Oocytes Research Articles

O-244 Mitochondrial fission factor (MFF) is required for female fertility and oocyte development

Abstract Study question To explore the role of Mitochondrial fission factor (MFF) in female fertility and oocyte development using a Mff knockout mouse model. Summary answer Mff deficiency influences mitochondrial dynamics in oocytes at germinal vesicle stage (GV), and results in premature ovarian failure (POF) and female infertility. What is known already Mff regulates mitochondrial quality control (MQC) by influencing mitochondrial dynamics. MQC has critical effects on oocyte development and POF. However, little is known about the role of Mff in oocyte development and female fertility. Study design, size, duration The Mff knockout (Mff-/-) mice were generated via the CRISPR-Cas9 system. Adult Mff-/- female mice were compared to wildtype (WT) female mice. Female mice at 2-month old from each group (Mff-/- or WT, n = 5) were used to assess the fecundity for 10 months. The follicle development (n = 4 mice), the number of GV oocytes (n = 5 mice) and metaphase II (MII) oocytes (n = 5 mice) were determined. Participants/materials, setting, methods Female mice from each group were mated with adult WT males to assess the fecundity. Follicle development was evaluated in ovaries after fixation, paraffin embedding, and sectioning, followed by hematoxylin and eosin staining. MFF and DRP1 protein expression was evaluated by western blot. Oocyte mitochondria were stained with the Mito-Tracker Red CMXRos. Main results and the role of chance The Mff deficient ovaries had significantly lower levels of MFF (0.056 vs. 1, p < 0.01) than the WT ovaries. Mff-/- female mice were infertile (0 vs. 39, p < 0.05). Mature (8-week-old) Mff deficient ovaries had significantly lower weight (0.0043 vs. 0.011, p < 0.05) compared to the WT. The 2-month-old female Mff-/- mice had a lower number of primordial (220 vs. 775, p< 0.05), primary (128 vs. 580, p< 0.05), secondary (29 vs. 82, p< 0.05) and antral follicles (5 vs. 23, p< 0.05). The numbers of GV oocytes (18 vs. 37, p< 0.05) and MII oocytes (2 vs. 16, p< 0.001) were lower in the Mff-/- mice than the WT mice. In addition, the Mff-/- ovaries expressed significantly less mitochondrial fission key regulator DRP1 (0.54 vs. 1, p< 0.05). Mff-/- oocytes contained elongated and aggregated mitochondria compared to the discrete and evenly distributed mitochondria in the ooplasm of WT oocytes. Herein, we found that the Mff played a concerted role in oocyte and follicle development. Limitations, reasons for caution The present observations in mice may not be applicable to humans. The results are derived global knockout of Mff. It is not known which kind of cell plays a vital role in fertility. Further investigation is needed to explore the regulatory mechanism of Mff in oocyte development and fertility regulation. Wider implications of the findings This study provides important insights into the relationship between mitochondrial dynamics and female fertility. Trial registration number not applicable

TRPS1: A Marker of Follicular Differentiation.

The trichorhinophalangeal syndrome type 1 (TRPS1) immunohistochemical (IHC) stain has increased in use in recent years as a marker for breast carcinomas. The TRPS1 gene is involved in various tissues, including the growth and differentiation of hair follicles. This article seeks to evaluate the IHC expression of TRPS1 in cutaneous neoplasms with follicular differentiation, such as trichoblastoma (TB), trichoepithelioma (TE), and basal cell carcinoma (BCC). IHC studies were performed on 13 TBs, 15 TEs, and 15 BCCs with an antibody against TRPS1. The study found a variable staining expression of TRPS1 in the tumor nests of TB, TE, and BCC. BCCs were distinct in that none of the BCCs demonstrated intermediate or high positivity, while TBs and TEs showed intermediate-to-high positivity in 5/13 (38%) and 3/15 (20%) of cases, respectively. We observed a distinct staining pattern among the mesenchymal cells of TB and TE. We found that TRPS1 highlighted perifollicular mesenchymal cells adjacent to the nests of TB and TE tumor cells. This staining pattern was absent in BCCs, where only scattered stromal cells were positive for TRPS1. Papillary mesenchymal bodies were also highlighted by TRPS1 in TB and TE. TRPS1 stained various parts of the normal hair follicle, including the nuclei of cells in the germinal matrix, outer root sheaths, and hair papillae. TRPS1 may be a useful IHC marker for follicular differentiation.

AMELOBLASTOMA IN A 2-YEAR-OLD BOY: A CASE REPORT

A 2-year-old boy presented a normochromic swelling on the left cheek for an unknown period. Panoramic radiography revealed a well-circumscribed, unilocular, radiolucent area extending from body to angle and ramus of mandible, measuring around 4 cm. The exploratory puncture was negative, and an incisional biopsy was performed. In the histopathology, the sections revealed neoplastic proliferation of sheets and strings of odontogenic cells, in a solid arrangement. Epithelial cells had a hyperstained nucleus with a basaloid pattern. Peripheral cells occasionally showed reverse polarization and nuclei arranged in a palisade, and central cells were loosely arranged like the stellate reticulum of the enamel organ. The neoplasm is externally lined by a thin fibrous connective tissue capsule, and the presence of tumor cell nests was observed within the capsule. There were significant hypercellularity and multiple mitotic figures throughout the lesion. The patient was sent to treatment. A 2-year-old boy presented a normochromic swelling on the left cheek for an unknown period. Panoramic radiography revealed a well-circumscribed, unilocular, radiolucent area extending from body to angle and ramus of mandible, measuring around 4 cm. The exploratory puncture was negative, and an incisional biopsy was performed. In the histopathology, the sections revealed neoplastic proliferation of sheets and strings of odontogenic cells, in a solid arrangement. Epithelial cells had a hyperstained nucleus with a basaloid pattern. Peripheral cells occasionally showed reverse polarization and nuclei arranged in a palisade, and central cells were loosely arranged like the stellate reticulum of the enamel organ. The neoplasm is externally lined by a thin fibrous connective tissue capsule, and the presence of tumor cell nests was observed within the capsule. There were significant hypercellularity and multiple mitotic figures throughout the lesion. The patient was sent to treatment.

PATL2 regulates mRNA homeostasis in oocytes by interacting with EIF4E and CPEB1.

The accumulation and storage of maternal mRNA is crucial for oocyte maturation and embryonic development. PATL2 is an oocyte-specific RNA-binding protein, and previous studies have confirmed that PATL2 mutation in humans and knockout mice cause oocyte maturation arrest or embryonic development arrest, respectively. However, the physiological function of PATL2 in the process of oocyte maturation and embryonic development is largely unknown. Here, we report that PATL2 is highly expressed in growing oocytes and couples with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The germinal vesicle oocytes from Patl2-/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed that PATL2 phosphorylation occurs in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found that the S279D mutation decreased the protein level of PATL2 and led to subfertility in Palt2S279D knock-in mice. Our work reveals the previously unrecognized role of PATL2 in regulating the maternal transcriptome and shows that phosphorylation of PATL2 leads to the regulation of PATL2 protein levels via ubiquitin-mediated proteasomal degradation in oocytes.

PARP12 regulates mouse oocyte meiotic maturation.

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification of proteins that involves the transfer of ADP-ribose moieties, and plays important roles in many biological processes including DNA repair, gene expression, RNA processing, ribosome biogenesis, and protein translation. Though it is accepted that PARylation is crucial for oocyte maturation, little is known about how Mono(ADP-ribosyl)ation (MARylation) regulates this process. Here, we report that Parp12,a mon(ADP-ribosyl) transferase of poly(ADP-ribosyl) polymerase (PARP) family, was highly expressed at all stages of oocytes during meiotic maturation. At germinal vesicle (GV) stage, PARP12 was mainly distributed in cytoplasm. Interestingly, PARP12 formed granular aggregation near to spindle poles during metaphase I (MI) and metaphase II (MII). PARP12 depletion results in abnormal spindle organization and chromosome misalignment in mouse oocytes. Chromosome aneuploidy frequency in PARP12 knockdown oocytes was significantly increased. Importantly, PARP12 knockdown triggers activation of spindle assembly checkpoint as shown by active BUBR1 in PARP12-KD MI oocytes. Besides, F actin was significantly attenuated in PARP12-KD MI oocytes which may affect the asymmetric division process. Transcriptomic analysis demonstrated that PARP12 depletion disrupts transcriptome homeostasis. Collectively, our results showed that the maternally expressed mono(ADPribosyl) transferases PARP12 was essential for oocyte meiotic maturation in mouse.

Toward a Bio-Organon: A model of interdependence between energy, information and knowledge in living systems

What is an organism? In the absence of a fundamental biological definition, what constitutes a living organism, whether it is a unicellular microbe, a multicellular being or a multi-organismal society, remains an open question. New models of living systems are needed to address the scale of this question, with implications for the relationship between humanity and planetary ecology. Here we develop a generic model of an organism that can be applied across multiple scales and through major evolutionary transitions to form a toolkit, or bio-organon, for theoretical studies of planetary-wide physiology. The tool identifies the following core organismic principles that cut across spatial scale: (1) evolvability through self-knowledge, (2) entanglement between energy and information, and (3) extrasomatic “technology” to scaffold increases in spatial scale. Living systems are generally defined by their ability to self-sustain against entropic forces of degradation. Life “knows” how to survive from the inside, not from its genetic code alone, but by utilizing this code through dynamically embodied and functionally specialized flows of information and energy. That is, entangled metabolic and communication networks bring encoded knowledge to life in order to sustain life. However, knowledge is itself evolved and is evolving. The functional coupling between knowledge, energy and information has ancient origins, enabling the original, cellular “biotechnology,” and cumulative evolutionary creativity in biochemical products and forms. Cellular biotechnology also enabled the nesting of specialized cells into multicellular organisms. This nested organismal hierarchy can be extended further, suggesting that an organism of organisms, or a human “superorganism,” is not only possible, but in keeping with evolutionary trends.

Yolk granule fusion and microtubule aster formation regulate cortical granule translocation and exocytosis in zebrafish oocytes.

Dynamic reorganization of the cytoplasm is key to many core cellular processes, such as cell division, cell migration, and cell polarization. Cytoskeletal rearrangements are thought to constitute the main drivers of cytoplasmic flows and reorganization. In contrast, remarkably little is known about how dynamic changes in size and shape of cell organelles affect cytoplasmic organization. Here, we show that within the maturing zebrafish oocyte, the surface localization of exocytosis-competent cortical granules (Cgs) upon germinal vesicle breakdown (GVBD) is achieved by the combined activities of yolk granule (Yg) fusion and microtubule aster formation and translocation. We find that Cgs are moved towards the oocyte surface through radially outward cytoplasmic flows induced by Ygs fusing and compacting towards the oocyte center in response to GVBD. We further show that vesicles decorated with the small Rab GTPase Rab11, a master regulator of vesicular trafficking and exocytosis, accumulate together with Cgs at the oocyte surface. This accumulation is achieved by Rab11-positive vesicles being transported by acentrosomal microtubule asters, the formation of which is induced by the release of CyclinB/Cdk1 upon GVBD, and which display a net movement towards the oocyte surface by preferentially binding to the oocyte actin cortex. We finally demonstrate that the decoration of Cgs by Rab11 at the oocyte surface is needed for Cg exocytosis and subsequent chorion elevation, a process central in egg activation. Collectively, these findings unravel a yet unrecognized role of organelle fusion, functioning together with cytoskeletal rearrangements, in orchestrating cytoplasmic organization during oocyte maturation.

Molecular effect of holding equine oocytes: a road to cytoplasmic maturation?

The quality of oocytes has a direct effect on the outcome of in vitro embryo production (IVP). For successful IVP, oocytes need to undergo maturation, which requires coordinated nuclear (NM) and cytoplasmic maturation (CM). The lack of adequate cytoplasmic maturation has been proposed to be associated with a lower developmental competence of the oocyte. The improvement of in vitro techniques relies upon mirroring in vivo conditions. Therefore, acquiring a deeper knowledge of the process involved in CM and pathways leading to germinal vesicle breakdown and maturation can provide the necessary foundation to improve IVP results. Holding oocytes in commercial embryo holding media for 24hrs at room temperature has become a routine procedure for oocyte shipment and laboratory scheduling. However, the effects of this procedure on oocyte CM remain unresolved. In this study, we analyzed the changes induced by the holding period on the transcriptome of equine oocytes (OC) and the surrounding cumulus cells (CC). Cumulus-oocyte-complexes (COCs) were collected from six mares using transvaginal aspiration. Only non-expanded, non-degenerated COCs were randomly allocated to one of two groups: no holding (NH) or 24hrs holding (H). The COCs were denuded, and each single OC and CC were stored individually in RNAlater® at -20°C until further processing. RNA was extracted from 24 samples (NH/CC=6; NH/OC=6; H/CC=6; H/OC=6), followed by librarypreparation and sequencing (150 pb-PE). Reads were trimmed and mapped to the equine reference genome (Ecab 3.0). Mapped reads were quantified using featureCount and analysis of differentially expressed genes (DEGs) was performed using DESeq2 (FDR< 0.1 and log fold change >2). Overall, there were 26,077 genes expressed in CCs and 17,930 in OCs. When compared by holding time, there were 660 DEGs between the CCs (NH vs. H) and 205 between the OCs. In CC, 74 genes had a higher expression and 586 had a lower expression in H than NH groups. In OC, 7 genes were upregulated, and 198 were downregulated in H compared to NH group. Gene ontology analysis showed changes in genes related to glutathione (GSH) synthesis, AP-1 transcription factor complex and mitogen-activated protein kinase (MAPK) pathway in CC. In OC, DEGs were involved in α-tubulin detyrosination and tight junction proteins involved in magnesium transport. Overall, our results suggest modification in processes related to CM, including post-translational changes in tubulin, GSH composition, and activation of MAPK pathways during oocyte holding. Meanwhile, genes considered markers of NM and meiotic resumption remained unchanged, suggesting pre-maturation like changes occur prior to germinal vesicle breakdown. Further studies to understand the implications of this pre-maturation step in the developmental competence of oocytes can help improve the outcome of equine IVP. Funding: Center for Equine Health

Gene expression by equine oocytes and cumulus cells, a window into the unknown

In vitro maturation (IVM) of oocytes (OC) is a critical step for the in vitro production of equine embryos. However, the regulatory mechanisms, biological processes, cellular components, and molecular functions involved in IVM are not entirely understood. We previously analyzed the gene expression dynamics of gonadotropin receptors in cumulus cells (CC) during IVM, demonstrating an upregulation of the follicle-stimulating hormone receptor gene (FSHR) and downregulation of the luteinizing hormone choriogonadotropin receptor gene (LHCGR) in mature compared to immature CC (de la Fuente.et.al. Journal of Equine Veterinary Science 2022; 113). Moreover, we found that CC from in vivo maturing cumulus-oocyte complexes (COC) obtained from dominant gonadotropin-stimulated follicles (DF-CC) had a lower expression of FSHR and a higher expression of LHCGR compared to their in vitro counterparts. However, the effect of IVM medium composition on the expression of gonadotropin receptors has not been investigated. Thus, the aim of this study was to determine differences in the gene expression of OC and CC following IVM of three reported IVM media: A) EQ-IVM (IVF-Bioscience) a commercially available medium, B) DMEM/F12-based medium (Meyers.et.al. Reproduction, Fertility and Development. 2019; 31:1874-1884.) and C) M199-based medium (Choi.et.al. Theriogenology. 2002; 58:771-774) and their difference with DF-CC samples. Extracted total RNA from OC and CC was sequenced and raw reads were trimmed (trimmgalore) and mapped (STAR) to the current equine referencegenome. Mapped reads were quantified (featurecount) and compared between groups, using the DESeq2 package (FDR cutoff 0.1; Min (log2) fold change 1.5). Overall, there was a mapping rate of >85%, and an average of 14,000 expressed genes in OC and CC samples with ∼68% categorized as protein-coding genes. Based on gene expression dis-similarities, OC and CC samples were partially clustered according to their respective maturation media, but all samples showed a different gene expression profile compared to DF-CC samples. Comparing the gene expression of CC between the A and B media, we found 607 differentially expressed genes (DEGs), 1905 DEGs between A and C, and 957 DEGs between B and C. All three media showed a similar DEGs number when compared to DF-CC samples (∼10K DEGs). FSHR showed a higher expression in all IVM samples compared to DF-CC. However, LHCGR presented a lower expression only in two of the IVM media used (B & C) compared to DF-CC, showing no significant expression difference in the commercial IVM media tested. These data highlight the dynamics of gonadotropin receptors during IVM, which may contribute to the understanding of the physiology of oocyte maturation and allow optimization of IVM conditions in horses. Funding: The Theriogenology Foundation, the Population Health and Reproduction Department Seed Grant, and the Center for Equine Health.

Specific processing of meiosis-related transcript is linked to final maturation in human oocytes.

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.

Blastocyst production using a new equine FSH for in vitro maturation

Currently, the majority of the equine ART laboratories collect germinal vesicle (GV)-stage oocytes to perform ICSI, and the gonadotrophins used for in vitro maturation are mainly porcine, bovine, or human. In this study, the effect of two different concentrations of equine FSH (eFSH) in the maturation medium was evaluated. Cumulus-oocytes complexes (COCs) were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to the groups. The control group was our standard maturation medium (BMM - TCM199-Earle's salts, 10% FBS, and additives) with human gonadotrophins added (Lazzari.et.al. Journal of Equine Veterinary Science. 2020; 89:103097); experimental groups were composed of BMM with eFSH added (0.01 and 0.05 IU/mL). After holding, COCs were transferred to maturation medium and cultured for 36h. ICSI was performed, and presumptive zygotes were cultured. The cleavage rate and number of cells were evaluated on D3, blastocyst formation and embryo quality, classified on A-B-C grade scale (adapted from Lewis.et.al. Reproduction, Fertility and Development. 2023; 35: 338-351), were evaluated from D6-10. Differences were analyzed by Fisher's Exact Test (significance level P<0.05). There was no difference (P>0.05) in degenerated, GV-stage, or MI oocytes after maturation for control, 0.01, and 0.05, respectively. The number of oocytes that reached the (meiosis) MII-stage was lower (P<0.05) for 0.01 U/mL (56.5%, 35/62)than the control (73.2%, 52/71), and also tended to be lower (P=0.0985) compared to 0.05 U/mL (71.2%, 47/66). There was no difference in cleavage or cell number among groups (Table 1). Group 0.01 U/mL had a higher (P<0.05) blastocyst rate (45.7%, 16/35) than control and 0.05 U/mL groups (28.8%,15/52; 25.5%, 12/47; respectively). No difference was found in embryo quality among groups. Although the 0.01 U/mL group showed a lower MII rate, it also showed numerically less 2-cell stage embryos, more 4-8 and 8-16 cells stage embryos, and more cleaved embryos. Additionally, the 0.01 U/mL group tended to have more high-quality embryos (grade A) than the control group and showed a higher total blastocyst rate compared to the other groups. Additional studies are required to establish an ideal maturation protocol using this new equine FSH.

Effect of pre-maturation oocyte timing on equine ICSI embryo development rates

Within the last 5 years, client adoption of ovum-pick-up (OPU) paired with equine intracytoplasmic sperm injection (ICSI) and in vitro embryo production (IVP) in Brazil has been rapid. Equine ICSI has facilitated the use of expensive semen be it imported, rare, poor-quality or low motility for IVP. A pre-maturation step occurs between OPU on the farm and transport to the lab. Oocytes are kept in 5 ml vials of pre-maturation medium, held at 22°C until placement in maturation medium. Oocytes in pre-maturation medium are kept at this low temperature to inhibit the onset of germinal vesicle breakdown and the onset of oocyte maturation. If maturation begins during transport, oocyte maturation rates and oocyte quality would be severely reduced. Recently, (Merlo, B et al 2022 Anim Reprod Sci 107071) reported different rates of equine ICSI embryo development based on timing of oocyte pre-maturation and maturation. In our IVP system, oocyte maturation time is tightly controlled between 28 and 32 hours, so we decided to evaluatethe timing of pre-maturation since it can vary between 0 and 24 hours. This variation is based on the time required for OPU completion by multiple teams, aspirating on the same day in different locations. We tested the potential impact of oocyte pre-maturation in 3-time intervals 0-8, 9-16 and 17-24 hours, in the 2021-2022 breeding season. The average blastocyst formation rate based on total oocytes collected was 17%. Embryo development from oocytes aspirated in the 3 pre-maturation time intervals was: 0-8 hours, 522 embryos produced from 3413 oocytes aspirated (15.3%); 9-16 hours, 471 embryos produced from 2667 oocytes aspirated (17.7%) and 17-24 hours, 3931 embryos produced from 22,896 oocytes aspirated (17.2%). There were no significant differences observed (Mann-Whitney test p >0.05), demonstrating that up to 24 hours, the holding time of pre-maturation oocytes, held at 22.5°C had no significant impact on embryo development in our equine ICSI embryo production system.

Expression pattern and subcellular localization of p62/SQSTM 1 during mouse oocyte maturation

Objective: Autophagy is a survival mechanism, and it is initiated by several factors such as oxidative stress. Cells give autophagy response against oxidative stress through Nrf2-Keap1-p62 pathway. p62 or Sequestosome 1 (SQSTM 1) which takes place during autophagy, and it is showed that p62 plays critical role during primordial follicle formation. During oocyte maturation, Germinal Vesicle (GV) oocytes resume meiosis with the beginning of puberty and progress through Metaphase I and Metaphase II stages. Metaphase II (MII) oocytes are ready to be fertilized and until fertilization they are arrested at metaphase II stages. It is known that oxidative stress is increasing during oocyte maturation. However, it is not shown that spatial and temporal expression of p62 throughout oocyte maturation. Thus, the aim of the study was to reveal expression pattern and the subcellular localization of the p62 protein in mouse GV, MI and MII oocytes. Methods: GV oocytes were received from female Balb/C mice at 4 weeks aged and GV oocytes were cultured for 8h to develop into MI oocytes and for 14h to mature into MII oocytes. Then, expression of p62 protein was observed in oocytes at GV, MI and MII maturation stages by using immunofluorescence method. Results: Our results showed that there is a significant increasing p62 expression when GV oocytes are compared to MI and MII oocytes, but there is no difference between MI and MII oocytes. Moreover, we revealed that p62 is localized in cytoplasm of GV oocytes, while it is localized around chromosomes in MI and MII oocytes. Conclusion: In conclusion, our results indicate that further study is mandatory to understand the participation of p62 during meiosis, and the impact of p62 during oocyte maturation.

Two Rare Cases of Paratubal Leydig Cell Nodules: Clinical Relevance and Debated Terminology of a Noteworthy Incidentaloma

Cells with cytologic and immunohistochemical features of Leydig cells are normally present in the ovary and the ovarian hilum, are testosterone-producing, and have been referred to as ovarian hilus cells. Rarely these cells form nests or nodules in extraovarian sites such as the mesovarium or mesosalpinx. Because they are so rare, these nodules can present a diagnostic challenge when first encountered. This report describes 2 such incidental nodules in the mesosalpinx associated with a small paratubal cyst and suggests that the term Leydig cell nodule be preferred over the nonspecific and confusing historical term ovarian hilus cell nest.

A rapidly-growing erythematous nodule on the right upper neck

A rapidly-growing erythematous nodule on the right upper neck

The developmental potential of mature oocytes derived from rescue in vitro maturation

To examine the developmental competence of immature oocytes in stimulated cycles, that matured after rescue invitro maturation (IVM) compared with their sibling invivo matured oocytes. Retrospective cohort study. IVF clinic. A total of 182 patients underwent 200 controlled ovarian stimulation cycles with intracytoplasmic sperm injection cycles in which immature oocytes were retrieved and at least one mature oocyte was obtained through rescue IVM. Invitro culture of immature germinal vesicle (GV) and metaphase I (MI) oocytes, retrieved in stimulated cycles. Fertilization rate, cleavage rate, blastulation rate, ploidy of embryos evaluated using preimplantation genetic testing for aneuploidy, morphokinetic parameters and pregnancy outcomes. In total, 2,288 oocytes were retrieved from 200 cycles. After denudation, 1,056 of the oocytes (46% ± 16%) were classified as metaphase II (MII). A total of 333/375 (89%) of MI oocytes and 292/540 (54%) of GV oocytes matured overnight and underwent intracytoplasmic sperm injection. The fertilization rates of matured oocytes from MI rescue IVM (R-MI) and from GV rescue IVM (R-GV) were comparable with those of their sibling MII oocytes (71% vs. 66%; 66% vs. 63%, respectively). Early cleavage rates (80% ± 35% vs. 92% ± 20%; 80% ± 42% vs. 95% ± 28%, respectively) and blastulation rates (32 ± 40% vs. 62 ± 33%; 24 ± 37% vs. 60 ± 35%, respectively) were significantly decreased in rescue IVM matured oocytes (R-oocytes)-derived zygotes, but the blastocyst (BL) euploidy rate and "good quality" BL rate were comparable with those of MII sibling-derived embryos. In addition, rescue IVM embryos showed significantly higher levels of multinucleation at the 2- and 4-cell stages, as well as higher rates of zygote direct cleavage from one to 3 to 4 cells. Overall, 21 transfers of rescue IVM embryos resulted in 3 healthy live births. For patients with a low maturation rate and/or low numbers of mature oocytes at retrieval, rescue IVM may contribute more competent oocytes and additional viable BLs for transfer from the same stimulation cycle, maximizing the chances for pregnancy and live birth.

Merkel Cell Hyperplasia Versus Intraepidermal Merkel Cell Carcinoma: A Comparative Study of 2 Cases.

Intraepidermal Merkel cell hyperplasia and Merkel cell carcinoma represent 2 histologically similar-appearing diagnoses with significant differences regarding prognosis and management. We present 1 case of each diagnosis to highlight characteristic histopathologic and immunohistochemical features. Our case of Merkel cell hyperplasia was identified by its small intraepidermal nest of monomorphic cells without atypia or mitoses, which demonstrated cytoplasmic, rather than perinuclear dot, patterning on CK20 staining. This can be contrasted with our case of intraepidermal Merkel cell carcinoma, which, despite a lack of dermal extension, demonstrated large nests of pleomorphic cells with frequent mitoses and apoptoses. The diagnosis was further confirmed by immunohistochemistry because CK20 staining showed classic perinuclear dot patterning. By presenting both diagnoses in parallel, this comparison aims to underscore crucial histopathologic and immunohistochemical similarities and differences.

A study on the seasonal cyclicity of ovarian development in adult Himalayan snow trout, Schizothorax plagiostomus.

Schizothorax plagiostomus, commonly known as snow trout, is a popular food fish found in parts of Central Asia and the Indo-Himalayan region. Despite such a broad range of distribution and potential financial value, it is a highly neglected cold-water ichthyofauna. Furthermore, an alarming decline in Schizothoracine population has been reported in the recent past due to climate change and uncontrolled anthropogenic interference. In this study, the seasonal variations in ovarian architecture and development were examined in adult S. plagiostomus from Garhwal Himalayan region, Uttarakhand, India. Ovarian-somatic index ranged from 16.86 ± 0.29 to 0.31 ± 0.56, with a maximal value in September and a minimal value in April. Ovarian histology revealed the abundance of primary growth oocytes in resting and preparatory stages; primary/secondary vitellogenic oocytes with numerous cortical alveoli were predominant in the developing stage of pre-spawning ovaries; secondary/tertiary vitellogenic oocytes were conspicuous in actively spawning ovaries; and atretic follicles/oocytes were discernible during the regressing stage of spent ovaries. Scanning electron microscopy of mature ova (mean diameter 2.003 ± 0.01 mm) prominently showed the structure micropyle (mean diameter 12.93 ± 3.38 μm). Fecundity analyses suggested that September was the principal breeding season, whereas residual spawning occurred with fresh rain in late winter during February-March. Collectively, this is the first comprehensive qualitative and quantitative report of the seasonal variations in the ovarian development and function for S. plagiostomus. These data may provide valuable information towards the captive breeding programme as well as conservation and management for Schizothoracine fishes in normal and altered climatic conditions.

Prolonged fasting induces significant germ cell loss in chickens after hatching

Germ cell loss is a crucial biological event during germ cell development. The number of female germ cells determines the reproductive performance and egg production of hens. Various intrinsic and extrinsic factors affect germ cell loss, such as germ cell nest breakdown in early life and nutritional deficiencies during daily husbandry. Here, we examined the effect of fasting on the germ cell number of chicks. The results showed that 72 h fasting resulted in a higher germ cell loss than that by 24 h fasting in chicks. The RNA-seq analysis revealed that the genes of ribosome pathway were down-regulated and the biological processes of protein processing in endoplasmic reticulum were inhibited in starved chicks. Furthermore, in female chicks treated with 72 h fasting, the qPCR of ovaries showed down-regulation of ribosome-related genes, and transmission electron microscopy imaging of ovaries showed fewer ribosomes. The blood biochemical indices indicated that 72 h fasting reduced the liver functions and affected the glucose metabolism, lipid metabolites and ion metabolites. In summary, the present results concluded negative impacts on the germ cell pool by prolonged fasting in the early life of chicks and manifested that adequate management should be cared for fasted time for breeding.

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development.

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.