Layer Of Follicular Cells Research Articles

Black rockfish（Sebastes schlegelii）nuclear progesterone receptor: Cloning, tissue distribution and potential role in ovoviviparity teleost pregnancy and parturition

Nuclear progesterone receptor (nPR) is a member of the nuclear receptor family known to play a critical role in gonad development, gametogenesis, and ovulation in vertebrates, and also important in pregnancy maintenance and parturition in mammals. However, its biological functions in ovoviviparity teleosts are less clear. This study aimed to determine the possible role of nPR in fish oogenesis, pregnancy and parturition using black rockfish as a model. The npr, which consists of 2076 bp and codes for a 691 amino acids protein, was cloned. Phylogenetic analysis revealed that the nPR of black rockfish is more conserved in teleosts. Quantitative real-time PCR analyses in various tissues showed that npr was mainly detected in the pituitary followed by the ovary, testis and brain, while it was very low or undetectable in the spleen, head kidney, intestine and gill. During the gonadal development process, the npr level showed no variation in the ovary. In testis, the mRNA level of npr showed varied expression in different developmental stages with the highest level in the maturation stage. Detection of npr expression during fertilization and the perinatal period further showed that nPR may play important roles in the regulation of pregnancy and the parturition. In situ hybridization further revealed that npr mRNA was observed in the follicular cell layer during the perinatal period. The results from in vitro experiments showed that npr level was significantly downregulated when the primary cultured follicular cells during vitellogenesis (stage Ⅳ) were exposed to hCG. Studies using primary culture of black rockfish ovarian cells during the perinatal period, treated in vitro revealed that genes related to PGs synthesis (pla2g4a, cox2) and matrix metalloproteinases (mmp2, mmp9) were upregulated in response to 17,20β-P. However, treatment with 17,20β-P downregulated npr expression. In conclusion, these data suggest that nPR plays an essential role in fertilization, pregnancy maintenance and parturition in black rockfish.

Studies on the hormone and cytokine producing function of human cumulus cells and its interrelation with fertility in polycystic ovarian syndrome

Cumulus cells (CC) are the specialized layer of follicular cells that are in close contact with the oocyte. They are considered as indirect markers of the oocyte quality. Changes of these cells suggest a damage of the ovary. Determination of cytokines in cumulus cell culture may predict the chance for the conception and development of pregnancy. The objective of the present study was to obtain a primary culture of CC from healthy donors and patients with polycystic ovarian syndrome (PCOS), to identify the most significant differences in production of key cytokines in the CC monocultures of patients and healthy individuals in order to predict the results of in vitro fertilization. Materials and methods: the cell culture technique was used, i.e., cumulus cells of healthy donors and patients with polycystic ovary syndrome were obtained by transvaginal puncture of follicles in the in vitro fertilization (IVF) program. This procedure does not affect the rights of embryo, since the CC are not used at the stage of IVF procedure and intracytoplasmic sperm injection (Ethical Committee Protocol No. 9 of May 16, 2022). IL-6, IL-10, IFN and progesteroneparameters were tested in adhesive cultures of CC by ELISA technique on days 1, 3, 7 of in vitro experiments. Results: We revealed a continuous secretion of progesterone, IL-6, IL-10, IFN in adhesive monocultures of CC. In the patients with PCOS, we have found a sharp increase of progesterone level in cultural media (p 0.01) on the 7th day, By contrast, the initially increased progesterone levels proved to be significantly decreased in donors on the 7th day of culture. Moreover, in the culture of CC from patients with PCOS (7th day of the experiment), the values of IL-6, IL-10 increased only two-fold compared with 30-fold increase of these cytokines in healthy donors (p 0.01). At the same term, we have observed a threefold decrease in IFN in the CC cultures of PCOS patients (p1-7 0.01; p 0.05) compared with cultured controls, which showed a 20-fold increase (p1-7 0.01), thus determining differences in total cytokine balance and, probably, influencing the pregnancy prognosis. Conclusion: Significant multidirectional changes of cytokine levels in the culture of cumulus cells of the patients with PCOS and in healthy individuals may be regarded as determining factors in formation of blastocyst and preservation of the embryo. A further in vitro research on the production of cytokines and sex steroids by CC is especially important on day 5 to 7, when the oocytes are selected for entry into the in vitro fertilization cycle. The study of morpho-functional properties of little-studied cumulus cells using the cell culture technique will enable us for a deeper study on the mechanisms of disturbed folliculogenesis in PCOS, and, thereby, improve the reproductive prognosis in this disorder.

Follicular cells protect Xenopus oocyte from abnormal maturation via integrin signaling downregulation and O-GlcNAcylation control

Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologous-expressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR) and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin β1 or Grb7 binding to the EGFR. In addition, we determined that oocytes surrounded by their follicular cells expressing EGFR-Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrin β1-EGFR complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT-O-GlcNAcase duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression.

A crustacean vitellogenin-derived peptide as an oocyte-specific delivery vehicle for gene silencing

Gene silencing by dsRNA is well documented in crustaceans, but RNA interference (RNAi) in developing oocytes is yet to be achieved. The main obstacle to RNAi in the oocytes of oviparous animals derives from their protective layers, including the cytosolic membrane, the vitelline envelope, and a layer of follicular cells. These layers form a barrier preventing the entry of large nonspecific molecules, such as double-stranded RNA (dsRNA). This article describes a sophisticated tool – designated OSDel [oocyte-specific delivery] – for the delivery of dsRNA for gene silencing in the developing oocyte. The methodology exploits the process of receptor-mediated endocytosis (RME) taking place in the crustacean female for internalizing vitellogenin (Vg) (the precursor of the major yolk protein, vitellin) into oocytes. In this process, the extracellular domain of the Vg receptor (VgR) interacts with a distinct amino acid sequence of Vg and internalizes it to form yolk droplets. Here, we tested the premise that a distinct 24 amino-acid peptide derived from Macrobrachium rosenbergii Vg (designated VgP) would interact with VgR to form particles of a size suitable for piggy-backing dsRNA into oocytes via RME. We found that fluorescently labeled VgP had a micromolar affinity for the ligand-binding domain (LBD) of the VgR and could indeed be delivered to and internalized in oocytes. As model system to illustrate the applicability of the OSDel, we injected vitellogenic females with dsRNA encoding the eye development gene PAX6 piggybacked on VgP. Proof that the dsRNA had been successfully internalized into the developing oocytes and had silenced the PAX6 gene was derived from impaired eye development in 87% of the embryos. The ability to manipulate embryos by simple injection into vitellogenic crustacean females may prove to be a powerful high throughput tool for functional genomics investigation in crustacean embryos and for silencing genes relevant to crustacean aquaculture and biotechnology.

The protective effect of co-administration of platelet-rich plasma (PRP) and pentoxifylline (PTX) on cyclophosphamide-induced premature ovarian failure in mature and immature rats

Cyclophosphamide (CP), as an antineoplastic agent, causes premature ovarian failure (POF) due to ovarian toxicity and subsequent infertility in women. Platelet-rich plasma (PRP) has accumulated significant attention in regenerative medicine. Pentoxifylline (PTX) as a methylxanthine derivative has been shown to have antioxidant and antiapoptotic properties. The aim of this study was to evaluate the protective effect of PRP and PTX on CP-induced POF. Fifty mature and immature female rats were assigned into five groups: control, CP (75 mg/kg, intraperitoneal [ip] on days 1 and 10 to induce POF), CP + PRP (200 μl, ip, half an hour after CP injection on day 1 and 10), CP + PTX (50 mg/kg, orally, half an hour after CP injection daily for 21 day), and CP + PRP + PTX. At the end of experiments on day 21, measurement of body weight, ovarian parameters (ovarian volume, follicular granulosa cell layers diameter, oocyte diameter, and the number of granulosa cells), measurement of ovarian hormone in sera for estradiol (E2), and anti-Mullerian hormone (AMH), as well as biochemical assessment were performed. The results showed that CP significantly reduced the ovarian parameters, E2, AMH, superoxide dismutase (SOD) activity and increased Malondialdehyde (MDA) levels compared to the control group (p < 0.001). Our results also indicated that all histomorphometric parameters and biochemical markers in CP-induced POF, were preserved close to normal by PRP and PTX treatments in both mature and immature rats (p < 0.001). Therefore, it is concluded that the co-administration of PRP and PTX can protect the ovary from CP-induced POF.

Inhibition of oocyte maturation by nitric oxide synthase 1 (NOS1) in zebrafish

It is well-documented that nitric oxide (NO) is an important regulator of oocyte maturation in mammals. Conversely, the function of NO during oocyte maturation has received little attention in nonmammalian vertebrates. NO is produced from L-arginine through the action of the enzyme NO synthase (NOS). Herein, we examined the expression, hormonal regulation, and involvement of NOS in meiotic signaling in zebrafish oocyte maturation. Three types of nos genes, nos1, nos2a, and nos2b, have been identified in zebrafish. We found that the expression of nos1 was highest in the ovary among the three nos genes, with maximal expression in full-grown (FG)-stage follicles during folliculogenesis. In addition, the concentration of NO was reduced during oocyte maturation and this corresponded with the decreased expression of nos1 in the follicular cell layers, suggesting that NOS1-derived NO may be one of the inhibitors of oocyte maturation in zebrafish. This is the first description of nos1 involvement in oocyte maturation in vertebrates. Moreover, the NO donor SNAP (S-nitroso-l-acetyl penicillamine) partially attenuates human chorionic gonadotropin (hCG)- and 17,20β-P-induced GVBD (germinal vesicle breakdown), perhaps by increasing cGMP levels during oocyte maturation. Finally, our results showed that SNAP and the cGMP analog 8-Br-cGMP inhibited hCG-induced mitogen-activated protein kinase (MAPK) activation, further indicating that NO and cGMP block oocyte maturation in zebrafish.

Molecular cloning, expression patterns and functional characterization of Gpr3 in the orange-spotted grouper (Epinephelus coioides)

G Protein-coupled receptor 3 (Gpr3) plays a critical role in maintaining oocyte meiotic arrest in mammals. To ascertain the function of Gpr3 in oocyte maturation in fish, the Gpr3 cDNA was cloned from orange-spotted grouper (Epinephelus coioides). The open reading frame of cloned sequence was 993 bp in length, encoding 330 amino acids. Gpr3 was primarily expressed in brain regions and ovary. During oocyte maturation induced by human chorionic gonadotropin (hCG), Gpr3 expression levels were dramatically elevated in follicular cell layers. Functional analysis showed that Gpr3 could constitutively active the cAMP/PKA signaling pathway, and hCG treatment activated Gpr3 promoter activities in cultured eukaryotic cells expressing luteinizing hormone receptors (LHRs). Finally, the role of Gpr3 in oocyte maturation was studied. Sphingosine 1-phosphate (S1P), an agonist of Gpr3, had no effect on oocyte maturation in vivo, suggesting that Gpr3 may be not involved in oocyte maturation in orange-spotted grouper.

Integrated lncRNA and mRNA Transcriptome Analyses in the Ovary of Cynoglossus semilaevis Reveal Genes and Pathways Potentially Involved in Reproduction.

Long non-coding RNAs (lncRNAs) have been reported to be involved in multiple biological processes. However, the roles of lncRNAs in the reproduction of half-smooth tongue sole (Cynoglossus semilaevis) are unclear, especially in the molecular regulatory mechanism driving ovarian development and ovulation. Thus, to explore the mRNA and lncRNA mechanisms regulating reproduction, we collected tongue sole ovaries in three stages for RNA sequencing. In stage IV vs. V, we identified 312 differentially expressed (DE) mRNAs and 58 DE lncRNAs. In stage V vs. VI, we identified 1,059 DE mRNAs and 187 DE lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DE mRNAs were enriched in ECM-receptor interaction, oocyte meiosis and steroid hormone biosynthesis pathways. Furthermore, we carried out gene set enrichment analysis (GSEA) to identify potential reproduction related-pathways additionally, such as fatty metabolism and retinol metabolism. Based on enrichment analysis, DE mRNAs with a potential role in reproduction were selected and classified into six categories, including signal transduction, cell growth and death, immune response, metabolism, transport and catabolism, and cell junction. The interactions of DE lncRNAs and mRNAs were predicted according to antisense, cis-, and trans-regulatory mechanisms. We constructed a competing endogenous RNA (ceRNA) network. Several lncRNAs were predicted to regulate genes related to reproduction including cyp17a1, cyp19a1, mmp14, pgr, and hsd17b1. The functional enrichment analysis of these target genes of lncRNAs revealed that they were involved in several signaling pathways, such as the TGF-beta, Wnt signaling, and MAPK signaling pathways and reproduction related-pathways such as the progesterone-mediated oocyte maturation, oocyte meiosis, and GnRH signaling pathway. RT-qPCR analysis showed that two lncRNAs (XR_522278.2 and XR_522171.2) were mainly expressed in the ovary. Dual-fluorescence in situ hybridization experiments showed that both XR_522278.2 and XR_522171.2 colocalized with their target genes cyp17a1 and cyp19a1, respectively, in the follicular cell layer. The results further demonstrated that lncRNAs might be involved in the biological processes by modulating gene expression. Taken together, this study provides lncRNA profiles in the ovary of tongue sole and further insight into the role of lncRNA involvement in regulating reproduction in tongue sole.

Congenital goiter with areas of signet ring cell differentiation in a juvenile giraffe: a very rare entity

BackgroundCongenital goiter is a common thyroid metabolic disorder characterized by low levels of thyroid hormone, subsequent secretion of excess thyroid-stimulating hormone (TSH) from the pituitary gland, and compensatory hyperplasia of the glands. The presence of signet ring cells (SRCs) does not provide sufficient evidence for the diagnosis of a thyroid tumor, making histopathological diagnosis challenging. In addition, SRCs can also appear in congenital goiter. Therefore, a comprehensive diagnosis of congenital goiter is warranted based on clinical symptoms, autopsy, histopathology, and laboratory examination.Case presentationA juvenile giraffe at the Ordos Zoo in Ordos presented with symptoms of loss of appetite, serious salivation, and slow growth rate since birth. Its height and weight were significantly lower than those of other giraffes of the same age. The animal ultimately died at 17 months of age. Autopsy revelaed that the thyroids were hard, with an uneven surface and with the presence of many small raised follicles, and dense in cross-section. Other organs were visibly atrophic. Histopathologically, diffuse follicles were irregular in size and shape in the hyperplastic goiter. Some follicles were collapsed due to lack of colloids. The follicles were lined by single or multiple layers of hyperplastic follicular cells (HFCs), some of which were exfoliated in the lumen. The HFCs were either cuboidal with eosinophilic cytoplasm and many red small granules or showed SRC differentiation, with nuclei pressed to one edge of the cell and distorted by cytoplasmic mucin that appeared as a single clear vacuole HFCs and as a foamy, multivesicular cytoplasmic material in others. Scattered necrosis of myocardial cells and hepatocytes, cerebral hemorrhage, necrosis of intestinal villi, and obvious atrophy of organs were also observed. Immunohistochemical tests were strongly positive for thyroglobulin and thyroid transcription factor-1 (TTF-1) in the cytoplasm of HFCs.ConclusionsHere we present a case of congenital goiter with areas of SRC differentiation in the thyroid of a juvenile giraffe.

Impact Of Continuous Treatment With Propylthiouracil On Renal And Hepatic Functions In Rabbits

This study was designed to investigate the effect of continuous treatment with the anti-thyroid drug, propylthiouracil (PTU), on renal and hepatic functions in rabbits as an experimental animal model. Animals were randomly divided into four different isolated groups (n = 10); Group I received normal saline. Group II, III, and IV were daily administrated with PTU in oral dosing of 50, 75, and 150 mg/kg, BWT, respectively, for three successive weeks. Serum T3 and T4 levels were measured in all groups. Increased serum creatinine and blood urea nitrogen levels (P<0.05) were also associated. Moreover, liver enzymes levels, aspartate aminotransferase, alanine aminotransferase, and total serum cholesterol levels showed a significant increase in a dose and time decedent manner. Thyroid glands of PTU-treated rabbits showed variable sized-follicles lined by multiple layers of follicular cells, which displayed signs of hyperactivity as the average follicular cell height. The diameter of its width was significantly increased compared with that in the control group. Besides, follicles were filled with a variable quantity of low-dense vacuolated colloids. Kidneys of such animals showed tubulointerstitial nephritis, glomerular atrophy, and multiple focal areas of mononuclear cell reaction. While the observed hepatic lesions were in the form of severe congestion in central vein and hepatic artery, hepatocellular necrosis, and granulocytic lymphoid cellular responses around portal areas associated with peri-portal fibrosis. Such lesions were dependent on doses of PTU. This study referred to that continuous treatment with an antithyroid drug PTU induced a hypothyroid state that was associated with impaired renal and hepatic functions in rabbits.

Evidence for a transcellular route for vitellogenin transport in the telotrophic ovary of Podisus nigrispinus (Hemiptera: Pentatomidae)

Vitellogenin is the main yolk precursor protein in insect oocytes. It is synthesized in the fat body and released into the hemolymph. To reach the oocyte surface, vitellogenin must cross a single layer of follicular epithelium cells. The transport of vitellogenin across the follicular epithelium has been suggested to occur through the enlarged intercellular spaces (patency) by a paracellular route or by endocytosis by follicular cells and release onto oocyte surface in a transcelluar route. In this study, we investigated whether vitellogenin transport in the meroistic telotrophic ovary of Podisus nigrispinus (Hemiptera) occurs via a paracellular or transcellular route. Light and transmission electron microscopies showed that short cell–cell contacts with well-developed occluding septate junctions were present in follicular cells with patency. Immunofluorescence microscopy revealed the presence of vitellogenin receptors in the plasma membrane and of vitellogenin in the cytoplasm of follicular cells. Data suggest that cell–cell contacts serve as a barrier to large vitellogenin molecules and that this protein is transported via a transcellular route of receptor-mediated endocytosis.

The regulation of oocyte maturation and ovulation in the closest sister group of vertebrates.

Ascidians are the closest living relatives of vertebrates, and their study is important for understanding the evolutionary processes of oocyte maturation and ovulation. In this study, we first examined the ovulation of Ciona intestinalis Type A by monitoring follicle rupture in vitro, identifying a novel mechanism of neuropeptidergic regulation of oocyte maturation and ovulation. Ciona vasopressin family peptide (CiVP) directly upregulated the phosphorylation of extracellular signal-regulated kinase (CiErk1/2) via its receptor. CiVP ultimately activated a maturation-promoting factor, leading to oocyte maturation via germinal vesicle breakdown. CiErk1/2 also induced expression of matrix metalloproteinase (CiMMP2/9/13) in the oocyte, resulting in collagen degradation in the outer follicular cell layer and liberation of fertile oocytes from the ovary. This is the first demonstration of essential pathways regulating oocyte maturation and ovulation in ascidians and will facilitate investigations of the evolutionary process of peptidergic regulation of oocyte maturation and ovulation throughout the phylum Chordata.

Ultrastructure of Ovarian Follicular Epithelium During Oogenesis in Lucilia silvarium (Diptera: Calliphoridae)

Lucilia silvarium (Diptera: Calliphoridae) is one of the medically important blowflies. The ovaries of this fly were examined at different stages using a light microscope and transmission electron microscopy. The gonadal cycle is elucidating eight stages of development. The ovaries are made up of ovarioles, each ovariole composed of a successive number of developing follicles. These follicles are arranged in the order that the younger follicles lie closer to the germinal cells, while the mature follicles lie posteriorly. The younger follicle consists of the germ cells enveloped by a single layer of epithelium. The germ cells differentiate into nurse cells and the oocyte. The follicular epithelium diversified itself during oogenesis and accommodate themselves to fit the increasing volume and growing of the oocyte. In the previtellogenic oocyte, the ovariole sections are spherical and show cubical follicular epithelial cell layer surrounding the oocyte and the nurse cells. The vitellogenic oocyte, has follicular epithelium elongated to become columnar surrounding the developing oocyte and squamous enclosing the nurse cells. In post vitellogenic oocyte, the follicular epithelial cells become very thin and hardly observed and the eggshell formation is completed.

Can constant light exposure affect the thyroid gland in prepubertal male albino rats? Histological and ultrastructural study.

Through scientific literature, there is evidence that light affects thyroid function in human, mice and rabbits. Constant light and sleep deprivation is also used as a form of human torture, as it has impact on cognitive performances. The present work was conducted to study the effect of constant light for short and long periods on the thyroid gland in the prepubertal male albino rats. A total of 30 prepubertal male albino rats were used. The rats separated into three groups: group I (control); group II were those rats put under steady encompassing light (24 h/day, light intensity of 600 lux) for 4 weeks; and group III were the rats maintained in constant light for 3 months. The rat thyroid gland was subjected to histological and ultrastructural examination. The rats exposed to light for long durations showed disturbed architecture; the follicles exhibited back to back arrangement (signs of hypertrophy with hyperplasia), lined by multiple layers of follicular cells or were lined by vacuolated cells. Few thyroid follicles exhibited cystic hyperplasia. Congested blood capillaries were demonstrated between the follicles. It can be concluded that the short-term exposure to constant light for 1 month had no apparent effect on thyroid gland tissues while longer exposure to light for 3 months had detrimental effects on the thyroid gland structure of male albino rats.

Prevalence, site of infection, and differentiating oocytes of the cymothoid isopod, Norileca indica (Milne Edwards, 1840) from its infected short mackerel, Rastrelliger brachysoma, in the Upper Gulf of Thailand

Norileca indica, a cymothoid isopod crustacean, is an obligate parasite infecting marine fishes in tropical areas. This study attempted to investigate the prevalence, site of infection and distinct stages of oogenesis of this parasitic isopod obtained from short mackerel (Rastrelliger bachysoma). All N. indica samples were collected and processed through histological techniques. N. indica showed higher prevalence during the dry season than the rainy season and its parasitic infections were more prevalent in the female R. brachysoma (60% prevalence) compared to males. Two distinct sites of N. indica attachment on the host (infection sites) including gill and integument were clearly observed. Interestingly, this isopod preferred to attach more on the integument surface than gill, especially in the female R. brachysoma (55% prevalence). Using histological observation, N. indica exhibited asynchronous development of oocyte in the ovary. The oocytes were enclosed by a thin layer of connective and muscular tissues. Three stages of oogenesis can be identified in the ovary: oogonium, primary oocyte, and secondary oocyte. Oogonium located inside the cyst near the germinal epithelium. A large nucleus of oogonium was surrounded by a weak basophilic cytoplasm. Unlike oogonium, multiple nuclei (2 - 3 nuclei) surrounded by basophilic cytoplasm was a key characteristic of the primary oocyte. The secondary oocyte contained spherical yolk granules with multiple layers of follicular cells, which might function to support yolk uptake into the ooplasm. In addition, early embryonic development of N. indica was also observed in this study. Taken together, this finding highlights parasitic prevalence and infection sites of N. indica in short mackerel, as well as a better resolution of its oogenesis, early development and reproductive cycle.

Morphological-metric, ultrastructural and immunohistochemical effects of gossypol on cultured granulosa cells and oocytes of ewes using MOEPF

Manipulation of oocytes enclosed in preantral follicles (MOEPF) allows for analyzing follicular development and use of this biotechnology in the pre-analysis of the beneficial or toxic effects of bio-products on granulosa cells and oocytes at different developmental stages. In this study, there was evaluation of the effects of gossypol by culturing granulosa cells and oocytes in ewe ovarian tissues. Ovarian tissues were cultured with gossypol at 37 °C, in humidified air and 5% CO2. Variables that were evaluated were morphology, morphometry, ultrastructure and abundance of estradiol receptor α (α-ER). There were no differences in developmental characteristics when there was treatment with any of the gossypol doses that were evaluated. Immunostaining indicated that when the gossypol dose increases, the abundance of α-ER also increases in the cytoplasm, nucleus, and granulosa cells. Findings with the ultrastructural analysis indicated that for granulosa cells there was fewer cells and greater disorganization and a lack of structural integrity of follicular cell layers as a result of all gossypol treatments. The culture of oocytes in preantral ovarian follicles in presence of gossypol did not affect the morphological-metric structure at the doses evaluated. The findings with evaluation of ultrastructural and immunohistochemical structures indicated granulosa cells and α-ER were affected by the treatments with gossypol indicating there were effects of this compound on ovarian function in sheep. This study indicated there is a toxic action of gossypol when using the biotechnology, MOEPF. Thus, gossypol negatively affects granulosa cell development and structural integrity of preantral follicles in sheep.

The dual role of cGMP in oocyte maturation of zebrafish

The roles of cyclic guanosine monophosphate (cGMP) signaling in oocyte maturation attracts much attention in mammals, but its roles in fish are still largely unknown. Using zebrafish as a model, we demonstrated for the first time in fish that cGMP is involved in oocyte maturation, and its functional model in oocyte maturation is different from that of mammals. The intracellular cGMP could be regulated by nitric oxide (NO), we found that all three NO synthase enzymes and four soluble guanylyl cyclases (sGC) are expressed in the zebrafish ovary. Intriguingly, either the activation or inhibition of the NO/sGC/cGMP pathway in fully grown follicles could lead to oocyte maturation. During oocyte maturation, cGMP levels increased in the follicular cell layer but decreased in oocytes, while NO levels increased in follicular cells but remained constant in oocytes. Based on these findings in zebrafish, we propose a hypothetical model on the dual role of cGMP in oocyte maturation: in follicular cells the LH signal could increase the level of NO and cGMP which induces oocyte maturation, while in the oocyte the decreased cGMP level can also induce oocyte maturation. These findings help us to understand the molecular mechanism of fish oocyte maturation.

Eicosapentaenoic Acid Improves Polycystic Ovary Syndrome in Rats via Sterol Regulatory Element-Binding Protein 1 (SREBP-1)/Toll-Like Receptor 4 (TLR4) Pathway.

BackgroundThis study aimed to investigate the protective effect of eicosapentaenoic acid (EPA) on rats with polycystic ovary syndrome (PCOS).Material/MethodsRats with PCOS were intraperitoneally injected with different doses of EPA. Levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were measured using corresponding kits. HE staining was used to observe lesions in ovarian tissue. Levels of inflammatory factors in ovarian tissue of rats were detected by ELISA. RT-PCR was to detect the expression of SREBP1 mRNA and Western blot was used to detect the expression of SREBP1 and TLR4 protein.ResultsThe levels of LH and T were significantly higher and FDH was significantly lower in the Model group compared with the Control group. EPA treatment increased the number of follicular cell layers and promoted maturation of oocytes. Levels of IL-1β, TNF-α, and IL-18 were significantly reduced after EPA treatment. Content of IL-10 was significantly increased after EPA treatment. Expression levels of SREBP1 and TLR4 were significantly deceased after EPA treatment.ConclusionsEPA can improve PCOS through the SREBP1/TLR4 pathway.

Virus-Induced Tubules: A Vehicle for Spread of Virions into Ovary Oocyte Cells of an Insect Vector.

Many arthropod-borne viruses are persistently propagated and transovarially transmitted by female insect vectors through eggs, but the mechanism remains poorly understood. Insect oocytes are surrounded by a layer of follicular cells, which are connected to the oocyte through actin-based microvilli. Here, we demonstrate that a plant reovirus, rice gall dwarf virus (RGDV), exploits virus-containing tubules composed of viral non-structural protein Pns11 to pass through actin-based junctions between follicular cells or through actin-based microvilli from follicular cells into oocyte of its leafhopper vector Recilia dorsalis, thus overcoming transovarial transmission barriers. We further determine that the association of Pns11 tubules with actin-based cellular junctions or microvilli of the ovary is mediated by a specific interaction between Pns11 and actin. Interestingly, RGDV can replicate and assemble progeny virions in the oocyte cytoplasm. The destruction of the tubule assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene strongly inhibits transovarial transmission of RGDV by its vectors. For the first time, we show that a virus can exploit virus-induced tubule as a vehicle to overcome the transovarial transmission barrier by insect vectors.

&lt;em&gt;Xenopus&lt;/em&gt; Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels. The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16. The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.