Folliculogenesis Research Articles

171 Not Just a Matter of Retained Embryonic Spinal Cord: Histopathology of the Filum Terminale in Tethered Cord Syndrome

INTRODUCTION: We present a large consecutive series of patients with tethered cord syndrome who underwent resection of the filum terminale (FT) and the histopathologic findings as analyzed by the neuropathologists. METHODS: FT specimens obtained from a consecutive series of 288 pediatric and adult patients that underwent spinal cord releases performed at a single academic institution from 2013-2021 were analyzed retrospectively (IRB #787945). Hematoxylin and eosin (HE) and immunohistochemistry staining was performed to study the spectrum of cells known to reside in connective tissues. RESULTS: The most common structures included peripheral nerves (97.6%), ependymal cells (70.8%), ganglion cells (38.5%), and neuropil (15.3%). Cases with excess ependymal cells were associated with ependymal canals (5.9%), ependymal cysts (3.1%), and one case with extensive ependymal “proliferation” (0.3%). With regards to the inflammatory cellular findings, eleven specimens (3.8%) contained scattered or accumulated lymphocytes, macrophages, mast cells, macrophage-derived giant cells, and a secondary lymph follicle in one patient. 36 patients (12.5%) had increased vascularity with enlarged capillaries. Nine patients (3.1%) had an associated type of vascular malformation. A group of specimens included dystrophic calcification within the FT (7.6%). CONCLUSIONS: Both vascular and inflammatory changes along with calcifications have been overlooked as possible associated histologic secondary to mechanical stress on the FT. Additionally, vascular anomalies could alter the filum’s mechanical properties due to venous congestion.

FTO attenuates the cytotoxicity of cisplatin in KGN granulosa cell-like tumour cells by regulating the Hippo/YAP1 signalling pathway.

Premature ovarian failure (POF) is a devastating condition for women under 40 years old. Chemotherapy, especially the use of cisplatin, has been demonstrated to promote the apoptosis of granulosa cells in primary and secondary follicles, leading to POF. Our previous studies demonstrated that fat mass- and obesity-associated (FTO) plays an essential role in protecting granulosa cells from cisplatin-induced cytotoxicity. Various studies have suggested that the Hippo/YAP signalling pathway plays a significant role in regulating cell apoptosis and proliferation. Additionally, YAP1 is the main downstream target of the Hippo signalling pathway and is negatively regulated by the Hippo signalling pathway. However, whether the Hippo/YAP signalling pathway is involved in the protective effect of FTO on granulosa cells has not been determined. In this study, we found that after cisplatin treatment, the apoptosis of granulosa cells increased in a concentration-dependent manner, accompanied by the downregulation of FTO and YAP1. Furthermore, overexpression of FTO decreased cisplatin-induced granulosa cell apoptosis, inhibited the Hippo/YAP kinase cascade-induced phosphorylation of YAP1, and promoted the entry of YAP1 into the nucleus. The downstream targets of YAP1 (CTGF, CYR61, and ANKRD1) were also increased. Si-RNA-mediated downregulation of FTO promoted cisplatin-induced granulosa cell apoptosis, activated the Hippo/YAP kinase cascade, and inhibited the YAP1 entry into the nucleus. These effects were completely reversed by the small molecule inhibitor of YAP1-verteporfin (VP). Taken together, these data suggested that FTO-YAP1 plays a positive role in regulating the proliferation of injured granulosa cells induced by cisplatin.

Secondary follicles enable efficient germline mtDNA base editing at hard-to-edit site

Efficient germline mtDNA editing is required to construct disease-related animal models and future gene therapy. Recently, the DddA-derived cytosine base editors (DdCBEs) have made mitochondrial genome (mtDNA) precise editing possible. However, there still exist challenges for editing some mtDNA sites in germline via zygote injection, probably due to the suspended mtDNA replication during preimplantation development. Here, we introduce a germline mtDNA base editing strategy: injecting DdCBEs into oocytes of secondary follicles, at which stage mtDNA replicates actively. With this method, we successfully observed efficient G-to-A conversion at a hard-to-edit site and also obtained live animal models. In addition, for those editable sites, this strategy can greatly improve the base editing efficiency up to 3-fold, which is more than that in zygotes. More important, editing in secondary follicles did not increase more the risk of off-target effects than that in zygotes. This strategy provides an option to efficiently manipulate mtDNA sites in germline, especially for hard-to-edit sites.

51 A preliminary study of the effect of dietary cottonseed on the follicular reserve of does artificially infected with Haemonchus contortus.

Abstract Numerous studies have reported the negative effect of cottonseed gossypol on the ovarian reserve of the follicles, impairing female fertility. Injuries resulting in the depletion of primordial follicles can lead to permanent infertility. At birth, female mammals possess a fixed number of ovarian primordial follicles, which develop into primary, secondary, and antral follicles in a continuous and irreversible process. In this study, we evaluated the potential effects of dietary cottonseed gossypol in combination with parasitic infection on ovarian follicular reserve in meat goats. A total of 40 Boer x Spanish cross does were randomly subjected to cottonseed (CS) and parasite (P: Haemonchus contortus) in a 2x2 factorial arrangement of does (n = 8) per treatment groups (CS-/P-; CS+/P-; CS-/P+, and CS+/P+). Does were artificially infected with Haemonchus contortus for 2 d, and after 25 d, they began shedding parasite eggs. Thereafter, does were fed a hay diet supplemented with cottonseed (0.5% BW; CS+) or concentrate (CS-), to meet the nutritional requirements. Ten weeks later, animals were slaughtered, and ovaries of each treatment group (n = 4) were fixed with formalin and subjected to a standard procedure of histology staining (Periodic acid–Schiff and Hematoxylin counterstaining). Cuts of one left and one right ovary of two does were placed on microscope slides, and 48 slides were subjected to a microscope visualization (EVOS FL Auto; 20x magnification). Primordial follicles were classified as healthy (stained nucleus), unhealthy (pycnotic), and undetermined. Data were analyzed with ANOVA 1 and expressed as mean ± sem. P < 0.05 indicates a significant difference. A total of 20,562 primordial follicles corresponding to 10,410 (50.6%) healthy; 3,729 (18.1%) unhealthy; and 6,909 (33.6%) undetermined were examined; of which 26%, 20%, 31%, and 23% of total primordial follicles belonged to the CS-/P-, CS-/P+, CS+/P-, and CS+/P+ treatment groups, respectively. A significantly (P < 0.05) greater number of follicles were counted on left vs. right ovary sections (11,584 vs. 8,978) corresponding to 56.3% vs. 43.7% of total follicle counts. Averages of 121 ± 6 vs. 101±6 follicles were calculated per left and right ovary sections. Diets containing cottonseed (CS+/P-) increased the average number of follicles per ovary section, while parasite infections significantly (P < 0.05; CS-/P+ and CS+/P+) decreased these numbers. Regardless of the treatment group, the proportions of healthy follicles remained comparable between treatment groups. We concluded that dietary cottonseed and parasitic infections inversely affect ovarian follicle reserve, while the proportions of healthy follicles were unaffected across treatment groups. Nevertheless, higher microscopic magnification is needed to better characterize the large proportion of undetermined follicles.

The Effects of Vitamin C on Histological Changes Induced by Acrylamide on Rat Ovary

Background and Aims: Acrylamide (ACR) adverse effects have been expressed in various tissues. Vitamin C protects the tissue from oxidative damage. The goal of this study was to examine the protective effects of vitamin C on histopathological changes induced by ACR on rat ovaries. Materials and Methods: In this experimental study, 28 adult female Wistar rats were used. The animals (n=7) were divided into 4 groups (1) Control group, (2) ACR (10 mg/kg) group, (3) vitamin C (200 mg/kg), (4) ACR [10 mg/kg) + vitamin C (200 mg/kg) daily for 35 days. After the last oral dose, the ovaries were removed placed in the tissue processor, and molded. To determine the volume of the ovary and the number of follicles, we used the Cavalieri principle and the physical dissector, respectively. Results: The results showed ACR increased the volume of the ovary (p ≤ 0.05), the volume of the medulla (p ≤ 0.01), and the decrease in the volume of the cortex (p ≤ 0.01) in comparison with the control group. These parameters were improved in the ACR + vitamin C group. The total number of primordial, primary, secondary, and graafian follicles showed a significant reduction in the ACR group compared to the other groups. The total number of follicles was increased in the ACR + vitamin C group. Conclusion: Based on this study, we concluded that vitamin C can improve histopathological changes of the ovary in ACR-exposed rats

Expression of IZUMO1 and JUNO in the gonads of domestic cats (Felis catus)

Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets.

Characterization of lymphocyte-rich hepatocellular carcinoma and the prognostic role of tertiary lymphoid structures.

Lymphocyte-rich hepatocellular carcinoma (LR-HCC) is largely unknown and a rare subtype of HCC with immune-rich stroma. Tertiary lymphoid structures (TLS), frequently observed in LR-HCC, are known to be prognostically significant in various malignancies; however, their significance in HCC remains unevaluated. Clinicopathologic data of 191 cases of surgically resected conventional HCC (C-HCC, n = 160) and LR-HCC (n = 31) were retrieved. Immunohistochemistry, multiplex immunofluorescence staining, RNA sequencing and proteomic analysis were conducted. Differences between the subtypes were statistically evaluated. LR-HCC was significantly correlated to larger tumour size, higher Edmondson-Steiner grade, presence of TLS and higher CD3-, CD8- and FOXP3-positive T cell, high PD-1 and PD-L1 expression (p < .001 for all) compared to C-HCC. Patients with LR-HCC exhibited significantly better overall survival (OS) (p = .044) and recurrence-free survival (RFS) (p = .025) than C-HCC. LR-HCC demonstrated TLS signatures with significantly higher proteomic-based immune scores in 14 of 17 types of tumour-infiltrating immune cells. Furthermore, C-HCC with secondary follicles, the most mature form of TLS, exhibited significantly better OS (p = .031) and RFS (p = .033) than those without. Across the global proteome, LR-HCC was well-differentiated from C-HCC and a map of protein-protein interactions between tumour-infiltrating lymphocytes and HCC in tumour microenvironment was completed. LR-HCC is clinicopathologically and molecularly distinct and shows better prognosis compared to C-HCC. Also, the presence of secondary follicle can be an important prognostic marker for better prognosis in both LR-HCC and C-HCC.

Enhanced solute transport and steady mechanical stimulation in a novel dynamic perifusion bioreactor increase the efficiency of the in vitro culture of ovarian cortical tissue strips.

Introduction: We report the development and preliminary evaluation of a novel dynamic bioreactor to culture ovarian cortical tissue strips that leverages tissue response to enhanced oxygen transport and adequate mechanical stimulation. In vitro multistep ovarian tissue static culture followed by mature oocyte generation, fertilization, and embryo transfer promises to use the reserve of dormant follicles. Unfortunately, static in vitro culture of ovarian tissue does not promote development of primordial to secondary follicles or sustain follicle viability and thereby limits the number of obtainable mature oocytes. Enhancing oxygen transport to and exerting mechanical stimulation on ovarian tissue in a dynamic bioreactor may more closely mimic the physiological microenvironment and thus promote follicle activation, development, and viability. Materials and Methods: The most transport-effective dynamic bioreactor design was modified using 3D models of medium and oxygen transport to maximize strip perifusion and apply tissue fluid dynamic shear stresses and direct compressive strains to elicit tissue response. Prototypes of the final bioreactor design were manufactured with materials of varying cytocompatibility and assessed by testing the effect of leachables on sperm motility. Effectiveness of the bioreactor culture was characterized against static controls by culturing fresh bovine ovarian tissue strips for 7days at 4.8 × 10-5m/s medium filtration flux in air at -15% maximal total compressive strain and by assessing follicle development, health, and viability. Results and Conclusions: Culture in dynamic bioreactors promoted effective oxygen transport to tissues and stimulated tissues with strains and fluid dynamic shear stresses that, although non-uniform, significantly influenced tissue metabolism. Tissue strip culture in bioreactors made of cytocompatible polypropylene preserved follicle viability and promoted follicle development better than static culture, less so in bioreactors made of cytotoxic ABS-like resin.

Improvement of Mouse Preantral Follicle Survival and Development following Co-Culture with Ovarian Parenchyma Cell Suspension.

The parallel and continued improvements in both infertility treatment and the management of malignancy cases have brought to the forefront the potential for fertility preservation. Using ovarian follicular resources can effectively improve reproductive capacity and prevent infertility. The primary aim of this research was to try to generate an appropriate in vivo environment for the growth of the mouse follicles. Hence, the possible effects of the ovarian parenchyma cell suspension were explored on the growth and maturation of preantral follicles in vitro. In this experimental study, ovarian parenchymal cells were mechanically dissociated from preantral follicles of 12-14 days-old NMRI mice and then divided into 5 experimental groups (G1: Control, G2: Fresh follicle with fresh parenchyma cell suspension, G3: Vitrified-warmed follicle with fresh parenchyma cell suspension, G4: Fresh follicle with frozen-thawed parenchyma cell suspension, and G5: Vitrified-warmed follicle with frozenthawed parenchyma cell suspension). The diameter of the follicles and immature oocytes, viability, antrum formation, resumption of meiosis, in vitro fertilization (IVF), and Gdf9, Bmp6, and Bmp15 gene expression were examined on different periods. The diameter of the follicles and the oocytes on days 4 and 8, as well as the survival rate of the follicles up to day 12, were significantly higher in G2 and G4 compared to the Ctrl group (G1: 73.66%, G2:87.99%, G3: 82.70%, G4: 94.37%, and G5: 78.59%). Expression of growth marker genes for G3, and G5 groups was significantly higher than other groups, which indicated the protective effects of parenchyma cell suspension on follicles damaged by vitrification solutions. The growth, survival, and maturation of preantral follicles could be enhanced by co-culturing them with ovarian parenchyma cells. Further studies are needed to optimize the conditions for a successful parenchyma cell suspension-induced in vitro maturation (IVM) to occur in infertility clinics.

Effects of Dietary Selenium Yeast Supplementation in Pregnant Cashmere Goats on the Development of Offspring Hair Follicles

The objective of this study was to investigate the effects of maternal dietary selenium yeast (SY) supplementation during pregnancy on the hair follicle development of kids. Sixty pregnant Hanshan white cashmere goats were randomly divided into the con group (fed with a basal diet) and the SY group (fed with a basal diet with 0.4 mg/kg SY). SY was supplemented during the pregnancy until the birth of the kids. The growth performance, cashmere performance, hair follicle characteristics, and serum antioxidant capacity of the kids were periodically determined. The results showed that the birth weight of the kids in the SY group was significantly higher than that in the con group (p &lt; 0.05), and the average weight at 15 days, 1 month, 3 months, and 5 months of age increased by 13.60%, 8.77%, 8.86%, and 3.90%, respectively (p &gt; 0.05). The cashmere fineness at early birth was dramatically reduced with SY supplementation (p &lt; 0.001), whereas cashmere length and production were significantly increased at 5 months of age (p &lt; 0.05). Histology assays indicated that the primary hair follicles were fully developed at birth, and there was no significant difference in the number of primary hair follicles between the two groups (p &gt; 0.05). The number of secondary hair follicles and the number and density of active secondary hair follicles in the SY group at 15 days were significantly higher than those in the con group (p &lt; 0.05) and were increased by 11.18%, 6.18%, and 22.55% at 5 months of age, respectively (p &gt; 0.05). The serum antioxidant capacity analysis revealed that the SY group had higher levels of T-AOC, SOD, CAT, and GSH-Px activities and lower levels of MDA (p &gt; 0.05). These results reveal that the maternal dietary supplementation of SY in gestation can promote the morphogenesis and maturation of secondary hair follicles and increase the number and density of secondary hair follicles by enhancing the body’s antioxidant capacity, contributing to the improvement of cashmere quality and yield.

The influence of carbofuran insecticide exposure on the number of ovarian follicles in mice (Mus musculus)

Objective: The aim of this research was to investigate the histopathological damage to the ovaries of mice (Mus musculus) caused by exposure to carbofuran. Method: Twenty female mice aged 10 weeks with a body weight ranging from 25-30 mg were used in this study. The mice were divided into four groups, with each group consisting of five replicates. The control group (P0) received 0.5% physiological NaCl, while the treatment groups (P1, P2, and P3) were exposed to carbofuran at doses of 0.0833 mg/kg BW, 0.0417 mg/kg BW, and 0.0208 mg/kg BW, respectively, for ten days. On the 12th day, ovarian necropsy and HE staining were performed to observe the number of primary, secondary, tertiary, and Graafian follicles. Data analysis was conducted using ANOVA and BNJ tests. Result: The results of this research showed that oral administration of carbofuran leads to a decrease in the number of secondary, tertiary, and Graafian follicles. High doses, such as 0.0833 mg/kg body weight, resulted in a more pronounced reduction in the number of secondary, tertiary, and Graafian follicles compared to the control group. Conclusion: A reduction in the number of secondary, tertiary, and Graafian follicles with increasing doses of carbofuran.

Protective Effect of Glutathione Administration on Ovarian Function in Female Rats with Cyclophosphamide-Induced Ovarian Damage

Objectives: We investigated the potential of glutathione to protect ovarian function in rats exposed to cyclophosphamide by measuring serum anti-Mullerian hormone (AMH) levels, follicle counts, and related parameters. Design: Forty-two adult female Sprague-Dawley rats were randomly divided into six groups and treated with various combinations of cyclophosphamide, glutathione, and sodium chloride. On day 21, the rats were anesthetized, and their ovaries were removed for examination. Participants/Materials, Setting, Methods: Histopathological examination, serum AMH concentrations, follicle counts, AMH-positive staining of follicle percentages were analyzed. Statistical analysis was performed using a one-way analysis of variance and Tukey’s test, with significance set at p < 0.05. Secondary measures encompassed histopathological examination and percentages of AMH-positive staining of follicles. Results: Significant differences were observed in follicle counts, AMH-positive follicle parameters, and serum AMH concentrations among the six groups. Group 2 (treated with cyclophosphamide) had the lowest primordial, primary, secondary, and antral follicle counts and the highest atretic count. Group 6, treated with cyclophosphamide and 200 mg/kg glutathione, showed improved follicle counts compared to those in group 2. Reducing the glutathione dose to 100 mg/kg was ineffective. Limitations: This was an experimental animal investigation with a comparatively modest sample size. Experimental studies should be conducted to determine the optimal dosage and duration of glutathione therapy. Information gathered from an experimental animal model may not yield precisely similar outcomes in humans; therefore, additional investigations are necessary to examine the impact of glutathione on women experiencing POI. Conclusions: The anti-oxidative protective effect of directly administered glutathione was demonstrated for the first time. Low-dose glutathione was ineffective, whereas a high dose yielded significant ovarian protection against cyclophosphamide. Our findings provide valuable insights for supplementing clinical trials on the protective effects of glutathione against ovarian damage.

Comparative effects of the antioxidant glutathione with metformin and Diane-35 on hormonal, metabolic, and inflammatory indicators in a DHEA-induced PCOS rat model

Objective Comparison of hormonal, metabolic and inflammatory markers of glutathione with metformin and Diane-35 in a rat model of PCOS induced by dehydroepiandrosterone. Methods Twenty-five female rats were randomized into four groups. Group 1 was administered a subcutaneous dose of 0.2 ml saline/day. Group 2 was given 0.2 ml of 1% carboxymethyl cellulose (CMC)/day orally for 28 days. A PCOS model was established with DHEA in rats. Group 3 was given 4.5 mg/kg/day of Diane-35 orally dissolved in 1% CMC for 28 days. Group 4 was given 300 mg/kg/day of metformin orally dissolved in 1 ml of saline for 28 days, and Group 5 was administered 100 mg/kg of glutathione intraperitoneally on days 35, 42, and 49. On day 56, the rats were sacrificed. Serum markers and follicle count were examined. Results Serum IL-6, hs-CRP, insulin, testosterone, SHBG, and MDA values were significantly lower in the glutathione group than in the PCOS group (p = 0.0006, p = 0.023, p = 0.0082, p = 0.0007, p = 0.0048, and p < 0.0001, respectively). The number of all follicles was similar between the control and glutathione groups (p < 0.05). When we compared the other groups with the PCOS group, the number of primary, secondary, atretic, and cystic follicles was significantly lower in the metformin and glutathione groups. The number of primordial and antral follicles was significantly higher than in the PCOS group. Conclusions Glutathione plays anti-inflammatory and antioxidant roles, similar to metformin, by lowering serum IL-6, insulin, testosterone, CRP, and MDA levels; decreasing atretic/cystic follicle count; and improving antral follicle count and folliculogenesis in PCOS patients.

Exploring the potential use of platelet rich plasma (PRP) from adult and umbilical cord blood in murine follicle culture

Ovarian follicle culture is a powerful tool to study follicular physiology and has potential applications in clinical and commercial settings. Despite remarkable progress, recreating folliculogenesis in vitro remains challenging for many mammalian species. This study investigates the impact of platelet-rich plasma (PRP) derived from adult blood (human platelet lysate, hPL) and umbilical cord blood (Umbilical cord plasma, UCP) on murine pre-antral follicle culture and oocyte maturation. Pre-antral follicles were cultured individually for 10 days with fetal bovine serum (FBS) serving as the control and two PRP sources (hPL and UCP) and their activated forms (Ac-hPL and Ac-UCP). The results suggest that neither hPL nor UCP, regardless of activation status, improved follicle culture outcomes compared to FBS. Interestingly, activation did not significantly impact the main functional outcomes such as maturation rates, survival, and growth. Oestradiol secretion and oocyte diameter, often considered hallmarks of follicle quality, did not show significant differences between matured and non-matured oocytes across the treatment groups. However, gene expression analysis revealed a significant upregulation of Gdf-9 and Bmp-15 mRNA levels in oocytes from the Ac-UCP group, regardless of maturation stage, suggesting that the accumulation of the mRNA could be due to potential challenges in translation in the Ac-UCP group. In conclusion, this study challenges the hypothesis that PRP, as a serum source, could improve follicle culture outcomes compared to FBS, the gold standard in murine follicle culture. Further research is needed to understand the species-specific effects of PRP and explore other potential factors affecting follicle culture and oocyte quality.

Bisphenol A triggers apoptosis in mouse pre-antral follicle granulosa cells via oxidative stress

BackgroundBisphenol A (BPA), an endocrine disrupting chemical with weak estrogenic and anti-androgenic activity, is widely present in various environmental media and organisms. It has certain reproductive toxicity and can cause a variety of female reproductive system diseases. Although BPA-stimulated apoptosis of granulosa cells has been widely elaborated, the effect of BPA on mouse pre-antral follicle granulosa cells (mpGCs) has not been well elucidated.ResultsIn this study, the results of live-dead cell staining showed that high concentrations of BPA severely impaired mpGCs growth viability and affected the cell cycle transition of mpGCs. We confirmed that BPA promotes the production of reactive oxygen species (ROS) and facilitates oxidative stress in mpGCs. In addition, immunofluorescence, transmission electron microscopy, and flow cytometry experiments demonstrated that BPA treatment for mpGCs resulted in apoptotic features, such as rounding, cytoplasmic crinkling, and mitochondrial damage. This was accompanied by a large production of ROS and apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. RNA-seq data showed that several apoptosis-related pathways were enriched in the high concentration BPA-treated group compared with the normal group, such as the p53 pathway, MAPK pathway, etc.ConclusionsThese results suggest that cells undergo oxidative stress effects and apoptosis after BPA treatment for mpGCs, which affects normal follicle development. The potential mechanism of BPA-induced female reproductive toxicity was elucidated, while providing a research basis for the prevention and treatment of female reproductive diseases.

Treatment of ovarian damage induced by chemotherapeutic drugs in female rats with G-CSF and platelet-rich plasma(PRP): an immunohistochemical study correlation with novel marker INSL-3

Objective To assess the impacts of Platelet-Rich Plasma(PRP) and Granulocyte Colony-Stimulating Factor(G-CSF) on a rat model with induced ovarian follicular damage caused by cyclophosphamide(Cy). Materials and Methods Forty-two Sprague–Dawley rats were randomly allocated into seven distinct groups as; Group 1(control): NaCl intraperitoneal (IP) injection was administered on days D1, D7, and D14. Group 2(Cy):Cy IP injection on D1 + NaCl IP injection on D7 and D14 were administered. Group 3(PRP): PRP IP injection on D1,D7 and D14 were administered. Group 4(Cy + PRP):Cy IP injection on D1 and PRP IP injection on D1, D7 and D14 were administered. Group 5(G-CSF): G-CSF IP injection on D1, D7 and D14 were administered. Group 6(Cy + G-CSF):Cy IP injection on D1+ G-CSF IP injection on D1, D7 and D14 were administered. Group 7(Cy + PRP + G-CSF):Cy IP injection on D1+ PRP IP injection on D1,D7 and D14+ G-CSF IP injection on D1,D7 and D14 were administered. Follicular number, histological scores of AMH and INSL3 stained follicles at different stages of follicular development, and serum Anti-Müllerian hormone(AMH) were evaluated. Results The primary, secondary, and antral follicle intensity scores for AMH-positive staining were most prominent in Groups 3 and 5. There was no significant difference between groups 4, 6 and 7 compared to group 1 in terms of follicule counts and AMH staining. The intensity scores of AMH-positive staining follicles were notably reduced in group 2 compared to groups 4, 6, and 7, with a significant difference (p < .01). Among the groups, group 2 exhibited the least intense antral follicle staining for INSL3, displaying a significant difference(p < .01) compared to the remaining groups. Conclusions Autologous PRP and G-CSF might protect ovarian function in the face of ovarian damage caused by Cy-induced effects.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Neurotrophin-4 promotes in vitro development and maturation of human secondary follicles yielding metaphase II oocytes and successful blastocyst formation.

Does a matrix-free culture system supplemented with neurotrophic factor 4 (NT4) improve human in vitro follicular development and meiotic maturation, ultimately resulting in fertilizable oocytes? NT4 supplementation of in vitro culture significantly enhances the growth, steroid hormone production, and maturity potential of human secondary follicles derived from fresh ovarian medulla (from post- and pre-pubertal patients), thereby yielding fertilizable oocytes. Reconstituting folliculogenesis in vitro is of paramount importance in the realms of fertility preservation, reproductive biology research, and reproductive toxicity assessments. However, the efficiency of in vitro culture systems remains suboptimal, as the attainment of fertilizable oocytes from in vitro growth (IVG) of human follicles remains unachieved, with the data being particularly scant regarding follicles from prepubertal girls. We have previously found that mouse oocytes from secondary follicles derived from IVG are deficient in neuroendocrine regulation. NT4 and its corresponding receptor have been identified in human follicles. Significantly, the addition of NT4 during the IVG process markedly enhances both follicle growth and oocyte maturation rates in mice. Fresh medulla tissue obtained during tissue preparation for ovarian tissue cryopreservation (OTC) were collected from 10 patients aged from 6 to 21 years old, all of whom had undergone unilateral oophorectomy as a means of fertility preservation. Isolated secondary follicles were individually cultured in vitro with or without NT4 in a matrix-free system. Secondary follicles, extracted via enzymatic digestion and mechanical disruption from each patient, were randomly allocated to either a control group or an NT4-supplemented group (100 ng/ml), followed by individual culture on an ultra-low attachment plate. Follicle growth and viability were assessed by microscopy. Levels of anti-Müllerian hormone (AMH), estradiol, and progesterone in the medium were quantified. An oocyte-specific marker was identified using confocal fluorescence microscopy following DEAD box polypeptide 4 (DDX4) staining. The competence of individual oocytes for maturation and fertilization were assessed after IVM and ICSI with donated sperm samples. Overall, isolated follicles from both groups survived up to 6 weeks with increasing diameters over the duration (P < 0.05), reaching terminal diameters of almost 1 mm with confirmed steroidogenesis and expression of oocyte marker (DDX4), and producing morphologically normal MII oocytes. When compared with the control group, the NT4 group had a similar initial follicular diameter (206 ± 61.3 vs 184 ± 93.4 μm) but exhibited a significant increase in follicular diameter from the ninth day of culture onwards (P < 0.05). From Week 3, estradiol and progesterone production were significantly increased in the NT4 group, while no significant difference was observed in AMH production between groups. The proportion of 'fast-growth' follicles in the NT4 group was significantly higher than that in the control group (13/23 vs 6/24, P < 0.05). An increased efficiency of MII oocyte maturation per live follicle in the NT4 group was also observed (control group vs NT4 group, 4/24 vs 10/23, P < 0.05). It is noteworthy that an MII oocyte obtained from the control group exhibited abnormal fertilization after ICSI. In contrast, an MII oocyte acquired from the NT4 group progressed to the blastocyst stage and showed potential for transfer. N/A. The cohort examined in this study was all patients diagnosed with beta-thalassemia major. Whether this culture system is effective for patients with other diseases remains unknown. Since the chosen dose of NT4 was established based on dose finding in mice, the optimal dose for use in a human IVG system needs further confirmation. The oocytes and embryos procured from this study have not been quantified for ploidy status or epigenetic signatures. Fresh medulla tissue obtained during tissue preparation for OTC may serve as a precious source of fertilizable oocytes for female fertility preservation, even for pre-pubertal girls, without the threat of tumour reintroduction. After further characterization and optimization of the system, this culture system holds the potential to provide a powerful future research tool, for the comprehensive exploration of human follicular development mechanisms and for conducting reproductive toxicity evaluations. This work was supported by the National Key R&D Program of China (grant number 2022YFC2703000) and National Natural Science Foundation of China (grant numbers 82271651 and 81871214). The medium used in human follicle in vitro culture in this study has been applied for a national invention patent in China (No. 202211330660.7). The inventors of the patent, in order, are: Y.G., C.F., and X.L.

CircERCC6 Positively Regulates the Induced Activation of SHF Stem Cells in Cashmere Goats via the miR-412-3p/BNC2 Axis in an m6A-Dependent Manner.

The cashmere, a kind of nature protein fiber, is one of the main use of cashmere goats. The induced activation of secondary hair follicle (SHF) stem cells by the dermal papilla cell-derived signals is a key biological process for the morphogenesis and growth of cashmere fiber in cashmere goats. Previously, the circRNA-ERCC6 (circERCC6) was identified from cashmere goat SHFs; however, its biological significance is unclear in the SHF physiology process of cashmere goats. In this study, we found that circERCC6 exhibited significantly higher expression at anagen SHF bulge compared with the counterpart of telogen and harbored three m6A modified sites (named m6A-685, m6A-862, and m6A-995) through methylation immunoprecipitation using a real-time quantitative polymerase chain reaction (Me-RIP-qPCR) technique. The knockdown experiments of circERCC6 in SHF stem cells showed that circERCC6 positively regulates the induced activation of SHF stem cells in cashmere goats. Through a dual-luciferase reporter assay, we demonstrated that m6A-modified circERCC6 (m6A-circERCC6) sponged miR-412-3p to upregulate the expression of BNC2 mRNA in SHFstem cells. Through m6A-deficient mutant assay in circERCC6 knockdown SHF stem cells, we further showed that m6A modification within circERCC6 is required to mediate the miR-412-3p/BNC2 axis to finally promote the proper induced activation of SHF stem cells in cashmere goats.

Single-Cell Transcriptome Sequence Profiling on the Morphogenesis of Secondary Hair Follicles in Ordos Fine-Wool Sheep.

The Ordos fine-wool sheep is a high-quality breed in China that produces superior natural textiles and raw materials such as wool and lamb meat. However, compared to the Australian Merino sheep, there is still a gap in terms of the wool fiber fineness and wool yield. The hair follicle is the main organ that controls the type of wool fiber, and the morphological changes in the secondary hair follicle are crucial in determining wool quality. However, the process and molecular mechanisms of hair follicle morphogenesis in Ordos fine-wool sheep are not yet clear. Therefore, analyzing the molecular mechanisms underlying the process of follicle formation is of great significance for improving the fiber diameter and wool production of Ordos fine-wool sheep. The differential expressed genes, APOD, POSTN, KRT5, and KRT15, which related to primary hair follicles and secondary hair follicles, were extracted from the dermal papillae. Based on pseudo-time analysis, the differentiation trajectories of dermal lineage cells and epidermal lineage cells in the Ordos fine-wool sheep were successfully constructed, providing a theoretical basis for breeding research in Ordos fine-wool sheep.