Effects of short and long - term nutrition and progesterone supplementation on the success of fixed - time artificial insemination in the ewe.

The success of fixed - time artificial insemination (AI) in the ewe is variable due to poor synchrony of estrus. We examined the effects of long-term nutrition (LTN; low, medium, high - 6 months), short-term nutrition (STN; 1.0 M, 1.5 M - 14 days) and progesterone supplementation (P; single pessary, replacement on Day 9) on synchrony and reproductive outcomes. High LTN advanced (P < 0.05) estrus, increased (P = 0.06) pregnancy (range 71.1 - 81.1%) and improved (P < 0.01) litter size (range 1.30 - 1.50). STN increased (P < 0.05) pregnancy (79.0 versus 72.3%) but not litter size or timing of estrus. A LTN x STN interaction (P < 0.01) for time of estrus indicates that the effects of LTN were moderated by STN depending on the level of LTN. Pessary replacement delayed (P < 0.05) the onset of estrus, improved synchrony but did not affect pregnancy or litter size. High LTN increased (P < 0.05) the number of large (≥ 3.8 mm) and medium - size follicles (2.0 - 3.7 mm) but the diameter of large follicles tended to be reduced (P = 0.08) on Day 12. STN did not affect follicle number or size whilst P reduced (P < 0.05) the diameter of large follicles on Day 12 (4.83 versus 5.10 mm) and increased the number of medium - size follicles (3.56 versus 2.74 mm). In conclusion, both LTN and STN are major sources of variability in AI programs whilst pessary replacement has potential to reduce variability.

Discovery of a novel and highly selective JAK3 inhibitor as a potent hair growth promoter.

JAK-STAT signalling pathway inhibitors have emerged as promising therapeutic agents for the treatment of hair loss. Among different JAK isoforms, JAK3 has become an ideal target for drug discovery because it only regulates a narrow spectrum of γc cytokines. Here, we report the discovery of MJ04, a novel and highly selective 3-pyrimidinylazaindole based JAK3 inhibitor, as a potential hair growth promoter with an IC50 of 2.03 nM. During in vivo efficacy assays, topical application of MJ04 on DHT-challenged AGA and athymic nude mice resulted in early onset of hair regrowth. Furthermore, MJ04 significantly promoted the growth of human hair follicles under ex-vivo conditions. MJ04 exhibited a reasonably good pharmacokinetic profile and demonstrated a favourable safety profile under in vivo and in vitro conditions. Taken together, we report MJ04 as a highly potent and selective JAK3 inhibitor that exhibits overall properties suitable for topical drug development and advancement to human clinical trials.

Lipopolysaccharide-binding protein in follicular fluid is associated with the follicular inflammatory status and granulosa cell steroidogenesis in dairy cows.

Metabolic stress and subsequent hepatic dysfunction in high-producing dairy cows are associated with inflammatory diseases and declining fertility. Lipopolysaccharide (LPS)-binding protein (LBP) is produced by hepatocytes and controls the immune response, suggesting that it is involved in the pathophysiology of inflammation-related attenuation of reproductive functions during metabolic stress. This study investigated the effect of LBP on the inflammatory status, oocyte quality, and steroidogenesis in the follicular microenvironment of dairy cows. Using bovine ovaries obtained from a slaughterhouse, follicular fluid and granulosa cells were collected from large follicles to evaluate the follicular status of metabolism, inflammation, and steroidogenesis. Cumulus-oocyte complexes were aspirated from small follicles and subjected to in vitro embryo production. The results showed that follicular fluid LBP concentrations were significantly higher in cows with fatty livers and hepatitis than in those with healthy livers. Follicular fluid LBP and LPS concentrations were negatively correlated, whereas LPS concentration showed a positive correlation with the concentrations of non-esterified fatty acids (NEFA) and ß-hydroxybutyric acid in follicular fluid. The blastulation rate of oocytes after in vitro fertilization was impaired in cows in which coexisting large follicles had high NEFA levels. Follicular fluid NEFA concentration was negatively correlated with granulosa cell expression of the estradiol (E2) synthesis-related gene (CYP19A1). Follicular fluid LBP concentration was positively correlated with follicular fluid E2 concentration and granulosa cell CYP19A1 expression. In conclusion, follicular fluid LBP may be associated with favorable conditions in the follicular microenvironment, including low LPS levels and high E2 production by granulosa cells.

Melatonin promotes proliferation of Inner Mongolia cashmere goat hair follicle papilla cells through Wnt10b.

The study demonstrated that melatonin (MT) can induce the development of secondary hair follicles in Inner Mongolian cashmere goats through the Wnt10b gene, leading to secondary dehairing. However, the mechanisms underlying the expression and molecular function of Wnt10b in dermal papilla cells (DPC) remain unknown. This research aimed to investigate the impact of MT on DPC and the regulation of Wnt10b expression, function, and molecular mechanisms in DPC. The findings revealed that MT promotes DPC proliferation and enhances DPC activity. Co-culturing DPC with overexpressed Wnt10b and MT showed a significant growth promotion. Subsequent RNA sequencing (RNA-seq) of overexpressed Wnt10b and control groups unveiled the regulatory role of Wnt10b in DPC. Numerous genes and pathways, including developmental pathways such as Wnt and MAPK, as well as processes like hair follicle morphogenesis and hair cycle, were identified. These results suggest that Wnt10b promotes the growth of secondary hair follicles in Inner Mongolian cashmere goats by regulating crucial factors and pathways in DPC proliferation.

ERß Regulation of Indian Hedgehog Expression in the First Wave of Ovarian Follicles.

Increased activation of ovarian primordial follicles in Erß knockout (ErßKO) rats becomes evident as early as postnatal day 8.5. To identify the ERß-regulated genes that may control ovarian primordial follicle activation, we analyzed the transcriptome profiles of ErßKO rat ovaries collected on postnatal days 4.5, 6.5, and 8.5. Compared to wildtype ovaries, ErßKO ovaries displayed dramatic downregulation of Indian hedgehog (Ihh) expression. IHH-regulated genes, including Hhip, Gli1, and Ptch1, were also downregulated in ErßKO ovaries. This was associated with a downregulation of steroidogenic enzymes Cyp11a1, Cyp19a1, and Hsd17b1. The expression of Ihh remained very low in ErßKO ovaries despite the high levels of Gdf9 and Bmp15, which are known upregulators of Ihh expression in the granulosa cells of activated ovarian follicles. Strikingly, the downregulation of the Ihh gene in ErßKO ovaries began to disappear on postnatal day 16.5 and recovered on postnatal day 21.5. In rat ovaries, the first wave of primordial follicles is rapidly activated after their formation, whereas the second wave of primordial follicles remains dormant in the ovarian cortex and slowly starts activating after postnatal day 12.5. We localized the expression of Ihh mRNA in postnatal day 8.5 wildtype rat ovaries but not in the age-matched ErßKO ovaries. In postnatal day 21.5 ErßKO rat ovaries, we detected Ihh mRNA mainly in the activated follicles in the ovaries' peripheral regions. Our findings indicate that the expression of Ihh in the granulosa cells of the activated first wave of ovarian follicles depends on ERß.

The Effect of Tamoxifen Citrate on Granulosa Cells of Ovarian Follicles in Adult Female Rats: Light Microscopic Study.

Background: Tamoxifen is a drug that has been used extensively as a chemotherapeutic agent for breast cancer. It should be taken for a long period, from few weeks up to many years, so it can induce gynecological and nongynecological complications. Aim: Present study was conducted to clarify the histopathological effects of tamoxifen intake on the ovarian follicles of rats and evaluate the promising recovery after drug withdrawal. Materials and Methods: Adult female albino rats (n = 24) were randomly divided into four groups. Group I: Control rats without treatment. Group II: Rats received olive oil vehicle. Group III: Rats received 5 mg/kg daily of tamoxifen dissolved in olive oil by oral administration for 4 weeks. Group IV: Rats received tamoxifen as in Group III then will be kept for another 4 weeks without treatment for recovery. Then, the rats were anaesthetized and the ovaries were removed and prepared for histological assessment by light microscope. Results: The ovarian histological findings in the ovary of Group III revealed an increase in atretic ovarian follicles, appearance of cystic ovarian follicles, and cystic corpus luteum. The granulosa cells of ovarian follicles were disorganized with vacuolation of their cytoplasm, increased number of pyknotic nuclei, fragmented nuclei, and apoptotic bodies. After the withdrawal of drug, the ovarian tissue showed slight improvement with the appearance of some atretic follicles with degenerated oocyte and stromal hyperplasia. Conclusion: Based on the results, tamoxifen induced marked histological changes in the ovary. If tamoxifen is mandatory for the prevention of breast cancer, frequent gynecological examination should be carried out to detect any side effects.

Upregulation of coagulation factor V by glucocorticoid in the preovulatory follicles of zebrafish.

Increased cortisol levels in the preovulatory follicular fluid suggests a role of glucocorticoid in human ovulation. However, the mechanisms through which cortisol regulates the ovulatory process remain poorly understood. In this study, we examined the upregulation of f5 mRNA by glucocorticoid and its receptor (Gr) in the preovulatory follicles of zebrafish. Our findings demonstrate a significant increase in 11ß-hydroxysteroid dehydrogenase type 2 (hsd11b2), a cortisol response gene, in preovulatory follicles. Additionally, hydrocortisone exerts a dose- and time-dependent upregulation of f5 mRNA in these follicles. Importantly, this stimulatory effect is Gr-dependent, as it was completely abolished in gr-/- mutants. Furthermore, site-directed mutagenesis identified a glucocorticoid response element (GRE) in the promoter of zebrafish f5. Interestingly, successive incubation of hydrocortisone and the native ovulation-inducing steroid, progestin (17α,20ß-dihydroxy-4-pregnen-3-one, DHP), further enhanced f5 expression in preovulatory follicles. Overall, our results indicate that the dramatic increase of f5 expression in preovulatory follicles is partially attributable to the regulation of glucocorticoid and Gr.

Histological, immunohistochemical and serological investigations of the ovary during follicular phase of estrous cycle in Saidi sheep.

BACKGROUND: Saidi sheep are the most abundant ruminant livestock species in Upper Egypt, especially in the Assiut governorate. Sheep are one of the most abundant animals raised for food in Egypt. They can convert low-quality roughages into meat and milk in addition to producing fiber and hides therefore; great opportunity exists to enhance their reproduction. Saidi breed is poorly known in terms of reproduction. So this work was done to give more information on some hormonal, oxidative, and blood metabolites parameters in addition to histological, histochemical and immunohistochemical investigations of the ovary during follicular phase of estrous cycle. The present study was conducted on 25 healthy Saidi ewes for serum analysis and 10 healthy ewes for histological assessment aged 2 to 5 years and weighted (38.5 ± 2.03 kg). RESULTS: The follicular phase of estrous cycle in Saidi sheep was characterized by the presence of ovarian follicles in different stages of development and atresia in addition to regressed corpus luteum. Interestingly, apoptosis and tissue oxidative markers play a crucial role in follicular and corpus luteum regression. The most prominent features of the follicular phase were the presence of mature antral (Graafian) and preovulatory follicles as well as increased level of some blood metabolites and oxidative markers. Here we give a new schematic sequence of ovarian follicles in Saidi sheep and describing the features of different types. We also clarified that these histological pictures of the ovary was influenced by hormonal, oxidative and blood metabolites factors that characterizes the follicular phase of estrous cycle in Saidi sheep. CONCLUSION: This work helps to understanding the reproduction in Saidi sheep which assist in improving the reproductive outcome of this breed of sheep. These findings are increasingly important for implementation of a genetic improvement program and utilizing the advanced reproductive techniques as estrous synchronization, artificial insemination and embryo transfer.

Dynamics of IGF signalling during the ovulatory peak in women undergoing ovarian stimulation.

CONTEXT: IGF signalling is known to affect human ovarian follicular function during growth and development. However, the role of the IGF system is unknown during the ovulatory peak, which is characterized by profound changes in granulosa cell (GCs) mitosis and function. OBJECTIVE: How is the IGF system expressed and regulated during the midcycle surge in women? DESIGN: Follicular fluid (FF) and granulosa cells (GCs) were collected during the ovulatory peak from two specific time-points. One sample was obtained before oocyte pick up (OPU): before ovulation trigger (OT) (T = 0 h) or at 12, 17, or 32 h after OT, and one sample was obtained at OPU 36 h after OT. SETTING: University hospital. PATIENTS/PARTICIPANTS: Fifty women undergoing ovarian stimulation were included. MAIN OUTCOME MEASURE: Gene expression profiles were assessed by microarray analysis of GCs. IGF-related proteins in the FF were assessed by using immunoassays or by determination of activity with a proteinase assay. RESULTS: Expression of proteins promoting IGF activity (i.e., IGF2, PAPPA, and IRS1) together with proliferation markers were downregulated on a transcriptional level in GCs after OT, whereas proteins inhibiting the IGF signal (i.e., IGFBPs, IGF2R, and STC1) were upregulated. STC1 gene expression and protein levels were greatly upregulated after OT with a parallel steep downregulation of PAPP-A proteolytic activity. CONCLUSIONS: These data suggest that downregulation of IGF signalling mediated by increased STC1 expression is instrumental for the sudden cessation in GC proliferation and onset of differentiation during the ovulatory peak.

Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle-Shaped Microfluidic Device.

Pluripotent stem cell-derived skin organoids (PSOs) emerge as a developmental skin model that is self-organized into multiple components, such as hair follicles. Despite their impressive complexity, PSOs are currently generated in the absence of 3D extracellular matrix (ECM) signals and have several major limitations, including an inverted anatomy (e.g., epidermis inside/dermis outside). In this work, a method is established to generate PSOs effectively in a chemically-defined 3D ECM environment. After examining various dermal ECM molecules, it is found that PSOs generated in collagen -type I (COLI) supplemented with laminin 511 (LAM511) exhibit increased growth compared to conventional free-floating conditions, but fail to induce complete skin differentiation due in part to necrosis. This problem is addressed by generating the PSOs in a 3D bioprinted spindle-shaped hydrogel device, which constrains organoid growth longitudinally. This culture system significantly reduces organoid necrosis and leads to a twofold increase in keratinocyte differentiation and an eightfold increase in hair follicle formation. Finally, the system is adapted as a microfluidic device to create asymmetrical gradients of differentiation factors and improves the spatial organization of dermal and epidermal cells. This study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially-controlling skin differentiation in the PSO framework.

The decline in cellular iron is crucial for differentiation in keratinocytes.

Iron is a vital metal for most biological functions in tissues, and its concentration is exquisitely regulated at the cellular level. During the process of differentiation, keratinocytes in the epidermis undergo a noticeable reduction in iron content. Conversely, psoriatic lesions, characterized by disruptions in epidermal differentiation, frequently reveal an excessive accumulation of iron within keratinocytes that have undergone differentiation. In this study, we clarified the significance of attenuated cellular iron content in the intricate course of epidermal differentiation. We illustrated this phenomenon through the utilization of hinokitiol, an iron chelator derived from the heartwood of Taiwanese hinoki, which forcibly delivers iron into cells independent of the intrinsic iron-regulation systems. While primary cultured keratinocytes readily succumbed to necrotic cell death by this iron chelator, mild administration of the hinokitiol-iron complex modestly disrupts the process of differentiation in these cells. Notably, keratinocyte model cells HaCaT and anaplastic skin rudiments exhibit remarkable resilience against the cytotoxic impact of hinokitiol, and the potent artificial influx of iron explains a suppressive effect selectively on epidermal differentiation. Moreover, the augmentation of iron content induced by the overexpression of divalent metal transporter 1 culminates in the inhibition of differentiation in HaCaT cells. Consequently, the diminution in cellular iron content emerges as an important determinant influencing the trajectory of keratinocyte differentiation.

3D culture applied to reproduction in females: possibilities and perspectives.

In vitro cell culture is a well-established technique present in numerous laboratories in diverse areas. In reproduction, gametes, embryos, and reproductive tissues, such as the ovary and endometrium, can be cultured. These cultures are essential for embryo development studies, understanding signaling pathways, developing drugs for reproductive diseases, and in vitro embryo production (IVP). Although many culture systems are successful, they still have limitations to overcome. Three-dimensional (3D) culture systems can be close to physiological conditions, allowing greater interaction between cells and cells with the surrounding environment, maintenance of the cells' natural morphology, and expression of genes and proteins such as in vivo. Additionally, three-dimensional culture systems can stimulated extracellular matrix generating responses due to the mechanical force produced. Different techniques can be used to perform 3D culture systems, such as hydrogel matrix, hanging drop, low attachment surface, scaffold, levitation, liquid marble, and 3D printing. These systems demonstrate satisfactory results in follicle culture, allowing the culture from the pre-antral to antral phase, maintaining the follicular morphology, and increasing the development rates of embryos. Here, we review some of the different techniques of 3D culture systems and their applications to the culture of follicles and embryos, bringing new possibilities to the future of assisted reproduction.

Loss of ERß Disrupts Gene Regulation in Primordial and Primary Follicles.

Loss of ERß increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERß in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erß knockout (Erßnull) mouse ovaries, and their transcriptomes were compared with those of control Erßfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erßnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERß has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erßnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erßfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erß-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erßnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erßnull PrFs were downregulated. Such deregulation of genes in Erßnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erßnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERß either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.

Integration Analysis of Hair Follicle Transcriptome and Proteome Reveals the Mechanisms Regulating Wool Fiber Diameter in Angora Rabbits.

Fiber diameter is an important characteristic that determines the quality and economic value of rabbit wool. This study aimed to investigate the genetic determinants of wool fiber diameter through an integration analysis using transcriptomic and proteomic datasets from hair follicles of coarse and fine wool from Angora rabbits. Using a 4D label-free technique, we identified 423 differentially expressed proteins (DEPs) in hair follicles of coarse and fine wool in Angora rabbits. Eighteen DEPs were examined using parallel reaction monitoring, which verified the reliability of our proteomic data. Functional enrichment analysis revealed that a set of biological processes and signaling pathways related to wool growth and hair diameter were strongly enriched by DEPs with fold changes greater than two, such as keratinocyte differentiation, skin development, epidermal and epithelial cell differentiation, epidermis and epithelium development, keratinization, and estrogen signaling pathway. Association analysis and protein-protein interaction network analysis further showed that the keratin (KRT) family members, including KRT77, KRT82, KRT72, KRT32, and KRT10, as well as CASP14 and CDSN, might be key factors contributing to differences in fiber diameter. Our results identified DEPs in hair follicles of coarse and fine wool and promoted understanding of the molecular mechanisms underlying wool fiber diameter variation among Angora rabbits.

Microscopic Focus on the Thyroid Follicles of the One-Humped Camel (Camelus dromedarius).

The microstructure of the thyroid gland of the one-humped camel (Camelus dromedarius) was described using morphometric, histological, immunohistochemical staining, and ultrastructural standard techniques. The follicular secretory units of the thyroid glands displayed comparable and variable sizes where the large follicles were situated at the peripheral margins; however, the small ones were located in the central region. Semisquamous epithelium (low cuboidal) lined the large thyroid follicles, while high cuboidal (columnar) epithelium lined the small ones. Our electron microscopic findings revealed that the low-sized cuboidal follicular cells lack organelles and are hypoactive. The high cuboidal follicular cells are active cells and rich in cellular organelles such as cisternae of rough endoplasmic reticulum, mitochondria, colloid droplets, scrolled Golgi apparatus, and secretory vesicles. A few degenerate follicular cells appeared on rare occasions. The parafollicular cells appeared with a more prominent and conspicuous nucleus than the follicular cells. The follicular cells were classified as active, inactive, or degenerated using transmission electron microscopy. The follicular and parafollicular cells showed calcitonin-positive immunoreactivity. Overall, the presented results showed particular convergences of the morphostructural aspects of the thyroid gland of C. dromedarius to that of other mammals with some distinctive features to cope with their physiology and harsh niche.

Erythropoietin effects on cryopreserved/transplanted cat ovarian tissue: A comparison of two incubation methods.

Many feline species are currently threatened with extinction. Therefore, germplasm bank establishment has become imperative. However, cryoinjury and ischemia-reperfusion injury pose significant obstacles to both cryopreservation and xenotransplantation. In this regard, erythropoietin (Epo) represents a potential alternative strategy due to its properties. This study aimed to assess the incubation of domestic cat ovarian tissue in Epo, both before and after cryopreservation, and investigate its effectiveness in promoting revascularization following xenotransplantation. Sixteen ovaries from 8 healthy cats were sliced following elective bilateral ovariohysterectomy (OHE). Subsequently, 8 fragments measuring 3 mm³ each were obtained from the cortical region of each ovary. The fragments were allocated into 3 treatment groups: Cryo group, fragments were cryopreserved, thawed and immediately transplanted; Cryo + Epo group, fragments were first cryopreserved in nitrogen, thawed, incubated in Epo (100 IU) for 2h and transplanted; and the Epo + Cryo group, in which fragments were first incubated in Epo (100 IU) for 2h, cryopreserved, thawed and immediately transplanted. The fragments were then xenotransplanted into the dorsal subcutaneous region of ovariectomized female nude mice and retrieved at 7, 14, 21, and 28 days post-transplantation. The results indicated that Epo effectively enhanced follicular survival, preservation of viability, and tissue revascularization. The Epo + Cryo group displayed better revascularization rates on D14 and D21 post-transplantation and an increase in primordial and growing follicles on D28, the Cryo + Epo group exhibited significantly more follicles on D14 and D21, with fewer degenerated follicles.

The close relationship between oocyte aging and telomere shortening, and possible interventions for telomere protection.

As women delay childbearing due to socioeconomic reasons, understanding molecular mechanisms decreasing oocyte quantity and quality during ovarian aging becomes increasingly important. The ovary undergoes biological aging at a higher pace when compared to other organs. As is known, telomeres play crucial roles in maintaining genomic integrity, and their shortening owing to increased reactive oxygen species, consecutive cellular divisions, genetic and epigenetic alterations is associated with loss of developmental competence of oocytes. Novel interventions such as antioxidant treatments and regulation of gene expression are being investigated to prevent or rescue telomere attrition and thereby oocyte aging. Herein, potential factors and molecular mechanisms causing telomere shortening in aging oocytes were comprehensively reviewed. For the purpose of extending reproductive lifespan, possible therapeutic interventions to protect telomere length were also discussed.

Investigating the impact of vitrification on bovine ovarian tissue morphology, follicle survival, and transcriptomic signature.

PURPOSE: Ovarian tissue cryopreservation is vital for fertility preservation, yet its effect on ovarian tissue follicle survival and transcriptomic signature requires further investigation. This study delves into the effects of vitrification on tissue morphology, function, and transcriptomic changes, helping to find possibilities for vitrification protocol improvements. METHODS: Ovarian cortex from 19 bovine animals were used to conduct pre- and post-vitrification culture followed by histological assessment, immunohistochemistry, and TUNEL assay. Follicles' functionality was assessed for viability and growth within the tissue and in isolated cultures. RNA-sequencing of ovarian tissue was used to explore the transcriptomic alterations caused by vitrification. RESULTS: Follicle density, cell proliferation, and DNA damage in ovarian stroma were unaffected by vitrification. However, vitrified cultured tissue exhibited reduced follicle density of primordial/primary and antral follicles, while freshly cultured tissue manifested reduction of antral follicles. Increased stromal cell proliferation and DNA damage occurred in both groups post-culture. Isolated follicles from vitrified tissue exhibited similar viability to fresh follicles until day 4, after which the survival dropped. RNA-sequencing revealed minor effects of vitrification on transcriptomic signatures, while culture induced significant gene expression changes in both groups. The altered expression of WNT and hormonal regulation pathway genes post-vitrification suggests the molecular targets for vitrification protocol refinement. CONCLUSION: Vitrification minimally affects tissue morphology, follicle density, and transcriptomic signature post-thawing. However, culture revealed notable changes in vitrified tissue samples, including reduced follicle density, decreased isolated follicle survival, and alteration in WNT signalling and ovarian hormonal regulation pathways, highlighted them as possible limitations of the current vitrification protocol.

Reconstruction of ovarian follicular-like structure by recellularization of a cell-free human ovarian scaffold with mouse fetal ovarian cells.

The present study assessed the supportive roles of the decellularized human ovarian tissue in homing of mouse fetal ovarian cells into the scaffold as well as the formation of the follicular-like structure. The human ovarian cortical tissues were decellularized by three freeze-thaw cycles and then, treated with Triton X-100 for 15 h and 0.5% sodium dodecyl sulfate for 72 h. After isolation and preparation of mouse fetal ovarian cells (19 dpc) they were seeded into the decellularized scaffolds and cultured for 7 days, then using a light microscope, laser confocal scanning microscope, and scanning electron microscope these scaffolds were studied. Analysis of gene expression related to oocyte and follicular cells such as Ddx4, Nobox, Gdf9, and Connexin37 was assessed by real-time RT-PCR and the DDX4 and GDF9 proteins were detected by immunohistochemistry. The result showed that the human ovarian tissue was decellularized properly and the tissue elements and integrity were well preserved. After 7 days of in vitro culture, the fetal ovarian cells attached and penetrated into different sites and depths of the scaffold. The formed organoid within the scaffold showed large round, small polyhedral, and elongated spindle cells similar to the follicle structure. The molecular analysis and immunohistochemistry were confirmed an increase in the expression of genes and proteins related to oocyte and follicular cells in these reconstructed structures. In conclusion, the recellularization of human ovarian scaffolds by mouse fetal ovarian cells could support the follicular-like structure formation and it provides an in vitro model for follicle reconstitution and offers an alternative approach for clinical usage.

Unraveling the Mystery of Multi-Oocyte Follicles: An Observational Study.

Introduction: Significant progress has been made in understanding oocyte fertilization and early developmental stages through in vitro fertilization (IVF) techniques. However, irregularities such as conjoined oocytes and binucleate giant oocytes, which are exceptions to the normal rule of one diploid female gamete per follicle, can potentially lead to chromosomal disorders in embryos and are recommended to be excluded from IVF attempts. The formation of primordial follicles during ovarian development, known as follicle assembly, is a critical process that establishes the ovarian follicle reserve. Multi-oocyte follicles (MOFs) containing two or more oocytes have been observed in various species, including humans, and their clinical significance on fertility and reproductive health remains unclear. Genetic and environmental factors, such as gene knockout and exposure to endocrine disruptors, have been implicated in MOF formation, but the mechanisms are not fully understood and require further investigation. Material & Method: In this Observational study, 350 slides of ovarian tissues were scanned using an AI-based automated microscope, Grundium Ocus 20, and the TIFF images were stored in cloud storage. The slides were examined using third-party software, Pathcore Seeden Viewer, for morphometry of binovular follicles. Results: In our observational study, we examined 350 ovarian tissue slides in detail by using an AI-based microscope, uncovering 22 slides from seven different tissues with binovular and multinovular oocytes. These rare multi-oocyte follicles (MOFs) challenge the conventional one-oocyte-per-follicle paradigm. MOFs are likely formed when oocytes fail to separate during cortical sex cord proliferation, regulated by factors several molecular factorsas well as environmental factors. Discussion: Multiple Ovarian Follicles (MOFs) are rare phenomena where two or more oocytes exist in one follicle. They arise when oocytes fail to separate during ovarian development, which is governed by hormones such as follicle-stimulating hormone (FSH), inhibin, BMP-15, GDF-9, and GCNF. MOFs can be caused by dysregulation and exposure to chemicals such as diethylstilbestrol (DES) and isoflavones. Binovular oocytes, which occur when two oocytes are released during ovulation and are fertilised by different sperm, can result in non-identical twins, which are influenced by genetic and environmen-tal factors such as maternal age, heredity, hormonal imbalances, and assisted reproductive techniques such as in vitro fertilisation (IVF). Polynuclear oocytes with many nuclei can develop as a result of meiotic spindle defects and environmental influences. Identifying these oocyte types may aid in improving ART results by improving knowledge of the reasons of infertility and devising appropriate interventions.