Layer Of Follicles Research Articles

Tandem mass tag-based proteomic analysis of granulosa and theca interna cells of the porcine ovarian follicle following in vitro treatment with vitamin D3 and insulin alone or in combination

This study was performed to investigate the proteomic basis underlying the interaction between vitamin D3 (VD) and insulin (I) within ovarian follicle using the pig as a model. Porcine antral follicles were incubated in vitro for 12 h with VD alone and I alone or in combination (VD + I) or with no treatment as the control (C). In total, 7690 and 7467 proteins were identified in the granulosa and theca interna compartments, respectively. Comparative proteomic analysis revealed 97 differentially abundant proteins (DAPs) within the granulosa layer and 11 DAPs within the theca interna layer. In the granulosa compartment, VD affected proteome leading to the promotion of cell proliferation, whereas I influenced mainly proteins related to cellular adhesion. The VD + I treatment induced granulosa cell proliferation probably via the DAPs involved in DNA synthesis and the cell cycle regulation. In the theca interna layer, VD alone or in co-treatment with I affected DAPs associated with cholesterol transport and lipid and steroid metabolic processes that was further confirmed by diminished lipid droplet accumulation. SignificanceThe application of quantitative proteomics demonstrated for the first time the complexity of VD and I interactions in porcine ovarian follicle, providing a framework for understanding the molecular mechanisms underlying their cross-talk. Although identified DAPs were related to crucial ovarian processes, including the granulosa cell proliferation and cholesterol transport in the theca interna layer, novel molecular pathways underlying these processes have been proposed. The identified unique proteins may serve as indicators of VD and I interactions in both follicle layers, and could be useful biomarkers of ovarian pathologies characterized by impaired VD and I levels, such as polycystic ovary syndrome.

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.

FcRY is a key molecule controlling maternal blood IgY transfer to yolks during egg development in avian species.

Maternal immunoglobulin transfer plays a key role in conferring passive immunity to neonates. Maternal blood immunoglobulin Y (IgY) in avian species is transported to newly-hatched chicks in two steps: 1) IgY is transported from the maternal circulation to the yolk of maturing oocytes, 2) the IgY deposited in yolk is transported to the circulation of the embryo via the yolk sac membrane. An IgY-Fc receptor, FcRY, is involved in the second step, but the mechanism of the first step is still unclear. We determined whether FcRY was also the basis for maternal blood IgY transfer to the yolk in the first step during egg development. Immunohistochemistry revealed that FcRY was expressed in the capillary endothelial cells in the internal theca layer of the ovarian follicle. Substitution of the amino acid residue in Fc region of IgY substantially changed the transport efficiency of IgY into egg yolks when intravenously-injected into laying quail; the G365A mutant had a high transport efficiency, but the Y363A mutant lacked transport ability. Binding analyses of IgY mutants to FcRY indicated that the mutant with a high transport efficiency (G365A) had a strong binding activity to FcRY; the mutants with a low transport efficiency (G365D, N408A) had a weak binding activity to FcRY. One exception, the Y363A mutant had a remarkably strong binding affinity to FcRY, with a small dissociation rate. The injection of neutralizing FcRY antibodies in laying quail markedly reduced IgY uptake into egg yolks. The neutralization also showed that FcRY was engaged in prolongation of half-life of IgY in the blood; FcRY is therefore a multifunctional receptor that controls avian immunity. The pattern of the transport of the IgY mutants from the maternal blood to the egg yolk was found to be identical to that from the fertilized egg yolk to the newly-hatched chick blood circulation, via the yolk sac membrane. FcRY is therefore a critical IgY receptor that regulates the IgY uptake from the maternal blood circulation into the yolk of avian species, further indicating that the two steps of maternal-newly-hatched IgY transfer are controlled by a single receptor.

Background and common lesions in the female reproductive organs of giant anteaters (Myrmecophaga tridactyla).

The giant anteater (Myrmecophaga tridactyla) is a vulnerable species in South America and is considered endangered or near extinction in Central America. Therefore, studies describing the reproductive characteristics of this species are pivotal for its conservation. Thus, this study aimed to provide a morphological description of the female reproductive tissues of this species. We collected tissue samples from six female giant anteaters and performed gross, morphological, and histochemical analyses. Five adult subjects and one juvenile were included in the study. In the ovary, classifications were made according to the follicle and oocyte sizes: primordial, primary, secondary, early antral, or antral. Typical follicles with a single oocyte surrounded by a simple or stratified layer of cubic epithelium, atretic follicles, corpora lutea, corpora albicans, and ovarian cysts were also observed. No ovarian lesions were observed. By contrast, endometritis, metritis, mucometra, and endometrial cysts were identified in the uterus. Uterine alterations in these subjects were frequent and could affect reproduction.

Transcriptome Analysis of Granulosa Cells Reveals Regulatory Mechanisms Related to Chicken Follicle Development.

In this study, we aimed to better understand the difference between the functions of the two types of granulosa cells and sought to discover more key genes involved in follicle development and follicle selection. Herein, we separately collected pre-hierarchical follicle granulosa cells (PHGCs) and preovulatory follicle granulosa cells (POGCs) for RNA extraction; the transcriptomes of the two groups were compared via RNA-seq. A total of 5273 differentially expressed genes (DEGs) were identified between the PHGCs and POGCs; 2797 genes were up-regulated and 2476 were down-regulated in the PHGCs compared with the POGCs. A qPCR analysis confirmed that the expression patterns of 16 randomly selected DEGs were highly consistent with the RNA-seq results. In the POGCs, many of the genes with the most significant increase in expression were related to steroid hormone synthesis. In addition, the genes with the most significant decline in expression, including AMH and WT1, were related to the inhibition of steroid hormone synthesis. These results suggest that steroid hormones play a key role in follicle development. Furthermore, a Gene Ontology (GO) analysis revealed that these DEGs were mainly involved in the primary metabolic process, the carbohydrate metabolic process, the cellular process, ribosomes, the cytoplasm, and intracellular processes. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were mainly enriched in steroid biosynthesis, the cell cycle, ribosomes, the TGF-beta signaling pathway, focal adhesion, and so on. We also observed the morphology of the follicles at different developmental stages, and the results showed that the thickness of the granular layer of the small yellow follicles (SYFs) decreased significantly with further development. In addition, we also found that the thickness of the granulosa layer of hens over 300 days old was significantly lower than that of 200-day-old hens. In short, these data indicate that the tissue morphology and function of granulosa cells change throughout follicle development.

Ovary of Zebrafish during Spawning Season: Ultrastructure and Immunohistochemical Profiles of Sox9 and Myostatin.

This study sought to examine the ovarian cellular and stromal components of the zebrafish (Danio rerio) throughout the spawning season using light and electron microscopic tools. The ovaries of zebrafish showed oocytes in all stages of follicular development and degeneration (atresia). Six stages of oogenesis were demonstrated: oogonia, early oocytes, late oocytes, vacuolated follicles, the yolk globule stage (vitellogenesis), and mature follicles. The SOX9 protein was expressed in the ooplasm of the primary and previtellogenic oocytes and the theca cell layer of the mature follicles. Myostatin was expressed in the granulosa and theca cells. Many stem cells in the ovarian stroma expressed myostatin and SOX9. During the spawning season, the EM results indicated that the zona radiata increased in thickness and was crossed perpendicularly by pore canals that contained processes from both oocytes and zona granulosa. The granulosa cells contained many mitochondria, rER, sER, and vesicles. Meanwhile, the thecal layer consisted of fibroblast-like cells. Atretic follicles could be demonstrated that involved both oocytes and their follicular walls. Several types of cells were distinguished in the ovarian stroma, including mast cells, telocytes, lymphocytes, fibroblasts, endocrine cells, macrophages, adipocytes, dendritic cells, and steroidogenic (stromal) cells. The ovary of the zebrafish serves as a model to investigate follicular development.

Involvement of cathepsin L in the degradation and degeneration of postovulatory follicle of the medaka ovary†.

Cathepsin L plays physiological and pathological roles in immune responses, cancer, metamorphosis, and oogenesis in several species. However, the function of Cathepsin L in medaka ovaries remains unclear. Therefore, here, we examined the physiological functions of Cathepsin L in the medaka ovaries. Cathepsin L mRNA transcripts and proteins were found to be constitutively expressed in the ovaries of Oryzias latipes over a 24-h spawning cycle. Expression was localized within the oocyte cytoplasm of growing follicles and the follicle layer of preovulatory and postovulatory follicles. Moreover, the active form of Cathepsin L was highly expressed in the follicle layer of periovulatory follicles and the ovaries 2-6h after ovulation. Recombinant Cathepsin L was activated under acidic conditions and exhibited enzymatic activity in acidic and neutral pH conditions. However, extracellular matrix proteins were degraded by recombinant Cathepsin L under acidic, not neutral pH conditions. Cathepsin L was secreted from preovulatory follicles, while active recombinant Cathepsin L was detected in the conditioned medium of a medaka cell line, OLHNI-2. Mechanistically, recombinant Cathepsin L activates recombinant urokinase-type plasminogen activator-1, which is expressed within the follicle layers post-ovulation. Meanwhile, the treatment of medakas with an E-64 or anti-Cathepsin L antibody effectively blocked follicular layer degeneration and degradation after ovulation, whereas in vitro ovulation was not inhibited by either. Collectively, the findings of this study indicate that although Cathepsin L does not impact ovulation in medakas, it contributes to the degeneration and degradation of the follicle layers following ovulation via activation of urokinase-type plasminogen activator-1, and not via the degradation of extracellular matrix proteins.

Identification of the Keratin-Associated Protein 22-2 Gene in the Capra hircus and Association of Its Variation with Cashmere Traits.

The Cashmere goat is an excellent local goat breed in Gansu Province of China, and it is expected to improve cashmere production and cashmere quality through selection and breeding to enhance its commercial value. Keratin-associated proteins (KAPs) play an important role in maintaining wool structure. The gene encoding the keratin-associated protein 22-2 (KAP22-2) gene has been identified in selected species other than goats, such as humans, mice, and sheep. In this study, the sequence of the sheep KAP22-2 gene (KRTAP22-2) was aligned into the goat genome, and the sequence with the highest homology was assumed to be the goat KRTAP22-2 sequence and used to design primers to amplify the goat gene sequence. A total of 356 Longdong Cashmere goats (Gansu Province, China) were used for screening of genetic variants. Four specific bands were detected by polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) analysis, and they formed a total of six band types individually or in combination. Four alleles were identified by DNA sequencing of PCR amplification products. A total of four single nucleotide polymorphic sites (SNPs) were detected in the four sequenced KRTAP22-2 alleles. Two of them are in the 5'UTR region and the other two are in the coding region, and the variants in the coding region are all non-synonymous mutations. In addition, there was a 6 bp length variation in allele C. The gene was expressed in the cortical layer of primary and secondary hair follicles, the inner root sheath, as well as hair papillae and hair maternal cells in goats. The results of the correlation analysis between genotypes and cashmere traits showed that after excluding genotypes with a gene frequency of less than 5%, the mean fiber diameter (MFD) of cashmere was significantly higher in the AB genotype than in the AA and AC genotypes. That is, the KRTAP22-2 gene variants are associated with mean fiber diameter in cashmere. The above results suggest that the goat KRTAP22-2 variant can be utilized as a molecular marker candidate gene for cashmere traits.

TFE3 nuclear expression as a novel biomarker of ovarian sclerosing stromal tumors and associated with its histological morphology

Sclerosing stromal tumors of the ovary are benign and tend to occur in youthful women with lobular structures at low frequencies. Three types of cells, including luteinized cells, short spindle myoid cells, and intermediate cells, are found in the lobules which abundant in the blood vessels. Currently, immunohistochemistry is used to detect normal follicles, sclerosing stromal tumors, granulosa cell tumors, and fibromas/thecomas. Our research results showed that transcription factor enhancer 3 (TFE3) was moderate to strong positive in the theca interna layer of normal follicles. TFE3 was expressed in seven out of eight sclerosing stromal tumors, mainly in luteinized cells. It did not express in 20 granulosa cell tumors. Of the nine fibromas/thecomas, TFE3 was weakly staining in 2 cases and negative in the remaining 7 cases. The expression of TFE3 was also weak in only one microcystic stromal tumor. 8 cases of sclerosing stromal tumors were analyzed by FISH using a TFE3 separation probe, and the results were negative. In short, as a nuclear transcription protein, TFE3 specifically expressed in sclerosing stromal tumors and could serve as a new marker for the diagnosis and differential diagnosis of sclerosing stromal tumors. Moreover, we speculate that TFE3 will promotes the formation of the vascular plexus after entry into the nucleus, which can further explain why sclerosing stromal tumors are different from other ovary sex-cord stromal tumors.

P-410 First wave of primordial follicle activation during human ovarian tissue manipulation for fertility preservation

Abstract Study question Does ovarian tissue biopsy, transportation and processing for fertility preservation and restoration trigger primordial follicle activation? Summary answer Early manipulation of ovarian tissue is sufficient to trigger follicle activation by stimulating PI3K/Akt and disrupting the Hippo pathway. What is known already Primordial follicle recruitment occurs continuously in physiological conditions by modulation of autocrine and paracrine factors, like the PI3K/Akt and Hippo pathways, to ensure follicle growth over time. During fertility preservation and restoration procedures, follicle activation may be disrupted and follow nonphysiological patterns. The ability to control activation dynamics by up- or downregulation of these pathways may enhance fertility restoration outcomes in a number of ways. Indeed, downregulation of follicle activation shortly after transplantation may protect the ovarian reserve from early depletion. Conversely, ovarian tissue in vitro culture may benefit from upregulation of primordial follicle activation to boost further growth. Study design, size, duration Fresh ovarian tissue was retrieved from nine women undergoing laparoscopic surgery for benign conditions. Three timepoints were investigated. One-third of collected tissue per patient was immediately fixed in the operating room, without any manipulation (time zero, T0). The remaining tissue was transferred to the laboratory and dissected to remove any surplus medulla. It was then cut into small cortical fragments, half of which were fixed after 25 minutes(T25) and the other half after 90 minutes(T90). Participants/materials, setting, methods All cortical fragments were fixed in 4%formaldehyde and embedded in paraffin for histology. In order to explore follicle activation,markers of the PI3K/Akt and Hippo signaling pathways were immunolabeled at each timepoint, targeting: (i) phospho-Akt (p-Akt) in primordial follicles by immunohistochemistry as a marker of early PI3K/Akt pathway activation; and (ii) Yes-associated protein (YAP) cellular localizationin the granulosa cell layer of primordial follicles by immunofluorescence as a marker of Hippo disruption. Main results and the role of chance An upturn in p-Akt expression was observed at T25 (22,34 ± 0.13%; p = 0.0233) and T90 (39,01 ± 0.22%, p = < 0.0001) compared to T0 (2,87 ± 0,03%). In terms of YAP cellular localization, a significant nucleus-to-cytoplasm shift was detected at T25 (1.11 ± 0.09; p = 0.0428) compared to T0 (0.97 ± 0.10), while T90 (1.07± 0.14) values were similar to T25. Our data prove that ovarian tissue manipulation triggers primordial follicle activation very early, involving both the PI3K/Akt and Hippo signaling pathways, which appear to cooperate in primordial-to-primary follicle transition. Our results indicate that the first stages of any fertility preservation or restoration procedure involving ovarian tissue manipulation contribute to dysregulation of the very mechanisms responsible for the ovarian reserve maintenance and follicle growth. Additional strategies are required to gain the control of follicle activation mechanisms in nonphysiological conditions (ex vivo ovarian tissue manipulation), in order to exploit ovarian reserve dynamics to serve the need of patients. Limitations, reasons for caution Analyses in the study were limited to histology and immunolabeling to acquire a descriptive picture of pathway activation kinetics over time. Further investigations using dynamic experimental models are essential to advance our understanding of signaling pathway synergy in vivo. Wider implications of the findings Since dysregulation of follicle activation in nonphysiological conditions appears to be associated with poor oocyte quality, enhancing our ability to control the relevant signaling pathways is crucial to optimizing fertility preservation procedures. Trial registration number not applicable

Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells1

Tissue factor pathway inhibitor 2 (TFPI2) plays a key role in female reproduction. However, its expression and function in chickens are still unclear. In the present study, ovarian tissues from 30 w and 15 w laying chickens were analyzed using RNA-seq to identify the differentially expressed gene TFPI2. The full-length cDNA of TFPI2 was obtained from adult chicken ovaries by rapid-amplification of cDNA ends (RACE), and the putative TFPI2 protein was found to share a highly conserved amino acid sequence with known bird homologs. In addition, TFPI2 was widely expressed in the tissues of adult chicken follicles according to quantitative real-time PCR (qRT–PCR) and Western blotting. Immunohistochemistry suggested that the TFPI2 protein existed in chicken ovary follicles at different developmental states, such as primordial follicles, the ovarian stroma, and the granulosa and theca layers of prehierarchical follicles (6-8 mm) and preovulatory follicles (F1). In vitro, Follicle stimulating hormone or Luteinizing hormone (FSH/LH) stimulated the expression of TFPI2 in chicken granulosa cells. FSH-/LH-induced TFPI2 mRNA expression was mediated by signaling pathways such as the PKA, PKC, PI3K, and mTOR pathways. Functionally, TFPI2 promoted the proliferation and viability of cultured granulosa cells and decreased the secretion of Progesterone (P4) and Estrogen (E2) and the mRNA abundance of key steroidogenic enzymes (STAR, Cyp17a1, Cyp19a1 and 3B-HSD) as well as MMPs (MMP2, 7, 9 and 11). Mechanistically, TFPI2 inhibited the expression of MMP7 via the Wnt signaling pathway. These findings indicate that TFPI2 may play an important role in regulating chicken follicular development and ovulation and suggest the molecular regulation mechanisms.

Global gene expression analysis of the turkey hen hypothalamo-pituitary-gonadal axis during the preovulatory hormonal surge

The preovulatory hormonal surge (PS) consists of elevated circulating luteinizing hormone (LH) and progesterone levels and serves as the primary trigger for ovarian follicle ovulation. Increased LH and progesterone, produced by the pituitary and the granulosa layer of the largest ovarian follicle (F1), respectively, result from hypothalamic stimulation and steroid hormone feedback on the hypothalamo-pituitary-gonadal (HPG) axis. The hypothalamus, pituitary, F1 granulosa, and granulosa layer of the fifth largest follicle (F5) were isolated from converter turkey hens outside and during the PS and subjected to RNA sequencing (n=6 per tissue). Differentially expressed genes were subjected to functional annotation using DAVID and IPA. A total of 12, 250, 1235, and 1938 DEGs were identified in the hypothalamus, pituitary, F1 granulosa, and F5 granulosa respectively (q<0.05, |fold change|>1.5, FPKM>1). Gene Ontology (GO) analysis revealed key roles for metabolic processes, steroid hormone feedback, and hypoxia induced gene expression changes. Upstream analysis identified a total of 4, 42, 126, and 393 potential regulators of downstream gene expression in the hypothalamus, pituitary, F1G, and F5G respectively, with a total of 63 potential regulators exhibiting differential expression between samples collected outside and during the PS (|z-score|>2). The results from this study serve to increase the current knowledge base surrounding the regulation of the PS in turkey hens. Through GO analysis, downstream processes and functions associated with the PS were linked to identified DEGs, and through upstream analysis, potential regulators of DEGs were identified for further analysis. Linking upstream regulators to the downstream PS and ovulation events could allow for genetic selection or manipulation of ovulation frequencies in turkey hens.

Cholesterol accumulation in ovarian follicles causes ovulation defects in Abca1a−/− Japanese medaka (Oryzias latipes)

ATP-binding cassette A1 (ABCA1) is a membrane protein, which exports excess cellular cholesterol to generate HDL to reduce the risk of the onset of cardiovascular diseases (CVD). In addition, ABCA1 exerts pleiotropic effects on such as inflammation, tissue repair, and cell proliferation and migration. In this study, we explored the novel physiological roles of ABCA1 using Japanese medaka (Oryzias latipes), a small teleost fish. Three Abca1 genes were found in the medaka genome. ABCA1A and ABCA1C exported cholesterol to generate nascent HDL as human ABCA1 when expressed in HEK293 cells. To investigate their physiological roles, each Abca1-deficient fish was generated using the CRISPR-Cas9 system. Abca1a−/− female medaka was found to be infertile, while Abca1b−/− and Abca1c−/− female medaka were fertile. In vitro ovarian follicle culture suggested that Abca1a deficiency causes ovulation defects. In the ovary, ABCA1A was expressed in theca cells, an outermost layer of the ovarian follicle. Total cholesterol content of Abca1a−/− ovary was significantly higher than that of the wild-type, while estrogen and progestin contents were compatible with those of the wild-type. Furthermore, cholesterol loading to the wild-type follicles caused ovulation defects. These results suggest that ABCA1A in theca cells regulates cholesterol content in the ovarian follicles and its deficiency inhibits successful ovulation through cholesterol accumulation in the ovarian follicle.

Basal cell carcinoma - principles of treatment

Basal cell carcinoma (BCC) is one of the most common malignant tumors in human medicine and the most common skin malignancy, with the largest number of lesions found on exposed parts of the skin, on the face, head, and neck. The average age of the patients is 60 years, with an increasing incidence in younger ages and an increased incidence in males. The incidence of BCC is increasing and doubles every 25 years. Annually, there are approximately 1,000,000 newly diagnosed cases worldwide. The frequency of malignant skin tumors depends on the influence of external factors such as ultraviolet radiation and other biological properties of the skin with a higher incidence in fair-skinned people (Fitzpatrick type I and type II skin types). BCC is a slow-growing malignant tumor that arises from the basal layer of the epidermis, the outer layer of hair follicles, or the sebaceous glands. BCC can be locally invasive and, if neglected, can infiltrate surrounding structures (muscles and cartilage) and vital structures, which can ultimately lead to death. The clinical presentation is very diverse and dependent on the histological subtype. Prevention is the most important and effective approach towards reducing the burden of BCC. The best treatment for BCC is surgical excision with confirmation and verification of surgical margins. The therapeutic goal is oncologic radical resection of the tumor, followed by reconstruction of the affected area for structure and optimal aesthetic result.

The anti-inflammatory cytokine IL-22 exhibited a proinflammatory effect in the ovary aiding with the parturition of ovoviviparous black rockfish (Sebastes schlegelii)

IntroductionAs a unique type of immunological process, pregnancy and subsequent parturition are associated with a series of inflammatory events. As an anti-inflammatory cytokine, IL-22 has been proven to participate in the parturition process in mammals. However, less is known about ovoviviparous teleosts. To investigate the role of IL-22 in fish parturition, black rockfish, an ovoviviparous teleost, was used. This fish is unique in that it provides over 60% nutrition supply for over 50 thousand embryos at the same time though a placenta-like structure during pregnancy while delivering all fries in two hours.MethodsSequence alignment, phylogenetic tree analysis and homology modeling were performed on IL-22 and its receptor. In situ hybridization demonstrated ovarian localization of il22 and il22r1. The protein of rIL-22 was obtained through the prokaryotic expression. RNA-seq analysis was performed on black rockfish ovarian cells treated with rIL-22.ResultsIn situ hybridization results showed that both il22 and il22ra1 were localized in the perinatal follicle layer and embryonic envelope. The il22 expression level was significantly increased during parturition compared to before and after parturition (P&amp;lt;0.05). To further understand the mechanism of IL-22 in parturition, rIL-22 was obtained. The downstream variation genes in primary cultured ovarian cells in the perinatal period were analyzed according to the transcriptomic results. A total of 168 differentially expressed genes (DEGs) were identified in the rIL-22 group compared with the PBS control group (padj &amp;lt; 0.05 and |log2FoldChange| 1). Of those, 134 DEGs were identified in the LPS and rIL-22 treatment groups, including chemokine, cytokine, and PG synthesis. 34 DEGs were mainly identified in immune-related pathways and reorganization of the cytoskeleton responded only to rIL-22 stimulation but not LPS. KEGG pathways mainly included immune response, PG synthesis, cell death and angiogenesis (p value &amp;lt; 0.05).DiscussionTaken together, our results indicated that IL-22 plays an important role in the parturition of black rockfish by upregulating PG synthesis and increasing chemokine, proinflammatory factor, as well as PG synthetase levels.

Luteinizing hormone stimulates the expression of amphiregulin in human theca cells

BackgroundLuteinizing hormone (LH) can stimulate mural granulosa cells to produce Amphiregulin (AREG), which can induce the resumption of meiosis in oocytes. Theca cells are present in the outer layer of follicles, providing communication with the pituitary axis through the established vascular system around the follicle. As LH target cells, it is unknown whether theca cells can produce AREG after LH stimulation.MethodsPrimary cultured human theca cells were treated with LH (with or without the inhibitor of PKA, H89), or agonists of adenylate cyclase (forskolin or db-cAMP). The mRNA and protein levels of AREG were evaluated by RT-qPCR, immunochemistry, immunofluorescence, western blotting, and ELISA.ResultsImmunohistochemistry of normal ovarian tissue obtained in the early-mid follicle phase showed that AREG expression was absent in both the theca layer and the granulosa cell layer of antral follicles. Double immunofluorescent staining revealed colocalization of AREG and CYP17A1 in human theca cells and colocalization of FSHR and AREG in human granulosa cells isolated from follicular fluid collected during IVF/ICSI after hCG trigger. LH significantly increased the mRNA and protein levels of AREG in human theca cells and the concentration of AREG in the culture medium. Forskolin and db-cAMP, activators of the cAMP/PKA signalling pathway, also significantly increased the mRNA and protein levels of AREG in human theca cells and the concentration of AREG in the culture medium. H89 antagonized the stimulating effect of LH on AREG expression in human theca cells. In addition, the concentration of AREG was lower in polycystic ovarian syndrome (PCOS) follicular fluid than in normal follicular fluid. The mRNA levels of AREG were significantly lower in PCOS granulosa cells and theca cells than in normal granulosa cells and theca cells.ConclusionLH can stimulate the expression of AREG in human theca cells, and the adenylate cyclase/cAMP/PKA cascade may mediate this process. Expression of AREG is decreased in PCOS theca cells compared to normal theca cells, with or without LH stimulation.

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.

BMP4/SMAD8 signaling pathway regulated granular cell proliferation to promote follicle development in Wanxi white goose

Granular cells proliferation in goose regulated by bone morphogenetic proteins (BMPs) signaling pathway is still unknown. In this experiment, BMPs and their receptor, and receptor activated mothers against decapentaplegic homologs (SMADs) were quantitatively expressed in granular cell layer of pre-hierarchycal and hierarchycal follicles in Wanxi White goose. The screened BMP was then used for construction of overexpressed and knockdown vectors and transfected into granular cells of goose to assess the cell proliferation and apoptosis. Granular cells with BMP-overexpressed were then used for ChIP-Seq analysis to elucidate the molecular mechanism of BMP affecting granular cell proliferation. The results showed that the mRNA expression of BMP4 was significantly expressed in pre-hierarchical follicles, and also highly expressed in hierarchical follicles than other BMPs, while the Ⅰ and Ⅱ type of BMP receptors were expressed in basic level. The mRNA expression of SMAD8 was significantly elevated in pre-hierarchical follicles. Overexpression of BMP4 could promote the proliferation of granular cells and inhibited the expression of BMP4 caused a higher cell apoptosis. ChIP-Seq identified multiple regulatory targets of SMAD4, which were mostly related to cell cycle and lipid metabolism according to the GO and KEGG pathway enrichment. From the five most significant binding motif and quantitative expression verification, the activin membrane binding inhibitor (BAMBI) was down regulated in BMP4 overexpressed granular cells. In conclusion, the BMP4 was highly expressed in granular cells and phosphorylates SMAD8, the activated SMAD8 combined with SMAD4 transfers into nucleus to regulate the expression of BAMBI to promote lipid synthesis.

Gga-miR-449b-5p Regulates Steroid Hormone Synthesis in Laying Hen Ovarian Granulosa Cells by Targeting the IGF2BP3 Gene.

Simple SummaryThe aim of this study was to explore the regulatory effect of gga-miR-449b-5p on GC proliferation and steroidogenesis in laying hens. Our results showed that gga-miR-449b-5p had no effect on the proliferation of GCs, but regulated the expression of key genes involved in steroid synthesis and the secretion of P4 and E2. In addition, gga-miR-449b-5p could target IGF2BP3 and inhibit its mRNA and protein expression. Therefore, we concluded that gga-miR-449b-5p played an important role in the synthesis of steroid hormones in laying hens.MiRNAs have been found to be involved in the regulation of ovarian function as important post-transcriptional regulators, including regulators of follicular development, steroidogenesis, cell atresia, and even the development of ovarian cancer. In this study, we evaluated the regulatory role of gga-miR-449b-5p in follicular growth and steroid synthesis in ovarian granulosa cells (GCs) of laying hens through qRT-PCR, ELISAs, western blotting and dual-luciferase reporter assays, which have been described in our previous study. We demonstrated that gga-miR-449b-5p was widely expressed in granulosa and theca layers of the different-sized follicles, especially in the granulosa layer. The gga-miR-449b-5p had no significant effect on the proliferation of GCs, but could significantly regulate the expression of key steroidogenesis-related genes (StAR and CYP19A1) (p < 0.01) and the secretion of P4 and E2 (p < 0.01 and p < 0.05). Further research showed that gga-miR-449b-5p could target IGF2BP3 and downregulate the mRNA and protein expression of IGF2BP3 (p < 0.05). Therefore, this study suggests that gga-miR-449b-5p is a potent regulator of the synthesis of steroid hormones in GCs by targeting the expression of IGF2BP3 and may contribute to a better understanding of the role of functional miRNAs in laying hen ovarian development.

Immuno-Neutralization of Follistatin Bioactivity Enhances the Developmental Potential of Ovarian Pre-hierarchical Follicles in Yangzhou Geese.

Simple SummaryFollistatin involves in the regulation of ovarian follicular development in mammals; however, the role of follistatin in goose ovarian follicular development has not been investigated. In this study, following immuno-neutralization of follistatin bioactivity in geese, the number of ovarian pre-ovulatory follicles significantly increased, and mRNA levels of genes involved in ovarian steroidogenesis and yolk deposition were upregulated in the granulosa layer of pre-hierarchical follicles. These results suggest that follistatin plays a limiting role in the development of ovarian pre-hierarchical follicles into pre-ovulatory follicles. These results also expand our understanding of the mechanism of follistatin on ovarian follicular development in geese.In order to explore the role of follistatin (FST) in ovarian follicular development and egg production in Yangzhou geese, sixty-four egg laying geese of the same genetic origin were selected and divided into two groups with equal numbers. One group was immunized against the recombinant goose FST protein by intramuscular injection, whereas the control group received bovine serum albumin (BSA) injection. Immunization against FST significantly increased the number of pre-ovulatory follicles. Furthermore, immunization against FST upregulated Lhr, Star, Vldlr, Smad3, and Smad4 mRNA levels in the granulosa layer of pre-hierarchical follicles. The results suggest that FST plays a limiting role in the development of ovarian pre-hierarchical follicles into pre-ovulatory follicles by decreasing follicular sensitivity to activin in geese. The mechanism may be achieved by regulating the SMAD3 signaling pathway, which affects progesterone synthesis and yolk deposition in pre-hierarchical follicles.