F1 Follicles Research Articles

CircRNAs involved in the red light of effect on follicle selection in pigeons

Red light (RL) can enhance egg production in poultry. CircRNAs play a crucial role by serving as transcriptional regulators. However, their role in influencing follicle development in White King pigeons remains unexplored. In this study, 54 paired White King pigeons were chosen and divided into RL and white light (WL) groups, each with 3 subgroups. The egg production of paired pigeons in each replicate was recorded for 45 d, and the characteristics of follicle development were monitored during the laying interval (LI). The granulosa cell layer from follicles of the second-largest follicle (F2) was collected, and high-throughput sequencing was performed to elucidate the molecular mechanism of follicle development in pigeons. The study confirmed that RL enhances egg production in pigeons. Additionally, under RL, the F2 follicle was selected, while under WL, small follicles were kept on the third day (LI3). A total of 5,510 circRNAs were identified across all samples, revealing differentially expressed circRNAs (DECs) in various comparisons: 627 in RF1 vs. WF1, 900 in RF2 vs. WF2, 606 in RF1 vs. RF2, and 937 in WF1 vs. WF2. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that host genes of DECs were enriched in pathways like steroid hormone biosynthesis, oocyte meiosis, GnRH signaling pathway, and apoptosis pathway. Moreover, circRNA_5497, circRNA_2016, and circRNA_3328 were common DECs across 4 groups, sharing miRNA binding sites with follicle selection-associated genes. In conclusion, our findings suggest that RL promotes egg production by stimulating follicle selection during LI, offering insights into the regulatory mechanisms of circRNAs in follicle selection under RL. This knowledge can help enhance the reproductive performance of pigeons.

Alterations in aquaporin 4 expression in the chicken ovary following tamoxifen treatment

Previously, we demonstrated the follicular size- and a layer of the follicular wall-dependend expression of aquaporin 4 (AQP4) in the chicken ovary. In this study, we aimed to examine the mRNA and protein expression of aquaporin 4 (AQP4) in the chicken ovary during a pause in laying induced by a tamoxifen (TMX, oestrogen receptor modulator) treatment. Ovarian white and yellowish follicles and the granulosa and theca layers of the largest yellow preovulatory follicles were harvested from control and TMX-treated (daily until the cessation of egg-laying) hens. It was found that the TMX treatment lowered the AQP4 transcript abundance in the white follicles, but increased it in the theca layer of the F3-F1 follicles. Moreover, the TMX caused a decrease in AQP4 protein abundance in the theca layer of the F2 follicle. We propose that AQP4 plays a role in the ovarian follicle development and that there is a relationship between oestradiol action and the AQP4 gene and protein expression in chicken ovarian follicles.

Dynamic Expression Profile of Follicles at Different Stages in High- and Low-Production Laying Hens.

Improving the efficiency of hens and extending the egg-laying cycle require maintaining high egg production in the later stages. The ovarian follicles, as the primary functional units for ovarian development and oocyte maturation, play a crucial role in regulating the continuous ovulation of hens. The egg production rate of laying hens is mostly affected by proper follicle growth and ovulation in the ovaries. The objective of this study was to identify the key genes and signaling pathways involved in the development of ovarian follicles in Taihang hens through transcriptome screening. In this study, RNA sequencing was used to compare and analyze the transcriptomes of ovarian follicles at four developmental stages: small white follicles (SWF), small yellow follicles (SYF), F5 follicles, and F2 follicles, from two groups: the high continual production group (H-Group) and the low continual production group (L-Group). A total of 24 cDNA libraries were constructed, and significant differential expression of 96, 199, 591, and 314 mRNAs was detected in the SWF, SYF, F5, and F2 follicles of the H and L groups, respectively. Based on the results of GO and KEGG enrichment analyses, each stage of follicle growth possesses distinct molecular genetic features, which have important effects on follicle development and significantly promote the formation of continuous production traits through the biosynthesis of steroid hormones, cytokine-cytokine receptor interaction, and neuroactive ligand-receptor interaction. Additionally, through STEM analysis, we identified 59 DEGs, including ZP4, KCNH1, IGFs, HMGA2, and CDH1, potentially associated with follicular development within four significant modules. This study represents the first transcriptome investigation of follicles in hens with high and low egg-producing characteristics at four crucial developmental stages. These findings provide important molecular evidence for understanding the regulation of follicular development and its variations.

Expression and regulation of Noggin4 gene in chicken ovarian follicles and its role in the proliferation and differentiation of granulosa cells

As a member of Noggin family, Noggin4 is reported to play an important role in the formation of head structure during the embryo development of Xenopus laevis and chicken. We previously detected an increase of Noggin4 transcript in the granulosa cells of chicken hierarchal follicles (Post-GCs) compared to pre-hierarchal follicles (Pre-GCs) by ONT transcriptome sequencing. To further clarify the role of Noggin4 in chicken follicle selection, in this study, we investigated its expression, regulation and function in follicles and granulosa cells. The mRNA expression of chicken Noggin4 exhibited dynamic changes during follicle development. It was significantly higher in the small yellow follicles than in the small white, F6, F5 and F4 follicles, and also increased in Post-GCs than in Pre-GCs. The Noggin4 mRNA could be stimulated by follicle stimulating hormone (FSH) and bone morphogenetic protein 4 (BMP4) in both Pre-GCs and Post-GCs. However, the estrogen and progesterone could exert opposing transcriptional regulations on Noggin 4 mRNA in both Pre- and Post-GCs. In chicken Post-GCs, knockdown of Noggin4 by siRNA reduced the mRNA expression of steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), but increased that of Wnt family member 4 (Wnt4), while overexpression of Noggin4 significantly decreased the mRNA expression of Wnt4 but had no marked effects on that of STAR and CYP11A1. Moreover, Noggin4 significantly decreased the mRNA expression of BMP4 in both Pre-GCs and Post-GCs. Overexpression of Noggin4 inhibited the proliferation of both Pre-GCs and Post-GCs. These data collectively suggest an important role of Noggin4 in chicken follicle selection, especially on the proliferation of granulosa cells.

Correlation analysis of polyamine metabolism and reproductive hormone levels in goose ovarian follicles

To investigate the relationship between polyamine metabolism and reproductive hormones in ovarian follicles of Sichuan white geese, follicle polyamine content and reproductive hormone levels and gene expressions related to polyamine metabolism, steroidogenesis and steroid hormone receptors were detected by HPLC, ELISA and RT-qPCR. The results showed that the overall trend of spermidine and spermine levels increased first and then decreased as increasing follicle size, with the highest level in F3 and F5 follicles (P < 0.05). Putrescine and 17β-estradiol (E2) levels in hierarchical follicles were significantly lower than those in prehierarchical follicles (P < 0.05). Progesterone (P4) first increased and then decreased, with the highest level in the F5 follicle (P < 0.05). The expression levels of estrogen receptor 1 (ER1) showed an overall increase as increasing follicle size (except in F3 follicles), while estrogen receptor 2 (ER2) in hierarchical follicles was significantly lower than that in the prehierarchical follicles (P < 0.05). In addition, the overall expression level of progesterone receptor (PR) decreased, with no significant differences among F1, F2 and F3 follicles (P > 0.05). Yolk putrescine contents were positively correlated with yolk E2 concentrations and PR expression levels (P < 0.05), A significant positive correlation of spermidine levels with yolk P4 concentrations and PR expressions was also observed, as well as the spermine levels with yolk P4 concentrations (P < 0.05). In summary, polyamines were involved in the regulation of follicular development in geese, and this regulation played a role in affecting steroidogenesis and the expression of genes related to hormone receptors.

SNP screening of the HSD3B1 gene and its association with laying performance in Taihang chickens

The 3 beta- and steroid delta-isomerase 1 (HSD3B1) gene has a great influence on reproductive traits and therefore has been widely studied. However, little is known about the relationships between polymorphisms of the HSD3B1 gene and laying traits in Taihang chickens. Therefore, the HSD3B1 gene was selected for polymorphism detection and analysis of its relationship with laying performance traits. The mRNA expression levels of HSD3B1 in 16 tissues of Taihang chickens were detected, and the HSD3B1 gene was SNP screened and genotyped, and associated with laying traits. The results showed that the HSD3B1 gene was widely expressed. The HSD3B1 expression level was highest in F1 follicles, lowest in small white follicles, and differentially expressed in large follicles with small yellow follicles. The C274T allele was moderately polymorphic in Taihang chickens, and the age of opening day was significantly earlier for the CC genotype than for other genotypes. The present results demonstrate that the homozygous CC genotype can be considered a genotype for molecular marker-assisted selection (MAS) to improve the breeding efficiency of Taihang chickens.

Transcriptome Profiling of Goose Ovarian Follicle Granulosa Cells Reveals Key Regulatory Networks for 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.

MiR-128-3p regulates chicken granulosa cell function via 14-3-3β/FoxO and PPAR-γ/LPL signaling pathways

MicroRNAs (miRNAs) are class of 22 nt short RNA sequences which inhibit protein translation through binding to the 3’UTR of its target genes. The continuous ovulatory property of chicken follicle makes it a perfect model for studying granulosa cell (GC) functions. In this study, we found that large number of miRNAs including miR-128-3p, were differentially expressed in the GCs of F1 and F5 follicles of chicken. Subsequently, the results revealed that miR-128-3p inhibited proliferation, the formation of lipid droplets, and hormone secretion in chicken primary GCs through directly targeting YWHAB and PPAR-γ genes. To determine the effects of 14-3-3β (encoded by YWHAB) protein on GCs functions, we overexpressed or inhibited the expression of YWHAB, and the results showed that YWHAB inhibited the function of FoxO proteins. Collectively, we found that miR-128-3p was highly expressed in the chicken F1 follicles compared to the F5 follicles. In addition, the results indicated that miR-128-3p promoted GC apoptosis through 14-3-3β/FoxO pathway via repressing YWHAB, and inhibited lipid synthesis by impeding the PPAR-γ/LPL pathway, as well as reduced the secretion of progesterone and estrogen. Taken together, the results showed that miR-128-3p plays a regulatory role in chicken granulosa cell function via 14-3-3β/FoxO and PPAR-γ/LPL signaling pathways.

Orexin system in buffalo ovarian follicles and effect of orexin on estradiol production.

Orexin is a ligand for orexin receptors OX1R and OX2R; it is a neuropeptide with pleiotropic functions, including regulation of reproduction. The current study was carried out to investigate the mRNA expression of the prepro-orexin gene (PPO) and orexin receptors (OX1R and OX2R) in ovarian follicles during different stages of their development in the ovary of water buffalo (Bubalus bubalis) and to determine the role of orexin on estradiol production. Ovarian follicles were classified into four groups based on size and estradiol (E2 ) level in the follicular fluid (FF) as follows: (i) small or F1, (ii) medium or F2, (iii) large or F3 and (iv) dominant/pre-ovulatory follicle or F4. In follicles, the mRNA expression of PPO and OX1R was greater in F3 and F4 follicles in granulosa cells (GC) and theca interna (TI) cells. OX2R expression did not vary amongst the different follicular stages in GC. Orexin A and orexin receptors were localized in the cytoplasm of GC and TI, and intensity was higher in F3 and F4 follicles. In addition, we cultured GC and treated them at 0.1,1.0, and 10 ng/ml orexin A alone or in the presence of FSH (30 ng/ml) or IGF-I (10ng/ml) for 48h. There was a significant (p < 0.05) increase in estradiol (E2 ) secretion and cytochrome P0450 family 19 subfamily A member 1 (CYP19A1) expression from GC at 1.0 and 10.0 ng/ml orexin A in the presence of 30 ng/ml follicle-stimulating hormone (FSH) or 10 ng/ml insulin-like growth factor-I (IGF-I). In conclusion, the present study provided evidence that the orexin system is expressed in buffalo ovarian follicles, and orexin-A in the presence of FSH and IGF-I has a stimulatory effect on estradiol secretion from the GC of water buffalo.

Impact of fermented Broussonetia papyrifera on laying performance, egg quality, lipid metabolism, and follicular development of laying hens

Hybrid Broussonetia papyrifera (BP) has been widely planted and commonly used as ruminant forage source after fermentation in China. Very less information is available to know the impact of fermented BP on laying hens, thus, we have investigated effects of dietary supplementation of Lactobacillus plantarum-fermented B. papyrifera (LfBP) on laying performance, egg quality, serum biochemical parameters, lipid metabolism, and follicular development of laying hens. A total of 288 HY-Line Brown hens (age, 23 wk) were randomly assigned into 3 treatment groups: control group (Con, a basal diet), LfBP1 and LfBP5 group (a basal diet supplemented with 1% or 5% LfBP). Each group has 8 replicates of twelve birds each. The results demonstrated that dietary supplementation of LfBP increased average daily feed intake (linear, P < 0.05), feed conversion ratio (linear, P < 0.05), and average egg weight (linear, P < 0.05) during the entire experimental period. In addition, dietary inclusion of LfBP enhanced the egg yolk color (linear, P < 0.01) but decreased the eggshell weight (quadratic, P < 0.05) and eggshell thickness (linear, P < 0.01). In serum, the LfBP supplementation linearly decreased the content of total triglyceride (linear, P < 0.01) but increased the content of high density lipoprotein-cholesterol (linear, P < 0.05). The gene expression related to hepatic lipid metabolism including acetyl-CoA carboxylase, fatty acid synthase, and peroxisome proliferator-activated receptor (PPARα) was down-regulated whereas liver X receptor was up-regulated in LfBP1 group. Moreover, LfBP1 supplementation remarkably reduced the F1 follicle number and ovarian gene expression of reproductive hormone receptors including estrogen receptor, follicle stimulating hormone receptor, luteinizing hormone receptor, progesterone receptor, prolactin receptor, and B cell lymphoma-2. In conclusion, dietary inclusion of LfBP could improve feed intake, egg yolk color, and lipid metabolism, but may cause a decline in eggshell quality with higher inclusion level, herein, 1% is suggested.

Ovarian mRNA Expression and Regulation of Matrix Metalloproteinase 16 in the Domestic Hen

In mammals, membrane-bound matrix metalloproteinases (MT-MMPs) are thought to play an important role in ovarian remodeling. However, the role and regulation of these proteases in the ovary of birds remain largely unknown. One of MT-MMPs, i. e., MMP-16, has been found in the hen ovary; therefore, this study was undertaken to examine whether the transcript level of MMP-16 changes during follicle development and whether gonadotropins and estrogen are involved in the regulation of this enzyme expression. The relative expression of MMP-16 mRNA in the ovarian follicles (white, yellowish, small yellow, and the granulosa and theca layers of three of the largest yellow preovulatory [F3-F1]) was examined 22 h and 3 h before F1 follicle ovulation as well as following equine chorionic gonadotropin (eCG) or tamoxifen (estrogen receptor modulator, TMX) treatments by quantitative real-time polymerase chain reaction (qRT-PCR). MMP-16 transcripts were detected in all examined ovarian tissues of control and treated hens. The relative expression of MMP-16 depended on follicular size/maturation and the layer of the follicular wall. A relatively higher expression of MMP-16 mRNA in the granulosa layer at 3 h compared to 22 h before ovulation of F1 was found. The injections of eCG decreased transcript abundance of MMP-16 in white and small yellow follicles, as well as in the theca layer of F3-F2 and the granulosa layer of the F1 follicle. In turn, TMX caused an increase in mRNA expression of MMP-16 in the theca layer of the largest preovulatory follicles and a decrease in the granulosa layer of the F1 follicle. Our results provide the first mRNA expression analysis of MMP-16 in the hen ovary under different physiological states. In addition, results indicate a possible role of gonadotropins and estrogen in regulating the transcription of MMP-16 in the chicken ovary.

Corticosterone stage-dependently inhibits progesterone production presumably via impeding the cAMP-StAR cascade in granulosa cells of chicken preovulatory follicles

Stress can suppress reproduction capacity in either wild or domestic animals, but the exact mechanism behind it, especially in terms of steroidogenesis, remains under-investigated so far. Considering the important roles of progesterone in avian breeding, we investigated the modulation of corticosterone on progesterone production in cultured granulosa cells of chicken follicles at different developmental stages. Using enzyme immunoassays, our study showed that corticosterone could only inhibit progesterone synthesis in granulosa cells from F5-6, F4, and F3 follicles, but not F2 and F1 follicles. Coincidentally, both quantitative real-time PCR and western blotting revealed that corticosterone could downregulate steroidogenic acute regulatory protein (StAR) expression, suggesting the importance of StAR in corticosterone-related actions. Using the dual-luciferase reporter system, we found that corticosterone can potentially enhance, rather than inhibit, the activity of StAR promoter. Of note, combining high-throughput transcriptomic analysis and quantitative real-time PCR, phosphodiesterase 10A (PDE10A), protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2A) and cAMP responsive element modulator (CREM) were identified to exhibit the differential expression patterns consistent with cAMP blocking in granulosa cells from F5-6, F4, and F3, but not F2 and F1 follicles. Afterward, the expression profiles of these genes in granulosa cells of distinct developmental-stage follicles were examined by quantitative real-time PCR, in which all of them expressed correspondingly with progesterone levels of granulosa cells during development. Collectively, these findings indicate that corticosterone can stage-dependently inhibit progesterone production in granulosa cells of chicken preovulatory follicles, through impeding cAMP-induced StAR activity presumptively.

Transcriptomic analyses of the HPG axis-related tissues reveals potential candidate genes and regulatory pathways associated with egg production in ducks

BackgroundEgg production is one of the most important economic traits in the poultry industry. The hypothalamic-pituitary–gonadal (HPG) axis plays an essential role in regulating reproductive activities. However, the key genes and regulatory pathways within the HPG axis dominating egg production performance remain largely unknown in ducks.ResultsIn this study, we compared the transcriptomic profiles of the HPG-related tissues between ducks with high egg production (HEP) and low egg production (LEP) to reveal candidate genes and regulatory pathways dominating egg production. We identified 543, 759, 670, and 181 differentially expressed genes (DEGs) in the hypothalamus, pituitary, ovary stroma, and F5 follicle membrane, respectively. Gene Ontology (GO) analysis revealed that DEGs from four HPG axis-related tissues were enriched in the "cellular component" category. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the neuroactive ligand-receptor interaction pathway was significantly enriched based on DEGs commonly identified in all four HPG axis-related tissues. Gene expression profiles and Protein–Protein Interaction (PPI) network were performed to show the regulatory relationships of the DEGs identified. Five DEGs encoding secreted proteins in the hypothalamus and pituitary have interaction with DEGs encoding targeted proteins in the ovary stroma and F5 follicle membrane, implying that they were these DEGs might play similar roles in the regulation of egg production.ConclusionsOur results revealed that neuroactive ligand-receptor interaction pathway and five key genes(VEGFC, SPARC, BMP2, THBS1, and ADAMTS15) were identified as the key signaling pathways and candidate genes within the HPG axis responsible for different egg production performance between HEP and LEP. This is the first study comparing the transcriptomic profiles of all HPG axis-related tissues in HEP and LEP using RNA-seq in ducks to the best of our knowledge. These data are helpful to enrich our understanding of the classical HPG axis regulating the egg production performance and identify candidate genes that can be used for genetic selection in ducks.

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

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

The Influence of the Corpus Luteum Location on Hormonal and Vitamin C Composition of Follicular Fluid and Serum in Dromedary Camels (Camelus dromedarius)

The aim of this study was to compare hormonal composition of the predominant antral follicle coexisting with or without a corpus luteum (CL) in dromedary camels. Forty-seven genitalia and blood samples were collected from clinically healthy adult (7–12 years of age) non-pregnant female camels during the breeding season at a local abattoir. Follicles (0.7–1.8 cm in diameter) did not coexist with a CL were classified as follicles F1 and their blood serum labeled F1S (n=12), follicles coexist with CL on the same ovary were labeled F2 and their blood serum labeled as F2S (n=9), and follicles with contralateral CL on the other ovary labeled F3 and their blood serum named F3S (n=11). Follicular fluid (FF) and sera were subjected to biochemical and hormonal analysis. Results revealed greater (P<0.05) concentrations of progesterone in the FF of F1 follicles and F1S serum than that found in F3 follicles and F3S serum. The concentrations of cortisone and T3 were higher (P<0.05) in FF of F1 and F3, and serum of F1S and F3S in comparison to FF of follicles type F2 and serum of F2S. The thyroxine and vitamin C concentrations in FF from F1 and F2 follicles, and serum of F1S and F2S were higher (P<0.01) in comparison with the FF from F3 and serum of F3S. The presence of CL might exert a local effect on hormonal composition of FF and could indirectly influence follicular development and oocyte quality. It is better to use oocytes originated from ovaries without a CL in in-vitro fertilisation (IVF) in dromedary camels.

Molecular characterization, expression profile and transcriptional regulation of the CYP19 gene in goose ovarian follicles.

Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5'-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.

PSX-A-24 Late-Breaking: Testosterone production by theca cells of large preovulatory follicles is affected by growth hormone depending on the hen age and the presence of granulosa cells

Abstract Testosterone produced by theca cells may be involved in regulating of the growth and ovulation of hen preovulatory follicles (Rangel, Gutierrez, Gen Comp Endocrinol, 203:250, 2014). In the current research, we studied effects of growth hormone (GH), a known regulator of the hen ovarian function, on in vitro testosterone production by the theca layer (TL) from the two largest yellow follicles in relation to the hen age and the presence of the granulosa layer (GL). Young hens with long clutch (YLC, 32–33 week-old, &amp;gt;10 eggs per clutch) and old hens with short clutch (OSC, 74–76 week-old, 3–6 eggs per clutch) were used. After isolation, TL from F1 and F2 follicles (n = 8–9) was cultured for 18 h in two systems, separately or together with the corresponding GL, in the presence or absence of chicken GH (25 ng/ml). Concentrations of testosterone in the spent media were measured by ELISA. The data were analyzed by RM-ANOVA. In the case of separate TL culture, GH did not change significantly testosterone production in both follicles of YLC hens and reduced it from 338±105 to 152±52 fmol/mg tissue (P &amp;lt; 0.05) in F1 follicles of OSC hens. When TL was cultured in the presence of GL, GH enhanced 1.8-2.6-fold (P &amp;lt; 0.05) the secretion of testosterone in the case of F1 follicles and decreased it 1.8-2.5-fold (P &amp;lt; 0.05) in the case of F2 follicles in both young and old hens. Regardless of the treatment, follicular size or culture system, the production of testosterone in OSC hens was 2–5 times higher than in YLC hens. The results indicate that the interaction between TL and GL changes the steroidogenic response of theca cells from preovulatory follicles to GH in young and old hens. Furthermore, testosterone production is obviously increased with reproductive aging of laying hens. The study was supported by RFBR (19-016-00216).

Royal Jelly Improves the Morphology of the Reproductive Tract, Internal Egg Quality, and Blood Biochemical Parameters in Laying Hens at the Late Stage of Production.

Simple SummaryThe main targets of senescence in laying hens are the ovaries and mostly the follicles. Recently, there has been great attention on the investigation of beehive products, including royal jelly (RJ), for their various functional, biological, and pharmaceutical benefits. Therefore, the purpose of this study was to evaluate the impacts of RJ administration on the egg production rate, ovarian and follicular patterns, and blood chemistry of aged Lohman Brown laying hens. RJ treatment, either 100 or 200 mg kg−1 body weight, may improve the morphology of the reproductive tract (including follicular growth and oviduct morphology), egg production rate, and internal egg quality traits of aged laying hens.The purpose of this study was to evaluate the effects of royal jelly (RJ) on the morphology of the reproductive tract, egg production, and blood biochemical indices of aged laying hens. In total, 120 Lohman Brown laying hens at week 58 of age were randomly assigned into three equal groups. Pure RJ was dissolved in distilled water and injected subcutaneously as follows: the first treatment (R1; 100 mg RJ kg−1); the second treatment (R2; 200 mg RJ kg−1); the control treatment (CON; 1 mL distilled water). Both RJ-treated groups exhibited a significantly higher number of large yellow follicles (LYFs), small yellow follicles (SYFs), and large white follicles (LWFs) (p ˂ 0.05). Furthermore, RJ treatment significantly increased the diameter and weight of the F1 follicles. However, only the R2 group exhibited significantly greater ovary and uterus weights. RJ treatment did not affect the percentage of oviduct and weight of ovarian stroma. In addition, RJ increased the hen–day egg production rate compared with the CON group; however, only the R2 group showed greater egg weight (p = 0.032). RJ treatment also improved the albumen height, Haugh units, and yolk index. The administration of RJ significantly decreased the serum glucose, but increased the levels of serum albumen and cholesterol. In conclusion, RJ treatment may improve the morphology of the reproductive tract (including follicular growth and oviduct morphology), egg production rate, and internal egg quality traits of aged laying hens.

Molecular Characterization, Tissue Distribution, and Expression Profiling of the CTSD Gene during Goose Ovarian Follicle Development

Cathepsin D (CTSD) is known to be crucial for the degradation and utilization of yolk precursors in ovarian follicles. However, little is known about its expression profiles and physiological actions in avian ovarian cells. In this study, the intact coding sequence of the CTSD gene in geese was cloned for the first time, with a length of 1197 bp. It encoded a polypeptide of 398 amino acids (AA) consisting of a signal peptide and two conserved functional domains (i.e., A1_Propeptide and Cathepsin_D2). The AA sequence of goose CTSD had &gt; 96% similarities with the homologs of turkeys, chickens, and ducks. Results from real-time PCR showed that goose CTSD mRNA was present in all tissues examined, with higher levels in the adrenal gland, liver, heart, and reproductive organs. Furthermore, levels of CTSD mRNA were much higher in goose granulosa layers than in the theca layers in any follicular category. Significantly, its expression remained almost unchanged in the theca layers throughout follicle development, while it increased gradually in the granulosa layers from 2-4 mm to F5 follicles but declined there after. These results suggested that CTSD may regulate goose ovarian follicle development through its actions on both the degradation and absorption of yolk precursors and granulosa cell apoptosis.

Molecular Characterisation, Tissue Distribution, and Expression Profiling of the Cathepsin B Gene during Ovarian Follicle Development in Geese

ABSTRACT 1. Although there is evidence that Cathepsin B (CTSB) regulates the degradation and absorption of yolk precursors during avian ovarian follicle development, nothing is known about its molecular characteristics, tissue distribution or expression profiles in goose ovarian follicular compartments. 2. The intact 1023 bp coding sequence of the goose CTSB gene was obtained for the first time. It encoded a polypeptide of 340 amino acids (AA) containing two conserved functional domains (i.e., Propeptide_C1 and Peptidase_C1A_Cathpsin B) and three active amino acid residues (+108, +279, and +299). Both the nucleotide and AA sequences of goose CTSB gene showed more than 90% similarity with its respective homologs from other avian species. 3. The qRT-PCR results showed that CTSB mRNA was ubiquitously expressed in all examined goose tissues, with moderate to high levels in the reproductive organs including the ovarian stroma and oviduct. 4. Expression of goose CTSB mRNA in the granulosa layers increased gradually from the 2–4 mm F5 follicles but declined to relatively low levels in the F4-F1 follicles while remaining statistically unchanged in the theca layers throughout follicle development. 5. High sequence similarity of goose CTSB gene to other avian species suggested functional conservation of avian CTSB genes, and its fluctuating levels in the granulosa layers may be associated with the orderly progression of goose follicle development. These data laid a foundation for further elucidating the role of CTSB in the avian ovary.