhibited the expression of Wnt family member 4 (Wnt4) and steroidogenic acute regulatory protein (StAR) in the granulosa cells of prehierarchal follicles, while stimulated Wnt4 in hierarchal follicles. Overexpression of SOWAHA increased the expression of Wnt4 in the granulosa cells of prehierarchal follicles, decreased that of StAR and cytochrome P450 family 11 subfamily A member 1 in the granulosa cells of hierarchal follicles and inhibited the proliferation of granulosa cells. Conclusion Integrated analysis of chicken SY follicle transcriptomes identified SOWAHA as a network gene that is affected by FSH in granulosa cells of ovarian follicles. SOWAHA affected the expression of genes involved in chicken follicle selection and inhibited the proliferation of granulosa cells, suggesting an inhibitory role in chicken follicle selection.

The selection of follicles determines the reproductive performance of birds, but the process of follicle selection in geese is still elusive. This study focuses on Yangzhou geese during the egg-laying period and divides the follicular development process into three stages: small follicle development, follicle selection, and follicle maturation. Transcriptome sequencing was performed on granulosa cells from large white follicles, small yellow follicles, and F5 and F4 follicles. In addition, we selected the transcripts that remained unchanged during the development and maturation of small follicles but significantly changed during the follicular selection stage as the transcript collection that plays an important role in the follicular selection process. Then, we performed functional analysis on these transcripts and constructed a ceRNA network. The results showed that during the follicular selection stage, the number of differentially expressed mRNAs, miRNAs, and lncRNAs was the highest. In addition, miR-222-3p, miR-2954-3p, miR-126-5p, miR-2478, and miR-425-5p are potential key core regulatory molecules in the selection stage of goose follicles. These results can provide a reference for a better understanding of the basic mechanisms of the goose follicle selection process and potential targets for the precise regulation of goose egg production performance.

Follicle selection in chicken refers to the process of selecting a follicle to enter hierarchy from a cohort of small yellow follicles (SY) with a diameter of 6 to 8 mm. The follicle being selected will develop rapidly and ovulate. Follicle selection is a key stage affecting chicken egg-laying performance. Our previous study showed that the phosphorylation level of lysine (K)-specific demethylase 1A (LSD1) at serine 54 (LSD1Ser54p) was significantly increased in F6 follicles compared to prehierarchal SY follicles, but its function was unclear. Here, the mechanism of this modification, the effect of LSD1Ser54p dephosphorylation on gene expression profile of chicken hierarchal granulosa cells and the function of fibroblast growth factor 9 (FGF9) that is regulated by LSD1Ser54p were further investigated. The modification of LSD1Ser54p was predicted to be mediated by cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). Treatment of chicken hierarchal granulosa cells with CDK5 inhibitor significantly decreased LSD1Ser54p level (P < 0.05) and LSD1Ser54p interacted with CDK5, suggesting that, in the granulosa cells of chicken hierarchal follicles, LSD1Ser54p modification was carried out by CDK5. When the LSD1Ser54p level decreased in the granulosa cells of chicken hierarchal follicles, both the mRNA expression of FGF9 and α-actinin 2 ( ACTN2 ) and the H3K4me2 level in their promoter regions significantly increased (P < 0.05), indicating that this phosphorylation modification enhanced the demethylation activity of LSD1. Moreover, in chicken hierarchal granulosa cells, overexpression of chicken FGF9 stimulated their proliferation and increased the mRNA expression of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 ( Hsd3b ) and steroidogenic acute regulatory protein ( StAR ). This study collectively revealed that phosphorylation of LSD1 at serine 54 by CDK5 enhanced its demethylation activity in chicken ovarian granulosa cells and regulated genes including FGF9 that is engaged in chicken follicle selection. [ABSTRACT FROM AUTHOR]

Magang geese exhibit a unique characteristic of follicular development, with eight largest orderly arranged pre-ovulatory follicles in the abdominal cavity. However, little is known about the mechanisms underlying this follicular development. This study aimed to compare gene expression profiles of granulosa cells (GCs) at different stages of follicular development and provide comprehensive insights into follicle selection and the mechanisms underlying the well-defined follicle hierarchy in Magang geese. GCs of large white follicles (LWFs), small yellow follicles (SYFs), F8, F4, and F1 were used for RNA-seq analysis; 374, 1117, 791, and 593 genes were differentially expressed in stages LWFs to SYFs, SYFs to F8, F8 to F4, and F4 to F1, respectively, suggesting that these genes contribute to follicle selection and development. Reliability of sequencing data was verified through qPCR analysis of 24 genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways revealed a complex mechanism that remodels the extracellular matrix and turnover of extracellular matrix components in follicular development and ovulation and involves multiple pathway, such as focal adhesion, adherens junction, and extracellular matrix–receptor interaction. Some unique characteristics were observed during the different follicular development stages. For instance, some differentially expressed genes were enriched in progesterone-mediated oocyte maturation and steroid biosynthesis from stage SYFs to F8, whereas others were enriched in actin cytoskeleton regulation and vascular smooth muscle contraction from stage F4 to F1. These findings enhance our current understanding of GC function and ovarian follicles during the key stages of follicular development.

Granulosa cells (GCs) are decisive players in follicular development. In this study, the follicle tissues and GCs were isolated from the goose during the peak-laying period to perform hematoxylin-eosin staining and RNA-seq, respectively. Moreover, the dynamic mRNA and lncRNA expression profiles and mRNA-lncRNA network analysis were integrated to identify the important genes and lncRNAs. The morphological analysis showed that the size of the GCs did not significantly change, but the thickness of the granulosa layer cells differed significantly across the developmental stages. Subsequently, 14,286 mRNAs, 3,956 lncRNAs, and 1,329 TUCPs (transcripts with unknown coding potential) were detected in the GCs. We identified 37 common DEGs in the pre-hierarchical and hierarchical follicle stages, respectively, which might be critical for follicle development. Moreover, 3,089 significant time-course DEGs (Differentially expressed genes) and 13 core genes in 4 clusters were screened during goose GCs development. Finally, the network lncRNA G8399 with CADH5 and KLF2 , and lncRNA G8399 with LARP6 and EOMES were found to be important for follicular development in GCs. Thus, the results would provide a rich resource for elucidating the reproductive biology of geese and accelerate the improvement of the egg-laying performance of geese. [ABSTRACT FROM AUTHOR]

Granulosa cells (GCs) are decisive players in follicular development. In this study, the follicle tissues and GCs were isolated from the goose during the peak-laying period to perform hematoxylin-eosin staining and RNA-seq, respectively. Moreover, the dynamic mRNA and lncRNA expression profiles and mRNA-lncRNA network analysis were integrated to identify the important genes and lncRNAs. The morphological analysis showed that the size of the GCs did not significantly change, but the thickness of the granulosa layer cells differed significantly across the developmental stages. Subsequently, 14,286 mRNAs, 3,956 lncRNAs, and 1,329 TUCPs (transcripts with unknown coding potential) were detected in the GCs. We identified 37 common DEGs in the pre-hierarchical and hierarchical follicle stages, respectively, which might be critical for follicle development. Moreover, 3,089 significant time-course DEGs (Differentially expressed genes) and 13 core genes in 4 clusters were screened during goose GCs development. Finally, the network lncRNA G8399 with CADH5 and KLF2, and lncRNA G8399 with LARP6 and EOMES were found to be important for follicular development in GCs. Thus, the results would provide a rich resource for elucidating the reproductive biology of geese and accelerate the improvement of the egg-laying performance of geese.

During an ovulatory follicular wave in the monovulatory species of heifers, mares, and women, the two largest follicles deviate in diameter at the end of a common follicle growth phase. The largest follicle before deviation becomes the future ovulatory follicle in most ovulatory waves. In 10–30% of the ovulatory waves, the destiny of the two follicles switches just before or at deviation so that the second-largest follicle becomes the future ovulatory follicle, and the largest follicle becomes a subordinate. In FSH-driven switching in heifers, mares, and women, the wave-stimulating FSH surge decreases to a low concentration before the largest follicle has developed the ability to utilize the low concentrations. The concentrations of FSH then increase (mares, women) or cease to decrease (heifers), and the next largest follicle acquires the capability of becoming the future ovulatory follicle. Luteolysis-driven switching has been reported in heifers but not in mares and women. The switching in heifers occurs during ovulatory wave 3 of three wave interovulatory intervals (IOI) when the wave of follicles is in the common growth phase in synchrony with the time of luteolysis. Regression of the CL during the common growth phase of ovulatory wave 3 is accompanied by decreased activity of follicles that are adjacent to the regressing CL but not when follicles and CL are separated or in opposite ovaries. The role of luteolysis in switching in heifers has been tested by treating with PGF2α when the largest follicle of wave 2 was near the end of the common growth phase. Switching in destiny of the largest follicle from the expected future dominant to a future subordinate occurred in most waves (10 of 17) when the largest follicle and regressing CL were in the same ovary and adjacent but not when separated in the same ovary or when in opposite ovaries (0 of 11). The newly selected future ovulatory follicle may develop in the opposite ovary. Thereby, frequency of the contralateral vs ipsilateral relationship between the preovulatory follicle and CL in heifers is greater in three-wave IOI than in two-wave IOI. In summary, the second largest predeviation follicle becomes the postdeviation dominant follicle when the decreasing FSH is out of phase with the largest predeviation follicle in heifers, mares, and women or when luteolysis and predeviation are in synchrony in heifers. • The second-largest follicle can be selected for dominancy in monovulatory species. • Switching occurs before the 2 largest follicles reach diameter deviation. • In FSH-driven switching, follicle diameter and FSH concentrations are out of phase. • Luteolysis-driven switching occurs in heifers in ovulatory wave three. • Point 3 is attributable to regression of both the CL and an adjacent follicle. [ABSTRACT FROM AUTHOR]

To elucidate the role of anti-müllerian hormone (AMH) in regulating the development of ovarian follicles in laying hens, the expressions of follicle-stimulating hormone receptor (FSHR), AMH receptor type 2 (AMHR2), steroidogenic-related genes steroidogenic acute regulatory protein (STAR), cytochrome P450 side-chain cleavage (CYP11A1), and 3β-hydroxysteroid dehydrogenase (HSD3B1) genes were measured from different sized follicles and granulosa cells. The results showed that the expressions of FSHR and AMHR2 genes were higher in small follicles and decreased after follicular selection. Oppositely, the expressions of STAR, CYP11A1, and HSD3B1 were significantly increased after follicular selection. It indicated that AMHR2 might mediate AMH suppression in the stimulating effects of follicle-stimulating hormone (FSH) on steroidogenic-related genes expression. To make sure the effects of AMH in this process, a total of 40 hens were treated (negative control, sham operation, 150 ng AMH/d or 300 ng AMH/d) for 25 d. We analyzed ovarian morphology, progesterone concentration in blood plasma, and the expressions of steroidogenic genes in ovaries and follicles. The AMH300 group had significantly lower weight of ovary and hierarchical follicles. Egg weight and ovary weight in AMH150 group were higher than those of sham operation and AMH300 groups, so did hierarchical follicles weight. The steroidogenic genes expressions showed an increase in ovarian tissue and the largest follicle of AMH150 and AMH300 groups. However, progesterone level in the blood was reduced by AMH injection with different concentrations. To further verify the above results, granulosa cells from 6 to 8 mm follicles were cultured with AMH (0, 5, 10, 20, 40, or 80 ng/mL). The results revealed that excessive AMH (80 ng/mL) exerted an inhibitory effect on progesterone synthesis and the expressions of STAR, CYP11A1, and HSD3B1. However, these genes expressions showed a significant increase in 20 ng/mL AMH-treated group. In summary, AMH inhibited the development of prehierarchical follicles in laying hens. The effects of AMH treatment with different concentrations on follicle development showed the follicle was selected by changing FSH responsiveness of prehierarchical follicles.

Ovarian follicle selection largely depends on the transition of granulosa cells from an undifferentiated to a fully differentiated state, which is accompanied by morphological and functional changes in follicles. The processes and transcriptional regulation of follicles during follicle selection are unclear; we thus used follicles from the prehierarchal to the hierarchal stage to investigate histology, reproductive endocrinology, and transcription. The morphology of follicles changed markedly during follicle selection. The numbers of large white, small yellow, and large yellow follicles (LWF, SYF, and LYF, respectively) were 11.83 ± 2.79, 6.83 ± 2.23, and 1.00, respectively, per ovary. LYF showed thicker granulosa cell layers than those of other prehierarchal follicles. Progesterone concentrations were significantly higher in LYF than that in LWF and SYF. In total, 16,823 genes were positively expressed in LWF, SYF, and LYF. Among follicle types, 1290 differentially expressed genes were enriched regarding cell differentiation, blood vessel morphogenesis, and response to steroid hormones. Candidate genes associated with follicle selection participated in the Wnt signaling pathway, steroid hormone biosynthesis, and the TGF-β signaling pathway. We produced insights into crucial morphological characteristics of transcriptional regulation in follicle development. Our results provide an important basis for revealing the mechanism of follicle selection and potential impact on the poultry industry.

Selection of a single dominant follicle is morphologically manifested by diameter deviation between the future dominant follicle (F1) and the future largest subordinate follicle (F2). Conventional deviation is defined as F2≥7 mm when F1 reaches ∼8.5 mm whereas, undersized deviation is if F2<7 mm when F1 reaches ∼8.5 mm. Greater frequency of undersized deviation has been temporally associated with greater circulating progesterone (P4) and greater FSH but reduced LH in observational studies. Experiment 1 was conducted to directly test if elevating P4 increased the likelihood of undersized deviation and altered circulating concentrations of LH and FSH. Experiment 2 was conducted to test if increasing LH action by treatment with exogenous porcine LH or human chorionic gonadotropin (hCG) in the presence of elevated P4, would stimulate growth of F2 and increase the likelihood of conventional deviation. Ovaries were evaluated by ultrasound and blood samples collected every 12 h after development of a new wave following follicle ablation on D6 (D0 = ovulation). Data were normalized to F1≥7.5 mm and compared using SAS software. In experiment 1 (n = 20), the CL was regressed by prostaglandin F2α treatment and heifers were randomized on D6 into control (no P4 treatment) or P4 treatment (75 mg every 12 h for 5.5 d) beginning when F1 reached ∼3 mm (P4-3 mm group) or ∼6 mm (P4-6 mm group). The P4 treatment significantly increased the frequency of undersized deviation from 0% (controls) to 54%, decreased LH by 44%, and increased FSH by 32%. In experiment 2 (n = 27) heifers were randomized on D6 into control (saline) or treatment with the LH analogs - pLH (1.25 mg porcine LH/12 h) or hCG (160 IU initially and subsequently 96 IU/24 h). Treatment with LH analogs significantly increased P4 (control, 4.6 ± 0.3 ng/mL; pLH, 6.6 ± 0.4 ng/mL; and hCG, 8.9 ± 0.4 ng/mL) and decreased FSH (control, 0.46 ± 0.03 ng/mL; combined-pLH/hCG, 0.34 ± 0.02 ng/mL). However, F1 and F2 diameter and frequency of conventional (37%) and undersized (48%) deviations were similar between the control and combined-pLH/hCG groups. In conclusion, elevated P4 was directly linked to undersized deviation but the P4 effect on decreasing F2 diameter occurred independently of the P4 effects on FSH and LH concentrations. • Increasing circulating P4 reduces follicle size (undersized) at diameter deviation. • Surprisingly, heifers with elevated P4 and undersized deviation had elevated FSH. • Elevated P4 is also associated with decreased circulating LH. • Using hCG to elevate LH action decreased follicle size at deviation and FSH. • Thus, undersized follicle deviation in high P4 is not explained by low LH or high FSH. [ABSTRACT FROM AUTHOR]

Follicle development is characterized by the recruitment, growth, selection, and dominance of follicles, and follicle selection determines the lifetime reproductive performance. However, in birds, the molecular mechanisms underlying follicle selection still remain elusive. This study analyzed genome-wide changes in the mRNA and miRNA expression profiles in both the granulosa and theca layers of geese ovarian follicles before selection (4–6- and 8–10-mm follicles) and after selection (F5). The sequencing results showed that a higher number of both differentially expressed (DE) mRNAs and DE miRNAs were identified between 8–10-mm and F5 follicles compared with those between the 4–6- and 8–10-mm follicles, especially in the granulosa layer. Moreover, a Short Time-series Expression Miner analysis identified a large number of DE mRNAs and DE miRNAs that are associated with follicle selection. The functional enrichment analysis showed that DE genes in the granulosa layer during follicle selection were mainly enriched in five pathways related to junctional adhesion and two pathways associated with lipid metabolism. Additionally, an interaction network was constructed to visualize interactions among protein-coding genes, which identified 53 junctional adhesion- and 15 lipid regulation-related protein-coding genes. Then, a co-expression network between mRNAs and miRNAs in relation to junctional adhesion was also visualized and mainly included acy-miR-2954, acy-miR-218, acy-miR-2970, acy-miR-100, acy-miR-1329, acy-miR-199, acy-miR-425, acy-miR-181, and acy-miR-147. Furthermore, miRNA–mRNA interaction pairs related to lipid regulation were constructed including acy-miR-107, acy-miR-138, acy-miR-130, acy-miR-128, and acy-miR-101 during follicular selection. In summary, these data highlight the key roles of junctional adhesion and lipid metabolism during follicular selection and contribute to a better understanding of the mechanisms underlying follicle selection in birds.

In hens, follicle selection is an important process affecting egg laying traits. This study investigated the role of parathyroid hormone-like hormone (PTHLH) in chicken follicle selection, its transcriptional regulation and genetic effects on egg laying traits. PTHLH and its receptor PTH1R were mainly expressed in follicles of 6–8 mm in diameter, exhibits differential expression pattern in the theca and granulosa cells of pre- and hierarchal follicles. PTHLH stimulates the proliferation of follicular granulosa and theca cells, the expression of StAR and CYP11A1 mRNA and the production of progesterone (P4) in pre-hierarchal follicles. Treatment with FSH increased PTHLH mRNA expression in pre-hierarchal follicular theca cells and hierarchal follicular granulosa cells. Two critical regions regulating chicken PTHLH transcription were revealed, each of which harbored a SNP: C>T (chr1: 72530014) for AP-1 and a SNP: A>G (chr1: 72531676). Hens with diplotype AC/GT were younger at first laying and laid more eggs at 32 weeks. The haplotype (G-1827T-165) with double mutations had the greatest promoter activity of chicken PTHLH transcription. Collectively, PTHLH plays an important role in chicken follicle selection by stimulating cell proliferation and steroidogenesis. Polymorphisms in chicken PTHLH promoter region are associated with egg laying traits by affecting the binding of transcription factor AP-1.

Ovarian follicle selection is the critical step which determines the oocyte development and ovulation. In avian species, the somatic cells in the follicles decide the process of follicle selection but the precise molecular regulation is not well defined. N6-methyladenosine (m6A) is a ubiquitous reversible epigenetic RNA modification that plays an important role in the gene expression regulation and cell functions. In this study, we profiled transcriptome-wide m6A methylation in chicken follicles during follicular selection process in order to identify key factors involved in the follicle selection. The chicken follicle transcriptome was extensively methylated by m6A and a negative correlation was found between the m6A methylation enrichment and gene expression levels. Interestingly, both the m6A methylation peaks and the m6A modified transcripts increased during follicle selection, which lead to the dynamic expression of many folliculogenesis relevant genes. Functional enrichment analysis indicated that m6A modification of key factors in Wnt pathway could play a major role in regulating follicle selection. This study is the first to comprehensively characterize the m6A patterns in the chicken transcriptome, and provides deep insights into the m6A topology and relevant molecular mechanisms underlying follicle selection. [ABSTRACT FROM AUTHOR]

Intraovarian effects on diameter of future dominant follicle (DF or F1) and future largest subordinate follicle (F2) during the few days before selection of the future DF and subordinate follicles were studied in 147 bovine interovulatory intervals. Follicle selection involves diameter deviation or the beginning of separation of growth rates between F1 and F2. Diameter deviation is classified as conventional (F2 ≥ 7.0 mm when F1 is 8.5 mm or at expected deviation) and as undersized (F2 < 7.0 mm when F1 is 8.5 mm). Diameter separation of F1 and F2 in conventional and undersized deviations is characteristically abrupt and gradual, respectively. The predeviation diameter of F2 when located in an ovary that later becomes the F1 intraovarian patterns of DF−CL, devoid (ovary without a DF or CL), DF alone, or CL alone and in left or right ovaries (LO, RO) was compared between conventional and undersized deviations. In conventional deviation, ovaries with the future DF (combined DF−CL and DF patterns) were associated with greater (P < 0.02) predeviation growth rate of F2 when the DF was in the right ovary (DF/RO, 1.6 ± 0.1 mm/d) than when in the left ovary (DF/LO, 1.2 ± 0.1 mm/d). The F2 was in DF/RO more frequently (75%, P < 0.002) than in non-DF/RO. When F2 was in the future devoid F1 pattern and F1 was 6 mm, F2 was smaller (P < 0.002) in the undersized class (5.3 ± 0.2 mm) than in the conventional class (6.3 ± 0.1) but not when F2 was in one of the other future F1 patterns. Only the devoid pattern was greater in frequency (P < 0.03) in the undersized class than in the conventional class. The novel hypothesis was supported that location of F2 in ovaries with different future F1 intraovarian patterns and on different sides affects the predeviation diameter and growth rate of F2 and thereby the frequencies of conventional and undersized deviations during follicle selection. • Growth rate of future F2 decreases when in an ovary without future F1 or CL. • Growth rate of future F2 increases when in right ovary with future F1. • Shorter intraovarian distance between future F1 and CL than between F2 and CL. [ABSTRACT FROM AUTHOR]

In hens, follicle selection is an important process affecting egg laying traits. This study investigated the role of parathyroid hormone-like hormone (PTHLH) in chicken follicle selection, its transcriptional regulation and genetic effects on egg laying traits. PTHLH and its receptor PTH1R were mainly expressed in follicles of 6–8 mm in diameter, exhibits differential expression pattern in the theca and granulosa cells of pre- and hierarchal follicles. PTHLH stimulates the proliferation of follicular granulosa and theca cells, the expression of StAR and CYP11A1 mRNA and the production of progesterone (P4) in pre-hierarchal follicles. Treatment with FSH increased PTHLH mRNA expression in pre-hierarchal follicular theca cells and hierarchal follicular granulosa cells. Two critical regions regulating chicken PTHLH transcription were revealed, each of which harbored a SNP: C>T (chr1: 72530014) for AP-1 and a SNP: A>G (chr1: 72531676). Hens with diplotype AC/GT were younger at first laying and laid more eggs at 32 weeks. The haplotype (G-1827T-165) with double mutations had the greatest promoter activity of chicken PTHLH transcription. Collectively, PTHLH plays an important role in chicken follicle selection by stimulating cell proliferation and steroidogenesis. Polymorphisms in chicken PTHLH promoter region are associated with egg laying traits by affecting the binding of transcription factor AP-1. [ABSTRACT FROM AUTHOR]

Heifers have two or three major follicular waves per interovulatory interval (IOI). In mares and women, the ovulatory wave is the only major wave in most (75%) IOI. The beginning of diameter deviation during follicle selection of the future dominant follicle (DF) is followed by continued growth of DF and decreasing growth of the future subordinate follicles. Diameter deviation in Bos taurus heifers, mares, and women begins when the future DF is a mean of 8.5, 22.5, and 10.5 mm, respectively. Selection of the ovulatory follicle occurs more frequently from right ovary (RO) in heifers and women and from left ovary (LO) in nulliparous mares with no difference between ovaries in parous mares. The RO predilection for ovulation is preceded by a predilection for more follicles in RO before the beginning of deviation as indicated by (1) in heifers and women, there were more predeviation follicles in RO than LO and ovulation occurred more frequently from RO whereas in mares there was no difference between ovaries in number of predeviation follicles and ovulation occurred with similar frequency between ovaries and (2) in heifers, the number of ovulatory waves with DF in the ovary that had more predeviation follicles was greater than the number of waves with DF in the ovary that had fewer follicles. In heifers, ovulation from RO occurs more frequently when the regressed CL is also in RO and is attributable to a positive intraovarian effect of the CL on predeviation follicles that were adjacent to the CL. The positive two-way effect between CL and future DF when adjacent is expressed by greater dimensions and vascular perfusion of CL and DF. This phenomenon awaits study in mares and women. An exception to more frequent RO ovulation in heifers occurs in wave 3 owing to a switch during predeviation in future dominance to a smaller follicle when the largest follicle is adjacent to the regressing CL. A preovulatory contralateral relationship (DF and CL in opposite ovaries) during the last wave of an IOI in heifers usually (eg, 88%) converts to an ipsilateral relationship during wave 1 of the next IOI in association with continuity in vascular perfusion and number of predeviation follicles per ovary. Alternating relationships between consecutive ovulations were not found in mares and is controversial in women. Applied potential of ovarian asymmetry is indicated by greater blastocyst rate for RO oocytes in cattle and greater pregnancy rate for RO ovulation in women. • Right ovary ovulation is more frequent in heifers and women but not in mares. • Right ovary ovulation is associated with greater oocyte quality. • A predilection for more follicles per wave favors right ovary ovulation. [ABSTRACT FROM AUTHOR]

Abstract Selection of the dominant follicle during a follicular wave is manifested by diameter deviation. At deviation (day 0), growth rate continues for the future dominant follicle (F1) and begins to decrease for the largest subordinate follicle (F2). The percentage of color-Doppler blood-flow signals in the wall of F1 and F2 and the temporality between FSH concentration and F1 and F2 diameter were determined daily in waves 1 and 2 in 24 Holstein heifers. Diameter and blood flow were compared among classes of deviation: (1) conventional (F2 ≥ 7.0 mm on day 0), (2) F2-undersized (F2 < 7.0 mm on day 0), and (3) F1,F2-switched (F2 larger than F1 on days −1 or 0). A class-by-day interaction for diameter of F2 (P < 0.004) and for blood-flow percentage of F2 (P < 0.02) represented greatest values on days −1 or 0 in the switched class and greater values in the conventional than undersized class. Changes were similar between diameter and blood flow in F1 and F2 before deviation. Blood flow in F2 decreased sooner than diameter after deviation indicating that a decrease in vascular perfusion preceded a decrease in diameter. Relationships between F1 and FSH in conventional deviation were similar between waves 1 and 2 for (1) growth rate of F1 on days −1 to 0, (2) interval from emergence of F1 at 4 mm to deviation, and (3) decrease in FSH on days −2 to 0. Relationships between F2 diameter and FSH were dissimilar between classes and between waves 1 and 2 indicating other hormones or factors are also involved in the complex control of F2. For example, the growth rate of F2 was greater (P < 0.05) for conventional than undersized class during wave 1 but similar between classes during wave 2. The FSH surge 2 was similar in profile and prominence between classes but the interval from the FSH peak of surge 2 to deviation was shorter (P < 0.05) in the undersized class (1.5 ± 0.3 d) than in the conventional class (2.3 ± 0.3 d). This was a novel finding and accounted for some of the dissimilarities in growth rate of follicles between classes in wave 2. Results did not support the hypothesis that the extent of blood flow in the wall of future dominant and largest subordinate follicles before deviation is an earlier indicator of follicle destiny than diameter. Results supported the hypothesis that follicle dynamics and FSH concentrations before deviation are temporally associated within conventional and undersized deviation classes but the temporality differs between classes. Highlights • Decrease in blood flow in follicle wall precedes decrease in diameter. • Interval from peak of FSH surge to diameter deviation effects follicle growth. • Differences in FSH surge do not account for differences in diameters. [ABSTRACT FROM AUTHOR]

Abstract Follicle diameter deviation has been identified as the pivotal morphological manifestation of follicle selection, however, many of the hormonal mechanisms leading to this key event and regulating variation between individual follicular waves remain undefined. This study compared circulating FSH, LH, and P4 with the follicular dynamics during three different physiological conditions. We hypothesized that these end-points would: 1) be similar for a spontaneous wave 2 vs one induced by follicular aspiration, 2) but would differ between wave 1 and 2, and 3) between conventional (F2 > 7.0 mm at deviation) vs undersized (F2 < 7.0 mm) deviations in either wave. Holstein dairy heifers (N = 24) were studied daily during an interovulatory interval. All heifers were evaluated during wave 1 and randomized 6 days after ovulation into an induced wave 2 and a spontaneous wave 2. Values were normalized to the day of expected diameter deviation (day 0) and compared for day −2 to 0 and 0 to 2. Hypothesis 1 was supported that an induced wave 2 from ablation of follicles of wave 1 and spontaneous wave 2 have similar follicle dynamics. However, the peak FSH surge was more prominent at emergence of an induced wave 2 (P < 0.003). Hypothesis 2 was supported that waves 1 and 2 differ in follicle and hormone dynamics. Circulating P4 was lower and LH was greater (P < 0.01) with no difference in diameter of F1 but with a greater (P < 0.01) diameter of F2 on day 0 in wave 1 (7.3 ± 0.2 mm) than in wave 2 (6.6 ± 0.2 mm). Differences between waves were not found when each follicular wave was categorized into conventional vs undersized deviation and analyzed separately. Hypothesis 3 was supported as there were differences in circulating hormones between conventional and undersized deviations. Growth rate of F2 differed (P < 0.0005) during days −2 to 0 (conventional, 2.6 ± 0.2 mm/2d; undersized, 1.4 ± 0.3 mm/2d). However, circulating FSH and P4 concentration on days −1 and 0 tended to be greater (P < 0.06) in undersized than conventional deviations. In conclusion, the effect of different hormonal conditions on follicle dynamics was observed for F2 and not for F1. Furthermore, understanding the physiology that produces conventional vs undersized deviations is crucial since these categories explained most differences in follicular dynamics and circulating FSH observed in these different physiological conditions. In addition, future studies of wave 2 may be facilitated by using an induced wave 2 since it was similar to a spontaneous wave 2. Highlights • Induced follicular waves have greater FSH surge but similar follicle growth dynamics. • Wave 1 & 2 have similar dominant (F1) but different subordinate (F2) follicle growth. • FSH & F2 differences wane when classified as conventional or undersized deviations. • Undersized deviations have slower F2 growth but greater FSH than conventional. • Conventional vs undersized deviation classes help explain follicular dynamics. [ABSTRACT FROM AUTHOR]

The majority of activated ovarian follicles undergo atresia during reproductive life in mammals, and only a small number of follicles are ovulated. Though hormone treatment has been widely used to promote folliculogenesis, the molecular mechanism behind follicle selection and atresia remains under debate due to inconsistency among investigation models. Using a high-throughput molecular pathology strategy, we depicted a transcriptional atlas of mouse follicular granulosa cells (GCs) under physiological condition and obtained molecular signatures in healthy and atresia GCs during development. Functional results revealed hypoxia-inducible factor 1 (HIF1) as a major effector downstream of follicle-stimulating hormone (FSH), and HIF1 activation is essential for follicle growth. Energy shortage leads to prevalent AMP-activated protein kinase (AMPK) activation and drives follicular atresia. FSHR-mTOR-HIF1 signaling helps follicles escape from the atresia fate, while energy stress persists. Our work provides a comprehensive understanding of the molecular network behind follicle selection and atresia under physiological condition. [Display omitted] • Geo-seq combined with TUNEL reveals complete transcriptome profile of follicle GCs • Distinct different transcription regulation exists in healthy and atresia follicles • Activation of HIF1 pathway promotes follicle development and inhibits atresia • FSHR-mTOR-HIF1 axis safeguards follicles' growth to escape their atresia fate The "norm" of a follicle is to die rather than to ovulate. By studying mouse ovaries with a high-throughput molecular pathology strategy, Liu et al. dissect the molecular regulation network behind follicle selection and atresia under physiological conditions. [ABSTRACT FROM AUTHOR]

The SLIT/ROBO pathway has been implicated in prehierarchical follicular development of hen ovary by an intrafollicular autocrine and/or paracrine fashion. SLIT3, one of the key components of the SLIT/ROBO family, serves as a ligand that potentially interacts with the four receptors, ROBO1, ROBO2, ROBO3 and ROBO4. But the exact roles and regulatory mechanism of SLIT3 in chicken ovarian follicle development remain largely unclear. The present study was conducted to investigate the potential roles and molecular regulation of SLIT3 in granulosa cell (GC) proliferation, differentiation and follicle selection within the prehierarchical follicles of hen ovary. We found that SLIT3 interacts physically with the four ROBO receptors, but the expression of the ROBO1 and ROBO2 genes are more susceptible to the regulation of SLIT3 ligand than that of the ROBO3 and ROBO4 genes. Moreover, the siRNA‐mediated knockdown of SLIT3 in the follicular GCs leads to a significant increase in cell proliferation. Conversely, overexpression of SLIT3 results in a remarkable reduction in GC proliferation. Furthermore, the overexpressed SLIT3 has notably decreased the mRNA and protein expression levels of follicle‐stimulating hormone (FSHR), growth and differentiation factor 9 (GDF9), steroidogenic acute regulatory protein (STAR) and cytochrome P450 11A1 (CYP11A1) in the GCs. These results indicated that SLIT3 may play an inhibitory effect on GC proliferation, differentiation and follicle selection, and these suppressive actions of SLIT3 in the GC proliferation can be prohibited by the siRNA‐mediated knockdown of ROBO1 and ROBO2 receptors. The current data provide a basis for further investigation of molecular mechanisms of SLIT3‐ROBO1/2 pathway in controlling the prehierarchical follicle development of the hen ovary. [ABSTRACT FROM AUTHOR]

Diameter deviation or follicle selection during a follicular wave begins with continuous growth rate of F1 (dominant or selected follicle; DF) and decreasing growth rate of F2 (largest subordinate). Intraovarian patterns based on presence or absence of the DF and CL are DF−CL, DF, CL, and devoid (neither DF nor CL). The DF and CL relationships in a pair of ovaries are ipsilateral (DF−CL pattern and devoid pattern) and contralateral (DF pattern and CL pattern). The effects of patterns and relationships on F1, F2, and FSH during deviation were determined in 21 mares. Diameters of F1 and F2 at expected beginning of deviation (F1, 22.5 mm; day 0) defined deviation classifications of conventional (F2 ≥ 18.5 mm on day 0), F2-undersized (F2 < 18.5 mm), and F1,F2-switched (F2 larger than F1 on day −1 or 0). During the decline in the FSH surge, an interaction (P < 0.001) of deviation classification and day indicated that FSH concentration was greater (P < 0.05) in undersized than in conventional deviations on days −3 and −1 and intermediate in switched deviation. Low FSH during days −3 to −1 in switched deviation began to increase on day −1 during switching in diameter rank between F1 and F2. The number of subordinate follicles per wave that attained a maximum of 11 to <16 mm was greatest in the DF−CL and devoid patterns and in ipsilateral (4.2 ± 0.5 follicles) than in contralateral (2.5 ± 0.7) relationships (P < 0.03). Concentration of FSH was greater (P < 0.04) on days −4 to 5 in ipsilateral (5.3 ± 0.2 ng/mL) than in contralateral (4.2 ± 0.4 ng/mL) relationships consistent with more follicles 11 to < 16 mm. In a survey (N = 63), an ipsilateral vs contralateral relationship was associated with 17 vs 17 conventional deviations and 7 vs 22 nonconventional deviations (P < 0.04), and conventional deviation (54%) was more frequent (P < 0.0001) than undersized (21%) or switched (25%) deviations. Results supported the hypotheses that (1) diameter deviation can be classified in mares into distinct categories based on diameter and rank of the future largest subordinate at expected deviation, (2) the number of subordinate follicles is influenced by ipsilateral vs contralateral relationships of DF and CL, and (3) conventional deviation is more common when the relationship between DF and CL is ipsilateral. Knowledge on follicle diameters for the different deviation classifications should be considered in studying follicle selection especially when terminal follicle sampling is done before expected deviation. [ABSTRACT FROM AUTHOR]

Follicle diameter deviation during a follicular wave in cattle is preceded by a common-growth phase and begins with continued growth rate of the future dominant follicle (DF or F1) and reduced growth rate of the future largest subordinate follicle (SF or F2). Follicle diameters were analyzed daily for wave 1 (n = 100) and wave 2 (n = 91). The beginning of expected deviation was designated day 0 and was defined as the day the largest follicle was closest to 8.5 mm. Conventional classification of deviation (F2 ≥ 7.0 mm at expected deviation) was more frequent (P < 0.0001) in wave 1 (59%) than in wave 2 (35%); deviation began abruptly. An F2-undersized classification (F2 < 7.0 mm at expected deviation) was greater (P < 0.0001) for wave 2 (40%) than for wave 1 (15%); separation of diameters of F1 and F2 occurred gradually during the last 2 days of the common-growth phase. The growth rate of F1 in conventional deviations of wave 1 was greater (P < 0.002) on days 0 to 1 (1.8 ± 0.1 mm/day) than on days −1 to 0 (1.5 ± 0.1 mm/day). Several other novel growth spurts of F1 were observed in association with deviation including during switching of diameter rank between F1 and F2 in each of waves 1 and 2. Hypotheses were supported that (1) conventional deviation is more common during wave 1 whereas F2-undersized deviation is more common during wave 2 and (2) a transient growth spurt occurs in F1 of wave 1 at the beginning of diameter deviation. [ABSTRACT FROM AUTHOR]

Quercetin (Que), widely found in a huge variety of plants, plays important roles in ovarian function. However, to data, there have been no reports about Que regulating granulosa cells (GCs) in prehierarchical follicles in chicken. Herein, GCs from follicles diameter from 4 to 8 mm in chicken were treated by Que in vitro culture to investigate how Que exerts its effect on follicular development. GCs treated by Que in concentrations of 10, 100, and 1,000 ng/mL were tested for cell proliferation and progesterone secretion. Eight cDNA libraries were constructed from GCs (4 samples per group) to explore transcriptome expression changes. The role of the MAPK/ERK signaling pathway was validated in this process. Treatment with 100 and 1,000 ng/mL levels of Que significantly promoted cell proliferation and progesterone secretion (P < 0.05). RNA-seq analysis data showed that 402 and 263 differentially expressed genes (DEGs) were up- and down-regulated, respectively. Functional enrichment analysis that the pathways related to follicular development included biosynthesis of amino acids, MAPK signaling pathway, and calcium signaling pathway. Notably, the function exerted in GCs of the different levels of Que was associated with the suppression of the MAPK pathway. In conclusion, our results proved that low levels of Que could promote MAPK signaling pathway, but high levels of Que inhibit MAPK signaling pathway in GCs from the prehierarchical follicles, promote cell proliferation and progesterone secretion, and benefit follicle selection. [ABSTRACT FROM AUTHOR]

Ovarian follicle selection is an important process impacting the laying performance and fecundity of hens, and is regulated by follicle-stimulating hormone (FSH) through binding to its receptor [follicle-stimulating hormone receptor (FSHR)]. In laying hens, the small yellow follicle (6–8 mm in diameter) with the highest expression of FSHR will be recruited into the preovulatory hierarchy during ovarian follicle development. The study of molecular mechanism of chicken follicle selection is helpful for the identification of genes underlying egg-laying traits in chicken and other poultry species. Herein, the transcriptomes of chicken small yellow follicles differing in the mRNA expression of FSHR were compared, and a total of 17,993 genes were identified in 3 pairs of small yellow follicles. The Wnt signaling pathway was significantly enriched in the follicles with the greatest fold change in FSHR expression. In this pathway, the expression level of Wnt4 mRNA was significantly upregulated with a log2(fold change) of 2.12. We further investigated the expression, function, and regulation of Wnt4 during chicken follicle selection and found that Wnt4 mRNA reached its peak in small yellow follicles; Wnt4 stimulated the proliferation of follicular granulosa cells (GCs), increased the expression of StAR and CYP11A1 mRNA in prehierarchical and hierarchical follicles, increased the expression of FSHR mRNA, and decreased the expression of anti-Müllerian hormone and OCLN mRNA. Treatment with FSH significantly increased Wnt4 expression in GCs. Moreover, Wnt4 facilitated the effects of FSH on the production of progesterone (P4) and the mRNA expression of steroidogenic enzyme genes in the GCs of hierarchical follicles, but inhibited the effects of FSH in the GCs of prehierarchical follicles. Collectively, these data suggest that Wnt4 plays an important role in chicken follicle selection by stimulating GC proliferation and steroidogenesis. This study provides a theoretical basis for improving the egg-laying performance of chicken and a reference for the elucidation of the molecular mechanism of follicular selection in mammals.

Diameter deviation is the beginning of a decrease in growth rate of the largest subordinate follicle (SF) and a continuing growth rate of the dominant follicle (DF). In wave 1 in cattle, deviation begins 2 or 3 days after ovulation when the future DF is about 8.5 mm. Gray scale and power-flow Doppler ultrasound examinations were done in experiment 1 (daily examinations, n = 13) and experiment 2 (examinations every 8 h, n = 15). Blood flow and an anechoic layer in the follicle wall were normalized to the beginning of diameter deviation (day 0 or hour 0). Only waves with conventional diameter deviation (68% of waves) were used as identified by: (1) future SF greater than 7.0 mm when DF was 8.5 mm and (2) future DF and SF did not switch in diameter rank. In experiment 1, deviations in the extent of blood-flow signals and in the extent of anechoic layer began on the same day as deviation in diameter. In experiment 2, deviations in diameter, surface area (πd 2 ), and anechoic layer began in synchrony, and deviation in blood-flow signals began 16 h earlier. Blood-flow deviation before diameter deviation was shown by (1) a first difference (P < 0.02) between follicles at hour −16 and (2) development during the hours −24 to 0 of a greater (P < 0.05) percentage difference between follicles in blood-flow signals (11.1 ± 2.3%) than in surface area (7.4 ± 0.7%) or diameter (4.5 ± 0.4%). Results supported the hypothesis that the extent of blood flow in the future dominant and subordinate follicles deviates before diameter deviates. A similar hypothesis for anechoic layer was not supported; diameter and anechoic layer deviated in synchrony. [ABSTRACT FROM AUTHOR]

The day of selection of the ovulatory follicle was first observed in any species by an equine practitioner during transrectal palpation in 1926. It was not until 1997 that ultrasonic imaging demonstrated the nature of selection. A wave-stimulating follicle stimulating hormone (FSH) surge reaches a peak on about day 13 postovulation when the growing follicles are about 13 mm. The FSH declines initially from secretion of inhibin by the multiple growing follicles. After about 2 days, estradiol is secreted by the future dominant follicle (DF) and synergistically contributes to a continuing FSH decline. On about day 16, the two largest follicles have grown to 23 and 20 mm, respectively, and begin to deviate in growth. The deviation process begins a day before diameter deviation as indicated in the future DF by (1) an increase in intrafollicular estradiol, inhibin-A, insulin-like growth factor 1 (IGF1), and vascular endothelial growth factor (VEGF) and (2) changes in echotexture and increased blood flow in the wall of the future DF. The IGF1 increases the sensitivity of the cells of the future DF to the reduced FSH, and VEGF presumably increases the vascular perfusion of the DF wall. The future DF is thus prepared to be the only follicle able to respond to the reduced levels of FSH. Ablation of various follicles at expected deviation indicates that several follicles have dominance capacity. And so, 90 years after the original observation of selection it can be proposed that the deviation process represents the theory of follicle selection in mares. [ABSTRACT FROM AUTHOR]

Mares are superb models for study of follicle selection owing to similarities between mares and women in relative follicle diameters at specific events during the follicular wave and follicle accessibility for experimental sampling and manipulation. Usually, only 1 major follicular wave with a dominant follicle (DF) greater than 30 mm develops during the 22 to 24 d of the equine estrous cycle and is termed the primary or ovulatory wave. A major secondary wave occasionally (25%) develops early in the cycle. Follicles of the primary wave emerge at 6 mm on day 10 or 11 (day 0 = ovulation). The 2 largest follicles begin to deviate in diameter on day 16 when the future DF and largest subordinate follicle (SF) are 23 mm and 20 mm, respectively. The deviation process begins the day before diameter deviation as indicated in the future DF but not in the future SF by (1) increase in prominence of an anechoic layer and vascular perfusion of the wall and (2) increase in follicular-fluid concentrations of IGF1, vascular endothelial growth factor, estradiol, and inhibin-A. A systemic component of the deviation process is represented by suppression of circulating FSH from secretion of inhibin and estradiol from the developing DF. Production of inhibin is stimulated by IGF1 and LH, and estradiol is stimulated by LH and not by IGF1 in mares. A local intrafollicular component involves the production of IGF1, which apparently increases the responsiveness of the future DF to FSH. The roles of the IGF system have been well studied in mares, but the effect of IGF1 on increasing the sensitivity of the follicle cells to FSH is based primarily on studies in other species. The greater response of the future DF than the SF to the low concentrations of FSH is the essence of selection. During the common growth phase that precedes deviation, diameter of the 2 largest follicles increases in parallel on average when normalized to emergence or retrospectively to deviation. Study of individual waves indicates that (1) the 2 follicles change ranks (relative diameters) during the common growth phase in about 30% of primary waves and (2) after ablation of 1, 2, or 3 of the largest follicles at the expected beginning of deviation, the next largest retained follicle becomes the DF indicating that several follicles have the capacity for dominance; therefore, it is proposed that the deviation process represents the entire mechanism of follicle selection in mares. [ABSTRACT FROM AUTHOR]

Follicle selection is an important step in the laying process of chicken, which is closely related to the laying performance and fecundity of hens. Follicle selection mainly depends on the regulation of follicle-stimulating hormone (FSH) secreted by pituitary gland and the expression of follicle stimulation hormone receptor. To uncover the role of FSH in chicken follicle selection, in this study, we analyzed the changes in the mRNA transcriptome profiles of FSH-treated chicken granulosa cells from prehierarchical follicles by long-read sequencing Oxford Nanopore Technologies (ONT) approach. Among the 10,764 genes detected, 31 differentially expressed (DE) transcripts of 28 DE genes were significantly upregulated by FSH treatment. These DE transcripts (DETs) were mainly related to the steroid biosynthetic process by GO analysis and enriched in pathways of ovarian steroidogenesis and aldosterone synthesis and secretion by KEGG analysis. Among these genes, the mRNA and protein expression of TNF receptor associated factor 7 ( TRAF7 ) was upregulated after FSH treatment. Further study revealed that TRAF7 stimulated the mRNA expression of steroidogenic enzymes steroidogenic acute regulatory protein ( StAR ) and cytochrome P450 family 11 subfamily A member 1 ( CYP11A1 ) genes and the proliferation of granulosa cells. This is the first study to investigate differences in chicken prehierarchical follicular granulosa cells before and after FSH treatment by using ONT transcriptome sequencing, which provides a reference for a more comprehensive understanding of the molecular mechanism of follicle selection in chicken. [ABSTRACT FROM AUTHOR]