Primary Follicles Research Articles

Characterizing oogenesis and programmed cell death in the eastern tree hole mosquito Aedes (Protomacleaya) triseriatus.

Oogenesis in flies manifests as a carefully orchestrated cascade of developmental gates and growth events, punctuated by programmed cell death (PCD) and follicular resorption events. In anautogenous mosquitoes, a blood meal stimulates growth of primary follicles, but the timing of developmental stages is species-specific, and few species have been characterized. Here, we characterize the first gonotrophic cycle of oogenesis in Aedes triseriatus (Diptera: Culicidae), the principal vector of La Crosse Virus (LACV), a major cause of pediatric encephalitis in North America. We note significant differences in the timing and appearance of developmental stages from previous studies of other mosquito species, particularly Aedes aegypti. We also describe the appearance and timing of PCD events including atresia, nurse cell death, and follicular epithelium death and show that the majority of follicular epithelium cells do not undergo apoptosis during oogenesis but persist in the ovariole at least until the second gonotrophic cycle. This thorough characterization of oogenesis and PCD in Ae. triseriatus, through which LACV must persist in order to achieve filial infection, also serves as a baseline to study host-pathogen interactions during transovarial transmission.

Cutaneous lymphomas—fast facts about an orphan disease—a short review

SummaryCutaneous lymphomas are a rare group of primary skin lymphoproliferative disorders, divided into T and B cell lymphomas. They differ substantially in clinical course and therapy. The two main subtypes of primary cutaneous T‑cell lymphomas include mycosis fungoides, which is the most common, and Sézary syndrome, the rare leukemic variant. Skin lesions seen in mycosis fungoides patients are erythematous patches, plaques, or tumors. Most patients remain at patch/plaque (early) stage, while some progress to tumor (advanced) stage during their clinical course. Sézary syndrome is characterized by erythroderma and involvement of lymph nodes and the peripheral blood. Treatment is dependent on the disease stage. Therapeutic options include skin-directed and systemic therapies. In localized, early stage mycosis fungoides, prognosis is usually good which changes in advanced stages. Significant progress has been made in recent years in the clinical management of progressive or relapsed cutaneous T‑cell lymphomas by the approval of new targeted therapies. Although there are no curative treatment options apart from allogeneic transplantation, response rates are often encouraging, in particular when using combination therapies. Primary cutaneous B cell lymphomas are rare and three main subtypes are recognized: primary cutaneous marginal zone lymphoma, primary cutaneous follicle center lymphoma, and primary cutaneous diffuse large B‑cell lymphoma, leg type. An accurate diagnosis of the subtype is important for therapeutic management. The most common clinical presentations are red-to-violaceous cutaneous nodules and papules. Primary cutaneous marginal and follicle center lymphoma have excellent 5‑year survival rates of 95–99%.

Regulatory T Cells Overexpressing Peli1 Show Better Efficacy in Repairing Ovarian Endocrine Function in Autoimmune Premature Ovarian Insufficiency

Regulatory T (Treg) cell dysfunction is involved in the pathogenesis of autoimmune premature ovarian insufficiency (POI). Adoptive transfer of Treg cells has been shown to be effective in the treatment of autoimmune POI in mice. However, the therapeutic effect of Treg cell therapy is limited because the phenotype and function of Treg cells is not properly maintained when they are reinfused in an inflammatory environment. Therefore, enhancing the function of Treg cells using genetic engineering is of great significance for improving the efficacy of Treg cells in the treatment of immune diseases. In this study, we investigated the role of the E3 ubiquitinated ligase Pellino 1 (Peli1) in the proliferation and immunosuppressive function of Treg cells and the therapeutic effect of Treg cells overexpressing Peli1 on autoimmune POI. The results showed that the overexpression of Peli1 promoted cell proliferation and enhanced the immunosuppressive function of Treg cells in vitro. After the adoptive transfer of Treg cells overexpressing Peli1 in autoimmune POI mice, the apoptosis rate of ovarian granulosa cells declined. The levels of the inflammatory inhibitors interleukin 10 and transforming growth factor-β as well as the ovarian hormone estradiol were elevated. The number of primordial, primary, secondary, and mature follicles was restored to a certain extent compared with those in control subjects. These results revealed that the adoptive transfer of Treg cells overexpressing Peli1 promoted its efficacy against zona pellucida protein 3 peptide-induced POI, which provides new insights into the treatment of autoimmune POI.

SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells.

The primordial to primary follicle transition (PPT) in the ovary is critical to maintain sustainable reproductive resources in female mammals. However, it is unclear how granulosa cells (GCs) of the primary follicle participate in regulating PPT. This study focused on exploring the role of transcription factor Sp1 (SP1)in regulating PPT based on the fact that SP1 is pivotal for pregranulosa cell proliferation before primordial follicle formation. The results showed that mice fertility was prolonged when Sp1 was specifically depleted from GCs (GC- Sp1 -/- ). Besides, the PPT in GC- Sp1 -/- mice was reduced, resulting in more primordial follicles being preserved. Single-cell RNA-seq also indicated that the level of cholesterol metabolism was downregulated in GC- Sp1 -/- mice. Additionally, the PPT was promoted by either overexpression of ferredoxin-1 (FDX1), one of the key genes in mediating cholesterol metabolism or supplementing cholesterol for cultured fetal ovaries. Collectively, SP1 in GCs participates in the metabolism of cholesterol partially by regulating the transcription of Fdx1 during the PPT.

PDE4 inhibitor Roflumilast modulates inflammation and lipid accumulation in PCOS mice to improve ovarian function and reduce DHEA-induced granulosa cell apoptosis in vitro.

This study was implemented to address the role of Roflumilast in polycystic ovary syndrome (PCOS) as well as to discuss its reaction mechanism in vivo and in vitro. In vivo, mice were administrated with 6 mg dehydroepiandrosterone (DHEA) per 100 g body weight and fed with 60% high fat diet to induce PCOS. The expression of phosphodiesterases 4 (PDE4) was assessed with RT-qPCR. The ovary pathology was observed by hematoxylin and eosin staining and follicles were counted. Enzyme-linked immunosorbent assay was adopted for the estimation of progesterone, testosterone and inflammatory factors and lipid accumulation was observed by Oil Red O staining. With the application of reverse transcription-quantitative PCR (RT-qPCR) and western blot, the messenger RNA (mRNA) and protein expressions of low-density lipoprotein receptor (LDLR) was resolved. In vitro, Cell counting kit-8 and flow cytometry analysis were applied for the assessment of cell proliferation and apoptosis. The proliferation- and apoptosis-related proteins were appraised with western blot. Additionally, the expressions of PDE-4 at both mRNA and protein levels were tested with RT-qPCR and western blot. Here, it was discovered that PDE4 was greatly elevated in PCOS mice and DHEA-induced ovarian granulosa cells (KGN). In PCOS mice, PDE4 was negative correlated with progesterone and had positive correlation with testosterone. Roflumilast could enhanced progesterone expression, increased the number of primary follicles, preantral follicles and antral follicles but reduced testosterone and decreased the number of cystic follicles in PCOS mice. It was also testified that Roflumilast could inhibit the release of inflammatory factors and lipid accumulation in PCOS mice. Besides, the proliferation of DHEA-induced KGN cells was enhanced while the apoptosis was declined by Roflumilast, accompanied by elevated contents of PCNA, Ki67 and antiapoptotic protein Bcl-2. Collectively, Roflumilast inhibited inflammation and lipid accumulation in PCOS mice to improve ovarian function and reduce DHEA-induced granulosa cell apoptosis.

Efficient establishment of an optimized culture condition for cashmere goat primary hair follicle stem cells.

Hair follicle stem cells (HFSCs) are an important basis for hair follicle morphogenesis and hair cycle growth. This cell type also represents an excellent model for studying the gene function and molecular regulation of the hair growth cycle, including proliferation, differentiation, and apoptosis. Basically, the functional investigation of hair growth-regulating genes demands a sufficient amount of HFSCs. However, efficient propagation of HFSCs in goats is a challenging process under the current culture conditions. Here, we investigated the effect of four components, including the Rho-associated protein kinase (ROCK) inhibitor Y-27632, leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and vitamin C, on cell growth and pluripotency in the basal culture medium (DMEM/F12 supplemented with 2% fetal bovine serum). We found that adding Y-27632, LIF, and bFGF independently increased the proliferation and pluripotency of goat HFSCs (gHFSCs), with Y-27632 having the most significant effect (P < 0.001). Fluorescence-activated cell sorting of the cell cycle revealed that Y-27632 promoted gHFSC proliferation by inducing the cell cycle from S to G2/M phase (P < 0.05). We further demonstrated that gHFSCs displayed superior proliferative capacity, clone-forming ability, and differentiation potential in the combined presence of Y-27632 (10 μM) and bFGF (10 ng/mL). We termed this novel culture condition as gHFEM, which stands for goat Hair Follicle Enhanced Medium. Taken together, these results indicate that gHFEM is an optimal condition for in vitro culture of gHFSCs, which will subsequently facilitate the study of HF growth and biology.

MiR-22-3p ameliorates the symptoms of premature ovarian failure in mice by inhibiting CMKLR1 expression.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. This study was aimed at exploring the improving effects of miR-22-3p on the symptoms of POF in mice by inhibiting chemokine-like receptor 1 (CMKLR1) expression. Female mice were intraperitoneally injected with cyclophosphamide to construct POF mice models. Lentiviral vectors containing miR-22-3p, short hairpin RNA (sh)-CMKLR1, and overexpression (oe)-CMKLR1, respectively, or in combination, were injected into the ovaries of both sides of POF mice. miR-22-3p and CMKLR1 expression in ovarian tissues of mice was assessed, and the targeting relationship between miR-22-3p and CMKLR1 was predicted and verified. Serum estradiol (E2), anti-Mullerian hormone, and follicle-stimulating hormone levels were assessed. Ovarian weight was weighed, and pathological changes and the number of primordial follicles, primary follicles, secondary follicles, and atresia follicles were observed. Apoptosis of ovarian tissues was determined. In ovarian tissues of POF mice, miR-22-3p expression was decreased while CMKLR1 expression was increased. miR-22-3p up-regulation or CMKLR1 down-regulation restored sex hormone levels, improved ovarian weight and the number of primordial follicles, primary follicles, and secondary follicles, and reduced the number of atresia follicle and ovarian granulosa cell apoptosis in POF mice. miR-22-3p targeted CMKLR1, and overexpressing CMKLR1 reversed the ameliorative effects of miR-22-3p overexpression on POF mice. Our research highlights that overexpressed miR-22-3p down-regulates CMKLR1 to ameliorate the symptoms of POF in mice. Therefore, the miR-22-3p/CMKLR1 axis could improve the symptoms of POF.

Activin Stimulates Follicle Formation and Activation and Modulates Steroidogenesis in Fetal Bovine Ovarian Tissue in Vitro 1

The earliest stages of ovarian follicular development, follicle formation and development of the capacity to activate, are critical to the size of the ovarian reserve. In primates and most domestic animals, these stages occur during fetal life and thus, are difficult to study. Our laboratory has used cattle, an excellent model for human ovarian development, to elucidate the regulation of these early stages in fetal bovine ovarian tissue in vitro. The experiments described here were designed to determine if fetal bovine ovaries have activin A and its receptors and if activin A affects follicular formation and/or activation to begin growth. During the second trimester, when follicles first form, activin increased the total number follicles and the number of primary (activated) follicles in cultured ovarian cortical pieces, compared to control medium; its effects were similar to the positive control, insulin. During the second trimester, activin decreased primordial follicle numbers and increased primary and secondary follicles, again mimicking the positive control. Our previous experiments showed that fetal ovaries produce ovarian steroids, particularly during the second trimester, and that estradiol and progesterone inhibit follicle formation and activation. In control and gonadotropin-treated ovarian pieces, activin A increased progesterone, but decreased androstenedione and estradiol accumulation in the medium. Messenger RNA for activin and its type II receptors was detected in fetal bovine ovaries, with ACVR2A receptor mRNA more abundant than ACVR2B. Immunohistochemistry localized mRNA for activin A and ACVR2A to germ cells of all stages, granulosa cells, and theca cells of early secondary follicles. Staining for ACVR2B was weaker and mostly confined to germ calls. Taken together, these results strongly suggest that activin A of fetal ovarian origin 3 plays a role in ovarian development by promoting follicle formation and activation and modulating ovarian steroidogenesis.

Visualization of Balbiani Body disassembly during human primordial follicle activation.

Dormant human oocytes contain a perinuclear super-organelle, called the Balbiani Body, which is not present in mature oocytes. Here, we use confocal imaging to visualize two Balbiani Body markers-mitochondria and the DEAD-box helicase DDX4-in preantral follicles isolated from a 20-year-old female patient. In primordial follicles, mitochondria were concentrated in a ring near the oocyte nucleus, while DDX4 formed adjacent micron-scale spherical condensates. In primary and secondary follicles, the mitochondria were dispersed throughout the oocyte cytoplasm, and large DDX4 condensates were not visible. Our data suggest that the Balbiani Body breaks down during the primordial to primary follicle transition, thus releasing mitochondria and soluble DDX4 protein into the oocyte cytoplasm.

Bax/Bcl-2 Expression Ratio Analysis of Rat Ovary Vitrified with Date Juice Concentrate as a Natural Extracellular Cryoprotectant.

The use of extremely low temperatures in vitrification is known to cause cryoinjury so that it can trigger the activation of the intrinsic apoptotic pathway, which can damage the structural integrity of the pre-antral follicle. Based on that, it is necessary to use an appropriate cryoprotectant to protect the preserved cell. This study aimed to identify the potential use of date juice concentrate (DJC) as a natural extracellular cryoprotectant to suppress the rate of apoptosis after vitrification. This experimental research uses 24 samples of ovarian rats. Rats were fed and drank an ad libitum. Ovaries were isolated in the proestrus phase, then processed into slides for immunohistochemistry (IHC) staining using anti-Bax and anti-Bcl-2 antibodies. IHC results were evaluated for the brown colour using ImageJ IHC Profiler. The results were analysed as an optical density and displayed in the Bax/Bcl-2 ratio. All data were statistically analysed with either parametric (analysis of various) or non-parametric (Kruskal-Wallis) tests. The combination of EG 7.5% + DJC 15% (KP2) showed the lowest Bax/Bcl-2 ratio in primordial and primary follicles. Meanwhile, the lowest Bax/Bcl-2 ratio in secondary follicles is found in KP4 (EG 15% + DJC 15%). The DJC is known to contain a dominant amount of glucose. The DJC shows antioxidant activity and contains antioxidant compounds, phenols and flavonoids. The sugar content and antioxidant compounds of DJC can protect against follicle membrane damage, so the rate of intrinsic apoptosis pathway is also suppressed initially with Bax protein suppression in the mitochondrial membrane.

The effect of kisspeptin on the maturation of human ovarian follicles in culture following vitrification-thawing processes.

Ovarian cryopreservation is one of the effective methods to preserve fertility for cancer patients. Still, this approach has some problems, namely ROS, resulting in adverse effects on oocytes and ovarian follicles. Kisspeptin as an antioxidant to control ovarian function, directly or indirectly. In this study, the effect of kisspeptin on follicle maturation was evaluated in culture following ovarian cryopreservation. Ovarian tissue samples of women between 20 and 35 years old (n=12) were laparoscopically collected. The samples were randomly divided into four groups: 1) control, 2) vitrification, 3) vitrified+1μM kisspeptin, and 4) vitrified+10μM kisspeptin. After vitrification and thawing processes, the tissues were cultured in DMEM medium for 7 days. H&E staining for histological evaluation, Real-Time PCR for GDF9 and BMP15 gene expression, and immunohistochemical staining for GDF9 and BMP15 protein expression were performed. In the vitrification group, ovarian tissue morphology was incoherent, and more primordial follicles than other follicle types were found. The expression of GDF9 and BMP15 genes and proteins were significantly decreased in this group compared with other groups (p<0.05). In the vitrification groups with kisspeptin (1 and 10 μM), the number of primary and secondary follicles was more than in the vitrification group. Besides, the expression of these genes and proteins was dramatically elevated in the vitrification groups with kisspeptin compared to the vitrification group alone (p<0.05). It seems that kisspeptin is an effective substance to improve the quality of the human ovarian cryopreservation medium by improving follicle maturation.

Cryoprotective Effects of Longan Honey on Preantral Follicle Integrity of Rat Ovary Post Vitrification

BACKGROUND: Longan honey (LH) has the potential as a natural extracellular cryoprotectant to maintain the integrity of intact preantral follicles against the cryoinjury during ovarian vitrification. OBJECTIVE: This research determined the cryoprotective effects of logan honey on preantral follicles integrity of rat ovary post-vitrification. MATERIALS AND METHODS: After vitrification, the follicle index was determined by observing the ovarian histological sections made using the paraffin method with hematoxylin-eosin staining and analyzed using Optilab 3.0 and Image Raster software. RESULTS: The results showed that the combination of ethylene glycol (EG) with LH and a dose variation of 7.5%-15% (KP1-KP4) increased the density of follicles, the number of intact follicles in G2 and G3, with a decrease in the atretic follicles in G1 better compared to the use of EG only (KKP1-KKP2). The differences in the histological structur e of preantral follicles affected the doses of LH needed by each type of follicle to maintain the integrity of the follicular structure from cryoinjury effects. The comparison of the G2 total follicle index values were KKP1 (90.7±18.3), KKP2 (115.6±9.9) &lt; KP1 (193.6±10.7), KP2 (189.3±10.5), KP3 (182.2±27.1) and KP4 (169.4±8.8). Among the variations in the dose of LH 7.5%-15%, the highest increase in the G3 index value was found in primary (51.653±9.791), tertiary (43.119±8.786), secondary (33.885±4.745), and primordial (28.670±2.516) follicles of KP3 (7.5% of LH). CONCLUSION: The primary and tertiary follicular stages maintain the best integrity and can be used after the vitrification of rat ovaries.

Expression of Angiopoietin 2 in ovarian follicles of crossbred Malabari goat

Angiopoietin-2 (Ang-2) is a local ovarian growth factor belonging to the angiopoietin/Tie (tyrosine kinase with Ig and EGF homology domains) signalling pathway, one of the main pathways involved in ovarian follicular angiogenesis. The main objective of this study is to assess the differential protein expression of Ang-2 in the developmental phases of ovarian follicles of crossbred Malabari goats employing an immunohistochemical technique. Ang-2 protein expression was found in all developmental phases of ovarian follicles, including primordial, primary, small, and large antral ovarian follicles. Ang-2 was found in the granulosa cells, theca interna cells, follicular fluid (granulosa cells in the FF), endothelial cells of capillaries and germinal epithelium of the goat ovaries. The Quantitative Method-Percentage Positivity of Ang-2 expression revealed that it was higher in the initial stages of ovarian follicular development and decreased with later stages of follicular development in cross-bred Malabari goats. Moreover, the protein expression of Ang2 was found to be higher in granulosa cells (GC) than in theca interna (TI) cells of antral follicles as evident from the higher staining intensity in GC cells compared to TI cells. It is therefore concluded that the Ang-2 protein expression in ovarian follicular cells decreased with an increase in follicle size and development and based on the combined scores for immunostaining (staining intensity and the number of positive staining cells), the Ang-2 was found to be expressed more in the granulosa cell layer compared to the theca interna cells of the antral follicle in crossbred Malabari goats.

Histochemical effects of brodifacoum on rat spleen

In this study, the histochemical effects of Brodifacoum, an anticoagulant used against rodents, on the spleen are examined under a light microscope using CD4 and CD8 histochemical staining methods. A single dose of 0.2 mg Brodifacoum was dissolved in Dimethyl Sulfoxide (DMSO) and was given orally to mature male rats. Spleen samples were collected under ether anesthesia after 24 h, 72 h, 14 days, and 30 days from the rats in the experimental groups and after 14 days from the rats in the control group. In this light microscope study, it was observed that the capsule, white pulp, and red pulp zones in the rat spleen were constructed normally and as their natural structures primary and secondary follicles (germinal center) they were few, and CD4 and CD8 lymphocytes were spherically structured. In the 24 h spleens of the rats, the diameters of germinal centers were expanded and deterioration of the structure of CD4 and CD8 cells was observed. Related to the increase in time (72 h and 14 days) it was determined that primary follicles increased in number and the diameters of germinal centers expanded. In addition to this, after30 days, the rate of CD4:CD8 of the brodifacoum applied rat spleens were approximately the rate of the control group, and the improvement of the structures of the cells was reported as an effect of regeneration. As a result of this study, it was found that Brodifacoum caused immunohistochemical abnormalities in the rat spleen, affected the morphological structure of CD4 and CD8 T lymphocytes and created an immune response in rats. It is thought that the obtained results will be a source for the studies on Brodifacoum.

Histopathological Alterations in Ovarian and Uterine Treated with Extract and Fraction of Kebar Grass (Biophytum petersianum)

Kebar grass (Biophytum petersianum) is being used traditionally by the people of West Papua to increase fertility. The effects of extracts and fractions of Kebar grass on the estrus cycle duration and estrus phase in rats were evaluated. The present work was designed to investigate the effects of administration of kebar grass extract and its fraction on the histopathological changes in the ovaries and uterine of rats. The female rats were treated with carboxy methyl cellulose sodium (CMC sodium) 1 % (group I), ethynile estradiol (group II), extract kebar grass (group III), n-hexane fraction (group IV), ethyl acetate fraction (group V), and water fraction (group VI) of kebar grass via gavage daily for 7 days. At days 7th and 14th, 3 rats in each group were euthanized. Uterine and ovarian collected and weighed. Hematoxylin-Eosin staining and the paraffin technique were used for histological preparation. Data were analyzed by One Way ANOVA followed by the Duncan test. The Kebar grass has an effect to increase ovarian and uterine weight, increasing most of the number of follicles especially primary follicle and uterine thickness. The treatment with ethyl acetate fraction of kebar grass (group V) constantly showed more effective results than the other kebar grass treatment.

Evaluation of goat ovaries and follicles by histological study

The present study evaluated slaughterhouse goat ovaries and follicles to know the effect of corpus luteum on the number and quality of ovarian follicles. For this study, 18 goat ovaries were collected and categorized as ovaries with corpus luteum (CL+) and without corpus luteum (CL–) group. Both groups were evaluated by morphometric measurements and histological observation. Among 18 ovaries, 12 ovaries contained CL (66.67%) and 6 ovaries were without CL (33.33%). Based on the morphometric measurements, the length and weight of the ovaries with CL were numerically higher (p&gt;0.05) than those of the ovaries without CL. But the diameter of the ovaries with CL was significantly higher (p&lt;0.05) than those of the ovaries without CL. In comparison to the CL– group, the CL+ group had a numerically higher (p&gt;0.05) number of follicles with a diameter of 2-6 mm and &gt; 6 mm. Histological analysis revealed that the total number of follicles (primary to antral) in CL– group (11.10±2.47) was numerically higher (p&gt;0.05) than that of the CL+ group (10.07±2.47). Presence of primary (4.44±1.59), secondary (1.45±0.83) and antral (6.27±1.31) follicles were found higher (p&gt;0.05) in ovaries without CL than that of with CL (4.05±1.61, 0.92±0.21, 5.08±1.21 respectively). On the other hand, the number of degenerated primary (1.29±0.56), secondary (0.52±0.14) and antral (3.38±0.66) follicles were found comparatively higher (p&gt;0.05) in the ovaries with CL than that of the ovaries without CL (1.14±0.69, 0.37±0.19, 2.40±0.40 respectively). Since, there was no statistical difference found among the number of total and different types of follicles between ovaries with or without corpus luteum; hence, it may be summarized that both type of ovaries may be used for the retrieval of oocytes for further reproductive biotechnological study. Bangladesh Journal of Animal Science, 51 (4): 145-151

Effect of apple vinegar on folliculogenesis and ovarian kisspeptin in a high-fat diet-induced nonalcoholic fatty liver disease in rat

BackgroundNon-alcoholic fatty liver disease (NAFLD) adversely affects reproduction. We aimed to study the effect of a high-fat diet (HFD), supplemented with apple vinegar, on folliculogenesis in a rat model of NAFLD.MethodsFemale rats were randomly divided into four groups (N = 28): Standard diet (SD), SD + vinegar, HFD, and HFD + vinegar groups. At the end of the study, biochemical tests were assessed in serum. HOMA-IR (Homeostatic model assessment-Insulin resistance) was calculated. Sex hormones were determined using an ELISA kit; ovary follicle counts were studied using histological methods. The proliferation index of granulosa cells was determined using immunohistochemistry. Kisspeptin expression in the ovary was detected using RT-PCR.ResultsThe HFD induced steatohepatitis and NAFLD. The ovaries in the rat model of NAFLD were atrophied. The ovaries had less count of developing follicles and corpus luteum, and more degenerated and cystic follicles in comparison with the SD group. Vinegar + HFD consumption decreased ALT, compared to the HFD group (P = 0.004). Steatohepatitis was reduced in the Vinegar + HFD group (P = 0.001). Vinegar + HFD considerably reduced HOMA-IR (p = 0.01). The HFD + vinegar diet could increase estradiol (P = 0.001), without significantly affecting progesterone or testosterone. In addition, an increase of primordial follicles as an ovarian reserve and also primary follicles were determined in the HFD + vinegar group. There were no statistical differences in the granulosa cell proliferation index in various follicle types between groups. HFD + vinegar significantly enhanced ovarian kisspeptin expression (p = 0.04).ConclusionsThe vinegar diet in a rat model of NAFLD raises estradiol, primordial, and small primary follicles, and increases ovarian kisspeptin expression indirectly. Insulin resistance and obesity were improved by apple vinegar, and anti-glycemic and anti-lipidemic effects were also determined. The supplementation of apple vinegar in NAFLD might be useful for ovary. However, it requires further investigation.

Cyclin-dependent kinase 6 (CDK6) as a potent regulator of the ovarian primordial-to-primary follicle transition.

Introduction: Ovarian follicle development requires tight coordination between several factors to initiate folliculogenesis to generate a mature and fertile egg. Studies have shown that cell cycle factors might contribute to follicle development, hover specific knowledge on individual CDKs and follicle activation has not been investigated. Among cell cycle regulators, CDK6 is a key player through binding to cyclin D resulting DNA synthesis and genome duplication. Interestingly, the CDK6 gene is differentially expressed in oocytes and granulosa cells from human primordial and primary follicles, which suggest a potential role of CDK6 in the primordial-to-primary transition. In this study, we investigated the potential regulatory role of CDK6 in progression of primordial to primary follicle transition using BSJ-03-123 (BSJ), a CDK6-specific degrader. Methods: In mouse ovarian in vitro culture, BSJ reduced the activation of primordial follicles, and reduced follicle development. As a next step, we examined the egg maturation read-out and found that BSJ-treated follicles matured to competent MII eggs with resumption of first meiosis, comparable with the control group. Results: Noteworthy, it appears that inhibition of CDK6 did increase number of apotoptic cells, articular in the granulosa cells, but had no impact on ROS level of cultured ovaries compared to control group, indicating that the cells were not stressed. Oocyte quality thus appeared safe. Discussion: The results of this study indicate that CDK6 plays a role in the primordial-to-primary transition, suggesting that cell cycle regulation is an essential part of ovarian follicle development.

Identification of oogonial stem cells in chicken ovary.

Oogonial stem cells (OSCs) are germ cells that can sustain neo-oogenesis to replenish the pool of primary follicles in adult ovaries. In lower vertebrates, fresh oocytes are produced by numerous OSCs through mitosis and meiosis during each reproduction cycle, but the OSCs in adult mammals are rare. The birds have retained many conserved features and developed unique features of ovarian physiology during evolution, and the presence of OSCs within avian species remain unknown. In this study, we investigated the existence and function of OSCs in adult chickens. The chicken OSCs were isolated and expanded in culture. We then used cell transplantation system to evaluate their potential for migration and differentiation in vivo. DDX4/SSEA1-positive OSCs were identified in both the cortex and medulla of the adult chicken ovary. These putative OSCs undergo meiosis in the reproductively active ovary. Furthermore, the isolated OSCs were expanded in vitro for months and found to express germline markers similar to those of primordial germ cells. When transplanted into the bloodstream of recipient embryos, these OSCs efficiently migrated into developing gonads, initiated meiosis, and then derived oocytes in postnatal ovaries. This study has confirmed the presence of functional OSCs in birds for the first time. The identification of chicken OSCs has great potential for improving egg laying and preserving endangered species.

Ligusticum chuanxiong promotes the angiogenesis of preovulatory follicles (F1–F3) in late-phase laying hens

Ligusticum chuanxiong (CX) is a traditional Chinese medicine that is widely planted throughout the world. CX is one of the most important and commonly used drugs to enhance blood circulation. The preovulatory follicles in laying hens have a large number of blood arteries and meridians that feed the follicles' growth and maturation with nutrients, hormones, and cytokines. With the extension of laying time, preovulatory follicles angiogenesis decreased gradually. In this study, we studied the mechanism of CX on preovulatory follicles angiogenesis in late-phase laying hens. The results show that CX extract can increase the angiogenesis of preovulatory follicles (F1-F3) of late-phase laying hens. CX extract can promote vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation in preovulatory follicles theca layers, promote the proliferation, invasion and migration through PI3K/AKT and RAS/ERK signaling pathways in primary follicle microvascular endothelial-like cells (FMECs). In addition, CX extract can up-regulate the expression of hypoxia inducible factor α (HIF1α) in granulosa cells (GCs) and granulosa layers through PI3K/AKT and RAS/ERK signaling pathways, thereby promoting the secretion of vascular endothelial growth factor A (VEGFA). In conclusion, the current study confirmed the promoting effect of CX extract on the preovulatory follicles angiogenesis, which sets the stage for the design of functional animal feed for late-phase laying hens.