Histological analysis of (antral) follicle density in ovarian cortex tissue attached to stripped endometriomas.

PURPOSE: When resecting endometriomas with the stripping technique, in the majority of cases, a thin line of adjacent ovarian cortex is attached to the endometrioma. In this study, we performed histological analysis to determine (antral) follicle density in the ovarian cortex tissue attached to stripped endometriomas and assessed patient- and surgical characteristics that could affect this. METHODS: Histological slides of previously removed endometriomas were assessed. Follicles in the attached ovarian tissue were classified according to maturation, and follicular density was determined. Immunofluorescent staining of antral follicles in a subset of endometriomas was also performed. RESULTS: In 90 out of 96 included endometriomas (93.7%), ovarian tissue attached to the cyst wall was observed. One thousand nine hundred forty-four follicles at different maturation stages were identified (3 follicles/mm3). Follicle density was negatively associated with age (p < 0.001). Antral follicles (< 7-mm diameter) were present in the ovarian tissue attached to 35 endometriomas (36.5%) derived from younger patients compared to endometriomas where none were detected (30 versus 35 years, p = 0.003). Antral follicle density was 1 follicle/mm3. Based on immunofluorescence, healthy antral follicles were identified in two out of four examined endometriomas. CONCLUSIONS: Ovarian tissue attached to stripped endometriomas holds potential as a non-invasive source for antral follicles. In theory, application of IVM could be an interesting alternative FP option in young patients with endometriomas who undergo cystectomy in order to transform the surgical collateral damage to a potential oocyte source. Our results encourage future research with fresh tissue to further assess the quality and potential of these follicles. TRIAL REGISTRATION: Clinical Trials.gov Identifier: B21.055 (METC LDD), date of registration 12-08-2021, retrospectively registered.

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.

Basal characteristics of patients who responded to Ovarian Fragmentation for Follicular Activation (OFFA) or In Vitro Activation (IVA): a systematic review and meta-analysis.

A systematic review and meta-analysis were performed to identify if there is a subset of patients with POI who are more likely to show follicular growth after ovarian fragmentation for follicular activation (OFFA) or in vitro activation (IVA). Five studies met inclusion criteria for meta-analysis with a total of 164 patients. Forty-three patients showed follicle development (26.21%). Of those, the pregnancy rate was 35.58% (11/43) and the live birth rate was 20.93% (9/43). Our meta-analysis showed that age was not associated with follicle growth. However, lower baseline FSH, lower duration of amenorrhea/diagnosis, and presence of follicles remaining in biopsy were statistically significant for follicle development. Patients with basal characteristics mentioned before may have more chances to show follicle growth after OFFA or IVA. Taking into account that approximately 20% of patients with follicle growth had live birth, these results are very promising. Given the overall certainty of evidence, future studies are needed to confirm said results.

Beyond apoptosis: evidence of other regulated cell death pathways in the ovary throughout development and life.

BACKGROUND: Regulated cell death is a fundamental component of numerous physiological processes; spanning from organogenesis in utero, to normal cell turnover during adulthood, as well as the elimination of infected or damaged cells throughout life. Quality control through regulation of cell death pathways is particularly important in the germline, which is responsible for the generation of offspring. Women are born with their entire supply of germ cells, housed in functional units known as follicles. Follicles contain an oocyte, as well as specialized somatic granulosa cells essential for oocyte survival. Follicle loss-via regulated cell death-occurs throughout follicle development and life, and can be accelerated following exposure to various environmental and lifestyle factors. It is thought that the elimination of damaged follicles is necessary to ensure that only the best quality oocytes are available for reproduction. OBJECTIVE AND RATIONALE: Understanding the precise factors involved in triggering and executing follicle death is crucial to uncovering how follicle endowment is initially determined, as well as how follicle number is maintained throughout puberty, reproductive life, and ovarian ageing in women. Apoptosis is established as essential for ovarian homeostasis at all stages of development and life. However, involvement of other cell death pathways in the ovary is less established. This review aims to summarize the most recent literature on cell death regulators in the ovary, with a particular focus on non-apoptotic pathways and their functions throughout the discrete stages of ovarian development and reproductive life. SEARCH METHODS: Comprehensive literature searches were carried out using PubMed and Google Scholar for human, animal, and cellular studies published until August 2022 using the following search terms: oogenesis, follicle formation, follicle atresia, oocyte loss, oocyte apoptosis, regulated cell death in the ovary, non-apoptotic cell death in the ovary, premature ovarian insufficiency, primordial follicles, oocyte quality control, granulosa cell death, autophagy in the ovary, autophagy in oocytes, necroptosis in the ovary, necroptosis in oocytes, pyroptosis in the ovary, pyroptosis in oocytes, parthanatos in the ovary, and parthanatos in oocytes. OUTCOMES: Numerous regulated cell death pathways operate in mammalian cells, including apoptosis, autophagic cell death, necroptosis, and pyroptosis. However, our understanding of the distinct cell death mediators in each ovarian cell type and follicle class across the different stages of life remains the source of ongoing investigation. Here, we highlight recent evidence for the contribution of non-apoptotic pathways to ovarian development and function. In particular, we discuss the involvement of autophagy during follicle formation and the role of autophagic cell death, necroptosis, pyroptosis, and parthanatos during follicle atresia, particularly in response to physiological stressors (e.g. oxidative stress). WIDER IMPLICATIONS: Improved knowledge of the roles of each regulated cell death pathway in the ovary is vital for understanding ovarian development, as well as maintenance of ovarian function throughout the lifespan. This information is pertinent not only to our understanding of endocrine health, reproductive health, and fertility in women but also to enable identification of novel fertility preservation targets.

Distocia em tigre d'água-de-orelha-vermelha, Trachemys scripta elegans, wied (1839): relato de caso / Dystocia in red-eared slider, Trachemys scripta elegans, wied (1839): case report / Distocia en tortuga de orejas rojas, Trachemys scripta elegans, wied (1839): reporte de caso

Atualmente muitos répteis se tornaram animais de companhia e são mantidos como pet's exóticos. A espécie Trachemys scripta elegans, Wied (1839) é um animal exótico da América do Norte, sua identificação é realizada pelas marcas avermelhadas encontradas lateralmente a sua cabeça. Na rotina clínica as principais enfermidades que acometem os quelônios são as de origem reprodutiva, como a estase folicular e distocia. O objetivo deste trabalho foi relatar um caso recorrente de distocia em um tigre d'água fêmea, para isso, a anamnese, o histórico da paciente, e seus sinais clínicos, em conjunto com os exames complementares de imagem foram essenciais para se obter diagnóstico definitivo. O tratamento foi realizado com a indução medicamentosa utilizando borogluconato de cálcio, seguida da aplicação de ocitocina, esta trouxe resultados positivos para a eliminação dos ovos. Porém devido ao histórico do paciente, optou-se pela intervenção cirúrgica de ovariossalpingectomia, sendo está a maneira permanente de resolução da patologia. O protocolo terapêutico escolhido proporcionou um resultado satisfatório e bem estar ao animal.(AU)

Currently, many reptiles have become companion animals and are kept as exotic pets. The species Trachemys scripta elegans, Wied (1839) is an exotic animal from North America, and its identification is based on the reddish markings found laterally on its head. In routine clinical practice, the main diseases that affect chelonians are those of reproductive origin, such as follicular stasis and dystocia. The aim of this study was to report a recurrent case of dystocia in a female red-eared slider turtle. For this purpose, the patient's anamnesis, history, and clinical signs, along with complementary imaging exams, were essential to obtain a definitive diagnosis. The treatment involved medical induction using calcium borogluconate, followed by the administration of oxytocin, which yielded positive results in egg elimination. However, due to the patient's history, surgical intervention in the form of ovariosalpingectomy was chosen as the permanent solution to the pathology. The chosen therapeutic protocol provided a satisfactory outcome and improved the animal's well-being.(AU)

Actualmente muchos reptiles se han convertido en animales de compañía y se mantienen como mascotas exóticas. La especie Trachemys scripta elegans, Wied (1839) es un animal exótico de América del Norte, su identificación se realiza por las marcas rojizas que se encuentran lateralmente a su cabeza. En la rutina clínica, las principales enfermedades que afectan a los quelonios son las de origen reproductivo, como la estasis folicular y la distocia. El objetivo de este trabajo fue reportar un caso recurrente de distocia en una hembra de tigre de agua, para ello la anamnesis, la historia de la paciente y sus signos clínicos, junto con los exámenes imagenológicos complementarios fueron fundamentales para obtener un diagnóstico definitivo. El tratamiento se realizó con inducción farmacológica con borogluconato de calcio, seguido de la aplicación de oxitocina, que arrojó resultados positivos con la eliminación de huevos. Sin embargo, debido a los antecedentes de la paciente, se optó por la intervención quirúrgica de ovarialpingectomía, que es la forma definitiva de resolución de la patología. El protocolo terapéutico elegido proporcionó un resultado satisfactorio y bienestar al animal.(AU)

Development of the human ovary: Fetal through pubertal ovarian morphology, folliculogenesis and expression of cellular differentiation markers.

A definition of normal human fetal and early postnatal ovarian development is critical to the ability to accurately diagnose the presence or absence of functional ovarian tissue in clinical specimens. Through assembling an extensive histologic and immunohistochemical developmental ontogeny of human ovarian specimens from 8 weeks of gestation through 16 years of postnatal, we present a comprehensive immunohistochemical mapping of normal protein expression patterns in the early fetal through post-pubertal human ovary and detail a specific expression-based definition of the early stages of follicular development. Normal fetal and postnatal ovarian tissue is defined by the presence of follicular structures and characteristic immunohistochemical staining patterns, including granulosa cells expressing Forkhead Box Protein L2 (FOXL2). However, the current standard array of immunohistochemical markers poorly defines ovarian stromal tissue, and additional work is needed to identify new markers to advance our ability to accurately identify ovarian stromal components in gonadal specimens from patients with disorders of sexual differentiation.

Expression and characterisation of Fmr1 splice variants during folliculogenesis in the rat.

CONTEXT: The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5'UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17. AIMS: To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat. METHODS: We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages. KEY RESULTS: We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17. CONCLUSIONS: The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15. IMPLICATIONS: Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI.

A screen of repurposed drugs identifies AMHR2/MISR2 agonists as potential contraceptives.

We identified the anti-Mullerian hormone (also known as Müllerian inhibiting substance or MIS) as an inhibitory hormone that induces long-term contraception in mammals. The type II receptor to this hormone, AMHR2 (also known as MISR2), represents a promising druggable target for the modulation of female reproduction with a mechanism of action distinct from steroidal contraceptives. We designed an in vitro platform to screen and validate small molecules that can activate MISR2 signaling and suppress ovarian folliculogenesis. Using a bone morphogenesis protein (BMP)­response element luciferase reporter cell­based assay, we screened 5,440 compounds from a repurposed drug library. Positive hits in this screen were tested for specificity and potency in luciferase dose­response assays, and biological activity was tested in ex vivo Mullerian duct regression bioassays. Selected candidates were further evaluated in ex vivo follicle/ovary culture assays and in vivo in mice and rats. Here, we report that SP600125, CYC-116, gandotinib, and ruxolitinib can specifically inhibit primordial follicle activation and repress folliculogenesis by stimulating the MISR2 pathway.

Aged mice ovaries harbor stem cells and germ cell nests but fail to form follicles.

BACKGROUND: We recently published evidence to suggest that two populations of stem cells including very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) in ovary surface epithelium (OSE) undergo proliferation/differentiation, germ cell nests (GCN) formation, meiosis and eventually differentiate into oocytes that assemble as primordial follicles on regular basis during estrus cycle. Despite presence of stem cells, follicles get exhausted with advancing age in mice and result in senescence equivalent to menopause in women. Stem cells in aged ovaries can differentiate into oocytes upon transplantation into young ovaries, however, it is still not well understood why follicles get depleted with advancing age despite the presence of stem cells. The aim of the present study was to study stem cells and GCN in aged ovaries. METHODS: OSE cells from aged mice (> 18 months equivalent to > 55 years old women) were enzymatically separated and used to study stem cells. Viable (7-AAD negative) VSELs in the size range of 2-6 µm with a surface phenotype of Lin-CD45-Sca-1+ were enumerated by flow cytometry. Immuno-fluorescence and RT-PCR analysis were done to study stem/progenitor cells (OCT-4, MVH, SCP3) and transcripts specific for VSELs (Oct-4A, Sox-2, Nanog), primordial germ cells (Stella), germ cells (Oct-4, Mvh), early meiosis (Mlh1, Scp1) and ring canals (Tex14). RESULTS: Putative VSELs and OSCs were detected as darkly stained, spherical cells with high nucleo-cytoplasmic ratio along with germ cells nests (GCN) in Hematoxylin & Eosin stained OSE cells smears. Germ cells in GCN with distinct cytoplasmic continuity expressed OCT-4, MVH and SCP3. Transcripts specific for stem cells, early meiosis and ring canals were detected by RT-PCR studies. CONCLUSION: Rather than resulting as a consequence of accelerated loss of primordial follicle and their subsequent depletion, ovarian senescence/menopause occurs as a result of stem cells dysfunction. VSELs and OSCs exist along with increased numbers of GCNs arrested in pre-meiotic or early meiotic stage in aged ovaries and primordial follicle assembly is blocked possibly due to age-related changes in their microenvironment.

The Key Role of Peroxisomes in Follicular Growth, Oocyte Maturation, Ovulation, and Steroid Biosynthesis.

The absence of peroxisomes can cause disease in the human reproductive system, including the ovaries. The available peroxisomal gene-knockout female mouse models, which exhibit pathological changes in the ovary and reduced fertility, are listed in this review. Our review article provides the first systematic presentation of peroxisomal regulation and its possible functions in the ovary. Our immunofluorescence results reveal that peroxisomes are present in all cell types in the ovary; however, peroxisomes exhibit different numerical abundances and strong heterogeneity in their protein composition among distinct ovarian cell types. The peroxisomal compartment is strongly altered during follicular development and during oocyte maturation, which suggests that peroxisomes play protective roles in oocytes against oxidative stress and lipotoxicity during ovulation and in the survival of oocytes before conception. In addition, the peroxisomal compartment is involved in steroid synthesis, and peroxisomal dysfunction leads to disorder in the sexual hormone production process. However, an understanding of the cellular and molecular mechanisms underlying these physiological and pathological processes is lacking. To date, no effective treatment for peroxisome-related disease has been developed, and only supportive methods are available. Thus, further investigation is needed to resolve peroxisome deficiency in the ovary and eventually promote female fertility.

miR-450-5p and miR-202-5p Synergistically Regulate Follicle Development in Black Goat.

Follicle maturation is a complex biological process governed by numerous factors, and researchers have observed follicle development by studying the proliferation and apoptosis of follicular granulosa cells (GCs). However, the regulatory mechanisms of GCs proliferation and death during follicle development are largely unknown. To investigate the regulatory mechanisms of lncRNAs, mRNAs, and microRNAs, RNA sequencing (RNA-seq) and small RNA-seq were performed on large (>10 mm) and small follicles (<3 mm) of Leizhou black goat during estrus. We discovered two microRNAs, miR-450-5p and miR-202-5p, which can target GCs in goats and may be involved in follicle maturation, and the effects of miR-450-5p and miR-202-5p on ovarian granulosa cell lines were investigated (KGN). Using cell counting kit-8 (CCK-8) assays, 5-Ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry, miR-202-5p overexpression could suppress the proliferation and induce apoptosis of GCs, whereas miR-450-5p overexpression induced the opposite effects. The dual-luciferase reporter assay confirmed that miR-450-5p could directly target the BMF gene (a BCL2 modifying factor), and miR-202-5p targeted the BCL2 gene. A considerable rise in phosphorylated Akt (p-AKT) protein was observed following the downregulation of BMF by miR-450-5p mimics. After BMF gene RNAi therapy, a notable elevation in p-AKT was detected. Mimics of miR-202-5p inhibited BCL2 protein expression, significantly decreasing p-AMPK protein expression. These results imply that during the follicular development in black goats, the miR-450-5p-BMF axis favored GC proliferation on a wide scale, while the miR-202-5p-BCL2 axis triggered GC apoptosis.

Molecular characterization, expression profile and transcriptional regulation of the CYP19 gene in goose ovarian follicles.

Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5'-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925 bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.

Evaluation of the Effects of Human Bone Marrow Mesenchymal Stem Cells Conditioned Medium on Growth and Maturation of Mouse Ovarian Follicle after Vitrification.

The aim of this research study is to evaluate the effect of human bone marrow mesenchymal stem cells conditioned medium (hBMSCs-CM) on growth and maturation of mouse ovarian follicle, and embryonic development after vitrification. The hBMSCs were cultured, and the derived CM was collected, concentrated, and stored. 14-day-old mice ovaries were collected and randomly divided into vitrified and non-vitrified groups. Then their isolated preantral follicles were cultured for 12 days in α-MEM supplemented with different concentrations of CM (2.5, 5, and 7.5%). Finally, the growth and diameter of follicles, maturation of oocytes, hormone level, and embryo developmental rate were assessed. The results showed the antrum formation, oocyte maturation, and hormone secretion were significantly higher in the presence of 7.5% CM (p < 0.001). In the vitrified group, the developmental rate of follicles was lower than the non-vitrified group, and the subgroup containing 7.5% CM showed better results than the 5%, and 2.5% CM subgroups. However, no changes in fertilization and embryonic development rates were observed. Supplementing follicle culture media with 7.5% CM could enhance follicle growth and oocyte maturation of follicles after vitrification.

Overexpression of Lin28a induces a primary ovarian insufficiency phenotype via facilitation of primordial follicle activation in mice.

Lin28a is an RNA binding protein and increasing evidence has indicated its role in regulating female fertility. Lin28a has been reported to be involved in ovarian follicle activation. However, its role and mechanisms in regulating primordial follicle activation have not yet been explored. To test whether overexpression of Lin28a activates ovarian primordial follicles, studies were conducted in wild type (WT) and Lin28a Tg mice. Female Lin28a Tg mice at 4-month old exhibited significantly smaller litter size and fewer ovulated oocytes when compared with the WT mice. By 6-month of age, these parameters in Lin28a Tg mice were less than 20% of the WT mice. At postnatal day (PD) 14, the number of primordial follicles was significantly decreased but the number of primary follicles was significantly increased in the transgenic mice. The number of primordial follicles, secondary and antral follicles in these mice were drastically reduced at PD21. In the ovary of Lin28a Tg mice, there were activation of Wnt/ß-catenin signaling and its downstream mTOR pathway. Interestingly, overexpression of Lin28a, which can also act as transcriptional activator, activated Wnt signaling through enhancing the transcription of Wnt co-receptor LRP5. In conclusion, overexpression of Lin28a induced a primary ovarian insufficiency phenotype in long term via facilitating Wnt/ß-catenin signaling leading to activation of primordial follicles.

[Histone methylation in mammalian follicular development].

The normal development of follicles involves a series of complex life processes such as ordered transcriptional activation and inhibition, which is crucial for female reproductive ability. Histone methylation can change the chromatin state in cells and affect the transcription activity of genes. Current studies indicate that epigenetic modifications such as histone methylation play an important regulatory role in follicular development in female mammals. This paper summarized the relationship between H3K4, H3K9 methylation and germ cell development, their regulatory effects, including their dynamical changes during follicular development, and the progress of H3K4me3 and other histone methylation binding to promoter regions of different genes to regulate gene expression and thus affect germ cell epigenetic reprogramming, oocyte transcription, meiosis and other processes. This review will provide a reference for the study of mechanisms related to histone methylation modification and the development and maturation of gonadal parenchymal cells.

Large-scale DNA demethylation occurs in proliferating ovarian granulosa cells during mouse follicular development.

During ovarian follicular development, granulosa cells proliferate and progressively differentiate to support oocyte maturation and ovulation. To determine the underlying links between proliferation and differentiation in granulosa cells, we determined changes in 1) the expression of genes regulating DNA methylation and 2) DNA methylation patterns, histone acetylation levels and genomic DNA structure. In response to equine chorionic gonadotropin (eCG), granulosa cell proliferation increased, DNA methyltransferase (DNMT1) significantly decreased and Tet methylcytosine dioxygenase 2 (TET2) significantly increased in S-phase granulosa cells. Comprehensive MeDIP-seq analyses documented that eCG treatment decreased methylation of promoter regions in approximately 40% of the genes in granulosa cells. The expression of specific demethylated genes was significantly increased in association with specific histone modifications and changes in DNA structure. These epigenetic processes were suppressed by a cell cycle inhibitor. Based on these results, we propose that the timing of sequential epigenetic events is essential for progressive, stepwise changes in granulosa cell differentiation.

Transcriptome analysis reveals that long noncoding RNAs contribute to developmental differences between medium-sized ovarian follicles of Meishan and Duroc sows.

Ovulation rate is an extremely important factor affecting litter size in sows. It differs greatly among pig breeds with different genetic backgrounds. Long non-coding RNAs (lncRNAs) can regulate follicle development, granulosa cell growth, and hormone secretion, which in turn can affect sow litter size. In this study, we identified 3554 lncRNAs and 25,491 mRNAs in M2 follicles of Meishan and Duroc sows. The lncRNA sequence and open reading frame lengths were shorter than mRNAs, and lncRNAs had fewer exons, were less abundant, and more conserved than protein-coding RNAs. Furthermore, 201 lncRNAs were differentially expressed (DE) between breeds, and quantitative trait loci analysis of DE lncRNAs were performed. A total of 127 DE lncRNAs were identified in 119 reproduction trait-related loci. In addition, the potential target genes of lncRNAs in cis or trans configurations were predicted. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that some potential target genes were involved in follicular development and hormone secretion-related biological processes or pathways, such as progesterone biosynthetic process, estrogen metabolic process, ovarian steroidogenesis, and PI3K-Akt signaling pathway. Furthermore, we also screened 19 differentially expressed lncRNAs in the PI3K-Akt signaling pathway as candidates. This study provides new insights into the roles of lncRNAs in follicular growth and development in pigs.

Role of Stem Cells in the Ovarian Tissue Cryopreservation and Transplantation for Fertility Preservation.

Although the cancer survival rate has increased, cancer treatments, including chemotherapy and radiotherapy, can cause ovarian failure and infertility in women of reproductive age. Preserving fertility throughout cancer treatment is critical for maintaining quality of life. Fertility experts should propose individualized fertility preservation methods based on the patient's marital status, pubertal status, partner status, and the urgency of treatment. Widely practiced fertility preservation methods, including ovarian transposition and embryo and oocyte cryopreservation, are inappropriate for prepubertal girls or those needing urgent initiation of cancer treatment. Ovarian tissue cryopreservation and transplantation, an emerging new technology, may be a solution for these cancer patients. The use of stem cells in ovarian tissue cryopreservation and transplantation increases oxygenation, angiogenesis, and follicle survival rates. This review discusses the recent advances in ovarian tissue cryopreservation and transplantation with special focus on the use of stem cells to improve fertilization techniques.

Impact of the antioxidant quercetin on morphological integrity and follicular development in the in vitro culture of Bos indicus female ovarian fragments.

We evaluated the effect of quercetin on the in vitro culture of bovine ovarian fragments in relation to morphology, development, and oxidative stress. Ovaries (n = 12) from Nelore heifers (n = 6) were used. Each pair of ovaries was divided into nine fragments, and one fragment from each animal was fixed in Bouin solution for 24 h (histology control) or frozen (- 80°C; control for oxidative stress). Other ovarian fragments (n = 8) were distributed into concentrations of 0, 10, 25, and 50 µg/mL of quercetin added to the culture medium for 5 or 10 d. Data were analyzed by chi-square test or ANOVA followed by Tukey's test (P < 0.05). Treatment with 25 µg/mL quercetin resulted in the highest proportion of total intact follicles for 5 (67.3%) and 10 d (57.1%); the concentration of 25 µg/mL also presented the best proportion of developing follicles for 5 d (68.7%) and 10 d (62.8%). Treatment with 25 µg/mL quercetin resulted in significant ferric reduction for 10 d of culture, but not for 5 d. No difference (P > 0.1) was observed in the production of reactive oxygen species or in the oxidative degradation of lipids between treatments and non-cultivated controls. Treatment with 25 µg/mL quercetin preserved the morphological integrity of the developing follicles for 5 and 10 d of culture, in addition to promoting the best antioxidant potential after 10 d of culture in bovine ovarian fragments.

Metabolic activity in cryopreserved and grafted ovarian tissue using high-resolution respirometry.

Cryopreservation of ovarian tissue followed by transplantation represents a strategy to restore ovarian function and fertility. Stress from cryopreservation-thawing processes can lead to alterations and/or damage to mitochondrial structure and functionality. High resolution respirometry and histological analysis were used to evaluate the effect of cryopreservation and transplantation on ovarian tissue. Four different conditions were performed: Fresh non-transplanted tissue, Fresh transplanted tissue, Cryopreserved non-transplanted tissue and Cryopreserved transplanted tissue. All groups were able to respond to the substrates-uncoupler-inhibitor protocol. We found a dramatic decrease in general oxygen consumption in hemi-ovaries submitted to cryopreservation and/or transplantation. The effect of cryopreservation on mitochondrial metabolism was less intense than effect of transplantation, since the transplantation affected all of the mitochondrial states. A total of 2644 follicles were analyzed. Of these, 2198 were classified as morphologically normal. The percentage of morphologically normal follicles was significantly lower in the Cryopreserved transplanted group when compared to the Cryopreserved non-transplanted group and the Fresh transplanted group (p-value < 0.05). Despite decreased follicular viability and mitochondrial activity, the cryopreservation followed by transplantation of ovarian tissue proved feasible for attempts to restore ovarian function.