Oocytes Research Articles

Terato-Neuroendocrine Tumour of Testis: Review and Update

Neuro-endocrine tumours commonly emanate from intestinal and respiratory epithelium. Neuro-endocrine tumours could also on rare occasions afflict various organs of the human body. Neuro-endocrine tumour afflicting the testis is an uncommon tumour which does account for far less than one percent (<1%) of all tumours of testis. To the knowledge of the author, less than 100 cases of primary neuro-endocrine tumour of the testis had been reported so far in the global literature. The reported cases of neuro-endocrine tumour of testis have ranged between 10 years and 83 years of age and the reported incidence rate had been noted to be higher within the fifth and sixth decade of life. Majority of patients who are afflicted by neuro-endocrine tumour of testis do tend to manifest with unilateral painless testicular mass. Sixteen percent (16%) of patients who are afflicted by neuro-endocrine tumour of testis do manifest with symptoms of neuroendocrine tumour syndrome. Eleven percent (11%) of primary neuroendocrine tumours of testis tend to be diagnosed initially with a contemporaneously-associated metastasis. Neuro-endocrine tumours of the testis occur as a primary testicular neuroendocrine tumour, or they may be metastatic neuroendocrine tumour to the testis with the primary tumour originating from elsewhere in the body especially from the gastrointestinal or cardiorespiratory tract system. Primary neuro-endocrine tumour of testis, could be further sub-divided into: (a) primary pure neuroendocrine tumour of testis; and (b) neuro-endocrine tumour of testis contemporaneously associated with teratoma or dermoid/epidermoid cysts. Once a neuro-endocrine tumour of testis is diagnosed, it is pivotal that clinicians should exclude a metastatic neuroendocrine tumour to the testis with the primary tumour originating from elsewhere within the human body. It has been iterated that the presence of teratomas elements within the neuro-endocrine tumour of testis does sufficiently rule out the primary site outside the testis. In order to accurately diagnose the neuro-endocrine tumour of the testis, it has been advised that it is pertinent to submit the tumour mass entirely and to look for any additional lineage of differentiation. Twenty five percent of primary neuro-endocrine tumours of testis are associated with teratoma. Histogenesis of pure neuroendocrine tumour of the testis is yet to be clarified. Two postulates had been propounded in relation to the mode origin of neuroendocrine tumour of testis including: (1) Emanation from a teratoma which is the germ cell origin postulate; (2) Emanating from argentaffin cells that are located within crypts of Lieberkühn .A study related to three pure testicular neuroendocrine tumours of testis had indicated that pure neuroendocrine tumour of testis might have different genetic background other than germ cell tumour.[11] Histopathology examination of specimens of neuro-endocrine tumour of testis has tended to demonstrate tumour cells that tend to be typified by the presence of nests of small round cells with uniform nuclei forming small acini and rosettes or sheets. The cells tend to contain eosinophilic granules within the cytoplasm and granular chromatin within the nuclei. The tumour cells do exhibit positive staining for neuroendocrine markers like chromogranin and synaptophysin as illustrated in detail in the article. Radical orchidectomy is the most adopted option of treatment; nevertheless, necessitation for the undertaking of provision of adjuvant treatment depending upon histological grading has not been clarified. Because of the possibility of development of local recurrence and or distant metastases after a long time, it is pivotal for all clinicians to carefully follow-up their patients over a long-period of time with clinical, laboratory testing and radiology-imaging testing follow-ups in order to establish early diagnosis of any local recurrence or distant metastasis to be able provide further treatment of curative intent early. There is also the possibility that neuro-endocrine tumours of testis had so far been under-reported due to mis diagnosis of the tumour upon pathology examination of the testicular tumour or the correct set of monoclonal and polyclonal antibodies had not been utilized in the immunohistochemistry study assessment of the tumours. It would be advised that all clinicians and pathologists globally should have a high index of suspicion for a neuro-endocrine tumour of testis in order to establish prompt diagnosis of the tumour and to provide appropriate assessment, treatment and follow-up of all their patients.

Bio‐Inspired Porous Microneedles Dwelled Stem Cells for Diabetic Wound Treatment

AbstractDiabetic wound healing is a serious, complex, and chronic process; one current promising and focusing technology in this area is stem cell treatment. Here, novel porous microneedle (MN) arrays is fabricated, which can highly mimetic stem cell niches, through template filling, and particle etching method. The human adipose‐derived stem cells (ADSCs) are encapsulated in Matrigel then loaded into the porous MN arrays by post‐perfusion. Because of the extracellular matrix‐mimicking, the biocompatible Matrigel offers a bionic microenvironment of stem cell nest suitable for growth. Benefiting from the numerous pore structures of MNs, the loaded ADSCs have enough space to fully absorb nutrients, proliferate greatly and exhibit prompted function. In addition, the cell‐loaded MN arrays have enough mechanical strength to penetrate the skin, allowing the ADSCs to get into the deep wound areas. Based on these features, the performance of the resultant MN arrays in promoting tissue regeneration is demonstrated, collagen deposition and angiogenesis in diabetes wounds of rat models. Thus, it is believe that the bioinspired porous MNs can act as excellent stem cell scaffolds and will find many practical values in clinic wound healing.

Characterization of insulin-like growth factor 3 and its potential role in the spotted steed Hemibarbus maculatus ovary development

As a new member of the insulin-like growth factors (Igfs), Igf3 was reported to play a vital role in fish reproduction. However, in spotted steed, the function of Igf3 remains largely unknown. In the present study, we identified and characterized Igf3 gene in spotted steed. Structural analysis showed that Igf3 contained five domains (B, C, A, D, E) and six conserved cysteine residues. The expression of Igf3 mRNA and protein were increased during ovary development and peaked in the maturation stage. The subcellular localization of IGF3 was highly expressed in granulosa cells and theca cells. Furthermore, recombinant IGF3 protein was produced and in vitro treatment with ovarian follicles significantly promoted the germinal vesicle breakdown (GVBD) rates of spotted steed follicles. The mRNA expression of cdc2 and cyclinB genes were significantly increased after IGF3 treatment, which were main components of maturation promoting factor (MPF). In addition, transcription levels of 3β-hsd, 20β-hsd, Cyp17a and Cyp19a1a were also changed. Taken together, these findings suggest that Igf3 is essential for ovary steroidogenesis and maturation in spotted steed.

Molecular Characterization, Expression Pattern, DNA Methylation and Gene Disruption of Figla in Blotched Snakehead (Channa maculata)

Figla is one of the earliest expressed genes in the oocyte during ovarian development. In this study, Figla was characterized in C. maculata, one of the main aquaculture species in China, and designated as CmFigla. The length of CmFigla cDNA was 1303 bp, encoding 197 amino acids that contained a conserved bHLH domain. CmFigla revealed a female-biased expression patterns in the gonads of adult fish, and CmFigla expression was far higher in ovaries than that in testes at all gonadal development stages, especially at 60~180 days post-fertilization (dpf). Furthermore, a noteworthy inverse relationship was observed between CmFigla expression and the methylation of its promoter in the adult gonads. Gonads at 90 dpf were used for in situ hybridization (ISH), and CmFigla transcripts were mainly concentrated in oogonia and the primary oocytes in ovaries, but undetectable in the testes. These results indicated that Figla would play vital roles in the ovarian development in C. maculata. Additionally, the frame-shift mutations of CmFigla were successfully constructed through the CRISPR/Cas9 system, which established a positive foundation for further investigation on the role of Figla in the ovarian development of C. maculata. Our study provides valuable clues for exploring the regulatory mechanism of Figla in the fish ovarian development and maintenance, which would be useful for the sex control and reproduction of fish in aquaculture.

A homozygous stop codon in HORMAD2 in a patient with recurrent digynic triploid miscarriage.

Recurrent miscarriage (RM) affects 1% to 5% of couples trying to conceive. Despite extensive clinical and laboratory testing, half of the RM cases remain unexplained. We report the genetic analysis of a couple with eight miscarriages and the search for their potential genetic etiology. Short tandem repeat (STR) markers, single nucleotide polymorphic (SNP) microarray, and human DNA methylation microarray were used to analyze the genotypes of two miscarriages. Exomes sequencing was performed on DNA from the two partners and identified variants were validated by Sanger sequencing. STR marker genotyping demonstrated that the two available miscarriages are triploid digynic and resulted from the failure of Meiosis II. SNP microarray analysis revealed an additional Meiosis I abnormality that is the segregation of the two maternal homologous chromosomes in one triploid miscarriage. Whole-exome sequencing on DNA from the two partners identified candidate variants only in the female partner in two genes with roles in female reproduction, a missense in EIF4ENIF1 (OMIM 607445) and a stop gain in HORMAD2 (OMIM 618842). EIF4ENIF1 is a eukaryotic translation initiation factor 4E nuclear import factor required for the oocyte germinal vesicle breakdown, and HORMAD2 is part of the synaptonemal complex that was hypothesized to act as a checkpoint mechanism to eliminate oocytes with asynapsis during meiotic prophase I in mice. While both genes may contribute to the phenotype, the Meiosis I abnormalities in the conceptions favor the causal role of HORMAD2 in the etiology of RM in this couple. This report illustrates the importance of comprehensively analyzing the products of conception to guide the search for the genetic causation of RM.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

GnRHr, LHr, and Vg gene expression levels and ovarian development of G5 transgenic mutiara female catfish (Clarias gariepinus) after exposure photoperiod induction

This study aimed to determine the relative expression ratios of the genes gonadotrophin-releasing hormone receptor (GnRHr), luteinizing hormone receptor (LHr), vitellogenin (Vg) and β-actin genes as expression control internal of the G5 fish using real-time PCR in a photoperiod experiment with designed treatments (A: 8L-16D; B: 12L-12D; C: 16L-8D for transgenic fish; and A*: 8L-16D; B*: 12L-12D; C*: 16L-8D for nontransgenic fish) for 60 days of rearing period. Ovary maturation was evaluated in G5 transgenic mutiara catfish during different photoperiod induction. A short photoperiod (8L-16D) induced an high expression of GnRHr, LHr, and Vg genes (mean, 4.42 ± 0.53, 5.63 ± 0.42, and 6.67 ± 0.31, respectively), indicating the role of dark cycle in increasing the gene expressions involved in ovarian maturation of G5 transgenic mutiara catfish. The lowest GnRHr, LHr, and Vg gene expression levels were found in nontransgenic fish (C*) (mean, 1.27 ± 0.13, 1.38 ± 0.24, and 2.42 ± 0.33, respectively). The exposure of transgenic fish (CgGH insert content) to a long photoperiod (16L-8D) resulted in lower expression levels of GnRHr, LHr, and Vg (mean, 2.31 ± 0.27, 2.34 ± 0.25, and 4.49 ± 0.30, respectively) and lower levels of hormones Vg and E2 (mean, 295.16 ± 21.71 μg/mL and 0.25 ± 0.03 ng/mL, respectively) and in non-transgenic fish (mean, 163.54 µg/mL and 0.14 ng/mL, respectively). Short photoperiods (8L-16D and 12l-12D) led to oocyte maturation and higher GSI values (mean, 12.24 ± 0.53 and 10.24 ± 0.38, respectively) compared to long photoperiods (16L-8D). Conversely, a long photoperiod led to decreased GnRHr, LHr, and Vg expression levels, and Vg and E2 hormone levels, leading to the growth of immature oocytes and decreased GSI (mean, 3.93 ± 0.29) in nontransgenic fish. The presence of CgGH in G5 transgenic mutiara female catfish can maintain the growth of primary oocytes to secondary oocytes during the 16L-8D photoperiod induction.

Epigen enhances the developmental potential of in vitro fertilized embryos by improving cytoplasmic maturation

Numerous growth factors contribute to oocyte maturation and embryonic development in vivo; however, only a few are understood. One such factor is epigen, a new member of the epidermal growth factor (EGF) family that is secreted by the granulosa cells of immature oocytes. We hypothesized that epigen may play a role in oocyte maturation, specifically in the nuclear and cytoplasmic aspects. This study aimed to investigate the effects of epigen on porcine oocyte maturation and embryo development in vitro. In this study, three different concentrations of epigen (3, 6, and 30 ng/mL) were added to tissue culture medium-199 (TCM-199) during in vitro maturation of porcine oocytes. A control group that did not receive epigen supplementation was also included. Mature porcine oocytes were fertilized, and the resulting zygotes were cultured until day 7. The levels of intracellular glutathione (GSH) and reactive oxygen species (ROS) were measured in the in vitro matured oocytes. At the same time, the expression patterns of genes related to apoptosis were detected in day 7 blastocysts (BLs) using real-time quantitative PCR Apoptosis was detected by annexin-V assays in mature oocytes. Data were analyzed using ANOVA and Duncan's test on SPSS, and results are presented as mean ± SEM. The group that received 6 ng/mL epigen had a significantly lower rate of germinal vesicle breakdown (GVBD) than the control group without affecting the nuclear maturation among the experimental groups. Among the treatment groups, the 6 ng/mL epigen group showed significantly higher levels of intracellular GSH and lower ROS production. Supplementation with 6 ng/mL epigen significantly improved blastocyst (BL) formation rates compared to those in the control and 3 ng/mL groups. Additionally, the blastocyst expansion rate was significantly higher with epigen supplementation (6 ng/mL). In the fertilization experiment, the group supplemented with 6 ng/mL epigen exhibited significantly higher levels of monospermy and fertilization efficiency and lower levels of polyspermy than the control group. This study indicated that adding epigen at a concentration of 6 ng/mL can significantly enhance the developmental potential of porcine oocytes fertilized in vitro. Specifically, the study found that epigen improves cytoplasmic maturation, which helps prevent polyspermy and emulates monospermic penetration.

In vitro maturation of African catfish (Clarias gariepinus, Burchell, 1822) oocytes results in viable larvae

In this study, we describe a protocol for in vitro maturation and ovulation of African catfish (Clarias gariepinus) postvitellogenic ovarian follicles, resulting in fertilizable and developmentally competent eggs. Such culture systems enable detailed studies of fish oocyte development and present a useful tool in assisted reproduction of fish. The maturation-inducing effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), human chorionic gonadotropin (hCG) and recombinant human insulin-like growth factor 1 (rhIGF-1) were evaluated based on their ability to promote ooplasm translucency and germinal vesicle breakdown (GVBD), which indicates meiosis resumption. Among the tested groups, only follicles exposed to DHP underwent GVBD in a time-dependent manner, with the highest rates (>80%) observed within 10 to 12 h of incubation. Furthermore, adding rhIGF-1 or hCG prior to or during incubations with DHP did not influence the GVBD outcome. This suggested that the oocytes acquired maturational competence prior to isolation, as high concentrations of DHP (1 μg/ml) were alone sufficient to induce complete nuclear and cytoplasmic maturation; however, only 4 ± 3% of these oocytes ovulated in culture. To promote ovulation, we incorporated prostaglandins (PGs) as an additional incubation step following DHP-induced maturation. Exposure to both prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) led to increased rupture of the follicular layer, most notably in groups with 5 μg/ml PGF2α (71 ± 8%). In addition to identifying optimal hormonal stimulation, effects of culture media pH in combination with media supplementation—in the form of bovine serum albumin (BSA) and fetal bovine serum (FBS)—were considered. Compared to results in media with pH of 7.5, significantly less follicles matured and ovulated in a more alkaline environment (pH 8.5), regardless of supplement type and concentration. Although the presence of FBS (5–20%) slightly improved ovulation rates, this benefit was not reflected in the fertilization outcomes. In vitro matured and ovulated oocytes obtained in non-supplemented media (pH 7.5) maintained their developmental competence and were successfully fertilized. The hatching rate was 39%, after which the survival rate of larvae was 8% at 72 h post fertilization (hpf).

Acidosis defense mechanisms in the preimplantation stages of embryos in BALB/c strain mice

Regulation of intracellular pH (pHi) is an important homeostatic function of cells. There are three major pHi regulatory mechanisms: the HCO3−/Cl− exchanger (AE), which alleviates alkalosis, and the Na+/H+ exchanger (NHE) and Na+,HCO3−/Cl− exchanger (NDBCE), both of which counteract acidosis. NHE activity, which is high at the germinal vesicle stage of oocyte, is inhibited during meiotic maturation, while this inhibition is abolished when the oocyte reaches the pronuclear (PN) stage of the zygote. On the other hand, we have previously found that NDBCE performs complementary regulation against acidosis during meiotic maturation. Additionally, we found that AE activity, which is a defense mechanism against alkalosis, gradually decreases during preimplantation period of embryonic development. Considering that NHE activity is inhibited during meiotic maturation and AE activity gradually decreases during embryonic development stages, we investigated whether NHE and NDBCE activities, both of which act against acidosis, functionally change from the PN zygote to the blastocyst stage of the embryo and identified these pH-regulating proteins at the molecular level in mice of the Balb/c strain. PN zygotes, two-cell (2-c), four-cell (4-c), morula and blastocyst stage embryos were obtained from 5-8-week-old, sexually mature female Balb/c mice by using the classical superovulation procedure. pHi was recorded by using the microspectrofluorometric technique on zygotes and embryos simultaneously loaded with the pH-sensitive fluorophore, 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The activities of NHE and NDBCE were determined from the recovery curve of induced-acidosis in bicarbonate-free and bicarbonate-containing media, respectively. Specific inhibitors such as cariporide (1 μM), S3226 (1 and 10 μM), EIPA (1, 5, and 25 μM), and amiloride (1 mM) were used to functionally identify NHE isoforms, and the nonspecific inhibitor 4,4′–diisocyanatostilbene-2,2′ disulphonic acid, disodium salt (DIDS) was used to confirm NDBCE activity. The isoforms of the pHi-regulatory proteins were also identified by molecular biology using real-time PCR. We found that NHE activity was high at all embryonic stages, and differences between stages were not significant. Functional and molecular findings indicated that isoforms of NHE 1 and 5 are present in the blastocyst, whereas isoforms of NHE 1, 3, and 4 are functional at earlier embryonic stages. Although the contribution of NDBCE activity to recovery from induced-acidosis was detected at all embryonic stages, it was significant only in the PN zygote and the 2-c embryo. This finding was confirmed by molecular analysis, which detected the expression of SLC4A8 encoding NDBCE at all embryonic stages. In conclusion, NHE is an active and important defense mechanism against acidosis and is encoded by at least two protein isoforms in all stages of the Balb/c strain of mice. NDBCE has a supportive function in all embryonic stages, especially in the PN zygote and the 2-c embryo. Preimplantation stage embryos have effective mechanisms to defend against acidosis in response to their metabolic end products (increased acid load) and the acidic environment in utero.

CD4+ T cell immunity against cutaneous melanoma encompasses multifaceted MHC II-dependent responses.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

The association of RBX1 and BAMBI gene expression with oocyte maturation in PCOS women

BackgroundPolycystic ovarian syndrome (PCOS) is a common endocrine disorder that affects 6–20% of women of reproductive age. One of the symptoms of PCOS is hyperandrogenism, which can impair follicular development. This disruption can cause issues with the development of oocytes and the growth of embryos. Although the exact cause of PCOS is not yet fully understood, studying the gene expression pattern of cumulus cells, which play a crucial role in the maturation and quality of oocytes, could help identify the genes associated with oocyte maturation in PCOS women. Through indirect activation of APC/Cdc20, RBX1 enables oocytes to bypass the GV (germinal vesicles) stage and advance to the MII (metaphase II) stage. our other gene is the BAMBI gene which stimulates WNT signaling, that is a crucial pathway for healthy ovarian function. This study aims to explore the expression level of the RBX1 and BAMBI genes between GV and MII oocytes of PCOS and non-PCOS groups.MethodsIn this experiment, we gathered the cumulus cells of MII (38 cases and 33 control) and GV (38 cases and 33 control) oocytes from women with/without PCOS. Besides, quantitative RT-PCR was used to assess the semi-quantitative expression of BAMBI and RBX1.ResultsAccording to our research, the expression level of RBX1 and BAMBI in MII and GV cumulus cells of PCOS patients was significantly lower than that in non-PCOS ones.ConclusionThis research raises the possibility of RBX1 and BAMBI involvement in oocyte quality in PCOS women.

Association between pathological infiltrative tumor growth pattern and prognosis in patients with resected lung squamous cell carcinoma

IntroductionLung squamous cell carcinoma (LUSC) usually shows expansive growth with large tumor nests; few reports on invasive growth patterns (INF) in LUSC have been associated with poor prognosis in gastrointestinal and urothelial cancers. In this study, we examine the association between INF and the prognosis of LUSC. Materials and methodsWe analyzed INF as a potential prognostic factor in 254 consecutive patients with LUSC who underwent complete surgical resection at our hospital between 2008 and 2017. INF was classified into 3 categories based on the structure of the tumor other than the large round solid nest of tumor cells. ResultsINF was categorized as INFa in 59 patients (23 %) with only well-demarcated large solid tumor cell nests, INFb in 89 patients (35 %) with medium to small, alongside large solid nests, and INFc in 98 patients (39 %) with cord-like/small nests or isolated cells plus large or medium solid nests. No significant lymph node metastasis differences were observed between INFc and INFa/b tumors. However, in patients with p-stage I, INFc had a poorer prognosis with regard to recurrence-free survival (RFS), with a 5-year RFS rate of 53.3 %, compared to 74.9 % for INFa/b (p = 0.010). ConclusionOur study highlights a novel pathological concept of INF in LUSC, and contributed to the proposal that it is a factor indicating an unfavorable prognosis in patients with early-stage LUSC. A prospective multicenter study is warranted for INFc patients, as careful follow-up and adjuvant chemotherapy might lead to the early detection and prevention of recurrence.

Phenotypic sorting of individual male and female intersex Cherax quadricarinatus and analysis of molecular differences in the gonadal transcriptome

Cherax quadricarinatus exhibit sexual dimorphism, with males outpacing females in size specification and growth rate. However, there is limited understanding of the molecular mechanisms underlying sex determination and sex differentiation in crustaceans. To study the differences between intersex individuals and normal individuals, this study counted the proportion of intersex individuals in the natural population, collected the proportion of 7 different phenotypes in 200 intersex individuals, and observed the differences in tissue sections. RNA-seq was used to study the different changes in the transcriptome of normal and intersex gonads. The results showed that: the percentage of intersex in the natural population was 1.5 %, and the percentage of different types of intersex ranged from 0.5 % to 22.5 %; the sections revealed that the development of normal ovaries was stagnant at the primary oocyte stage when intersex individuals with ovaries were present; We screened for pathways and genes that may be associated with gonadal development and sex, including ovarian steroid synthesis, estrogen signaling pathway, oocyte meiosis, progesterone-mediated oocyte maturation, etc. Relevant genes including tra2a, dmrta2, ccnb2, foxl2, and smad4. This study provides an important molecular basis for sex determination, sex-controlled breeding, and unisex breeding in red crayfish.

Dynamic methylation pattern of H19DMR and KvDMR1 in bovine oocytes and preimplantation embryos.

This study aimed to evaluate the epigenetic reprogramming of ICR1 (KvDMR1) and ICR2 (H19DMR) and expression of genes controlled by them as well as those involved in methylation, demethylation, and pluripotency. We collected germinal vesicle (GV) and metaphase II (MII) oocytes, and preimplantation embryos at five stages [zygote, 4-8 cells, 8-16 cells, morula, and expanded blastocysts (ExB)]. DNA methylation was assessed by BiSeq, and the gene expression was evaluated using qPCR. H19DMR showed an increased DNA methylation from GV to MII oocytes (68.04% and 98.05%, respectively), decreasing in zygotes (85.83%) until morula (61.65%), and ExB (63.63%). H19 and IGF2 showed increased expression in zygotes, which decreased in further stages. KvDMR1 was hypermethylated in both GV (71.82%) and MII (69.43%) and in zygotes (73.70%) up to morula (77.84%), with a loss of methylation at the ExB (36.64%). The zygote had higher expression of most genes, except for CDKN1C and PHLDA2, which were highly expressed in MII and GV oocytes, respectively. DNMTs showed increased expression in oocytes, followed by a reduction in the earliest stages of embryo development. TET1 was downregulated until 4-8-cell and upregulated in 8-16-cell embryos. TET2 and TET3 showed higher expression in oocytes, and a downregulation in MII oocytes and 4-8-cell embryo. We highlighted the heterogeneity in the DNA methylation of H19DMR and KvDMR1 and a dynamic expression pattern of genes controlled by them. The expression of DNMTs and TETs geneswas also dynamic owing to epigenetic reprogramming.

Expression characteristics of the cyp19a1b aromatase gene and its response to 17β-estradiol treatment in largemouth bass (Micropterus salmoides).

To investigate the regulatory role of the cyp19a1b aromatase gene in the sexual differentiation of largemouth bass (Micropterus salmoides, LMB), we obtained the full-length cDNA sequence of cyp19a1b using rapid amplification of cDNA ends technique. Tissue expression characteristics and feedback with 17-β-estradiol (E2) were determined using quantitative real-time PCR (qRT-PCR), while gonad development was assessed through histological section observations. The cDNA sequence of LMB cyp19a1b was found to be1950 base pairs (bp) in length, including a 5' untranslated region of 145 bp, a 3' untranslated region of 278 bp, and an open reading frame encoding a protein consisting of 1527 bp that encoded 508 amino acids. The qRT-PCR results indicated that cyp19a1b abundantly expressed in the brain, followed by the gonads, and its expression in the ovaries was significantly higher than that observed in the testes (P < 0.05). After feeding fish with E2 for 30 days, the expression of cyp19a1b in the pseudo-female gonads (XY-F) was significantly higher than that in males (XY-M) (P < 0.05), whereas expression did not differ significantly between XX-F and XY-F fish (P > 0.05). Although the expression of cyp19a1b in XY-F and XX-F fish was not significantly different after 60 days (P>0.05), both exhibited significantly higher levels than that of XY-M fish (P<0.05). Histological sections analysis showed the presence of oogonia in both XY-F and XX-F fish at 30 days, while spermatogonia were observed in XY-M fish. At 60 days, primary oocytes were abundantly observed in both XY-F and XX-F fish, while a few spermatogonia were visible in XY-M fish. At 90 days, the histological sections' results showed that a large number of oocytes were visible in XY-F and XX-F fish. Additionally, the gonads of XY-M fish contained numerous spermatocytes. These results suggest that cyp19a1b plays a pivotal role in the development of ovaries and nervous system development in LMB.

Maturation and culture affect the metabolomic profile of oocytes and follicular cells in young and old mares.

Introduction: Oocytes and follicular somatic cells within the ovarian follicle are altered during maturation and after exposure to culture in vitro. In the present study, we used a nontargeted metabolomics approach to assess changes in oocytes, cumulus cells, and granulosa cells from dominant, follicular-phase follicles in young and old mares. Methods: Samples were collected at three stages associated with oocyte maturation: (1) GV, germinal vesicle stage, prior to the induction of follicle/oocyte maturation in vivo; (2) MI, metaphase I, maturing, collected 24 h after induction of maturation in vivo; and (3) MIIC, metaphase II, mature with collection 24 h after induction of maturation in vivo plus 18 h of culture in vitro. Samples were analyzed using gas and liquid chromatography coupled to mass spectrometry only when all three stages of a specific cell type were obtained from the same mare. Results and Discussion: Significant differences in metabolite abundance were most often associated with MIIC, with some of the differences appearing to be linked to the final stage of maturation and others to exposure to culture medium. While differences occurred for many metabolite groups, some of the most notable were detected for energy and lipid metabolism and amino acid abundance. The study demonstrated that metabolomics has potential to aid in optimizing culture methods and evaluating cell culture additives to support differences in COCs associated with maternal factors.

Transient suppression of Wnt signaling in poor-quality buffalo oocytes improves their developmental competence.

One of the most evolutionary conserved communication systems, the Wnt signaling pathway is a major gene regulatory pathway that affects the developmental competence of oocytes and regulates most embryonic developmental processes. The present study was undertaken to modulate the canonical Wnt (Wingless/integration) signaling pathway in the poor-quality (colorless cytoplasm after Brilliant Cresyl Blue staining, BCB-) buffalo cumulus-oocyte complexes (COCs) to improve their in vitro maturation (IVM) and embryo production (IVEP) rates. The expression of key Wnt pathway genes was initially assessed in the good (blue cytoplasm after Brilliant Cresyl Blue staining, BCB+) and poor quality (BCB-) buffalo COCs to establish a differential activity of the Wnt pathway. The BCB- COCs were supplemented with the Wnt pathway inhibitor, Dickkopf-related protein 1 (DKK1) and later subjected to IVM and IVEP along with the BCB+ and BCB- controls. The cumulus expansion index (CEI), rate of nuclear maturation (mean percentage of oocytes in the MII stage) and embryo production, and the expression of developmentally important genes were evaluated to assess the effect of Wnt pathway inhibition on the development competence of these poor-quality oocytes. The Wnt pathway genes exhibited a significantly higher expression (p < 0.05) in the poor-quality BCB- oocytes compared to the good-quality BCB+ oocytes during the early maturation stages. The supplementation of BCB- COCs with 100 ng/mL DKK1 effectively inhibited the expression of the key mediators of the Wnt pathway (β-catenin and dishevelled homolog 1, DVL1). DKK1 supplemented BCB- COCs exhibited significantly improved cytoplasmic and nuclear maturation indices, development rates and significantly elevated expression (p < 0.05) of genes implicated in germinal vesicle breakdown (GVBD) and embryonic genome activation (EGA) vis-à-vis BCB- control COCs. These data indicate that inhibition of the Wnt pathway during the initial course of oocyte maturation can improve the development competence of poor-quality buffalo oocytes.

Pathogenic missense variation in PABPC1L/EPAB causes female infertility due to oocyte maturation arrest at the germinal vesicle stage.

Women undergoing controlled ovarian hyperstimulation prior to in vitro fertilization (IVF) are treated using various protocols to induce multiple follicular growths. Complete failure of all oocytes to mature during IVF cycles is rare; however, it is a known cause of primary female infertility. Recently, pathogenic variations in a few genes have been identified in women with oocyte maturation defects; however, the underlying genetic causes remain largely unknown.This study included a Turkish family comprising three sisters with recurring oocyte maturation arrest at the germinal vesicle stage after multiple ovarian stimulations. Exome sequencing revealed a homozygous missense variant (c.1037C>T, p.Ala346Val) in the EPAB gene (also known as PABPC1L) in all three affected sisters, which was either absent or heterozygous in the unaffected family members. Functional experiments confirming the pathogenicity of the variant were performed by transfecting HEK293T cells and demonstrated the instability and increased rate of proteolysis of the mutated PABPC1L/EPAB protein. The identified variant, located in the well-conserved fourth RNA recognition motif (RRM4), in silico 3D modelling suggested changes in the physical properties of the pathogenic variant of PABPC1L/EPAB.Our findings validate PABPC1L/EPAB as an essential genetic contributor to the oocyte maturation process in humans and have direct implications for the genetic counselling of patients and their family members.

Vertical transmission and prevalence of white spot syndrome virus (WSSV) in the wild spawning population of the Indian white shrimp, Penaeus indicus

White spot disease, caused by white spot syndrome virus (WSSV), has historically been the most devastating disease in shrimp aquaculture industry across the world. The mode of virus transmission is the most crucial stage in the dynamics and management of virus infection. This study explored the mechanism of vertical transmission of WSSV in Indian white shrimp, Penaeus indicus, potential native species for domestication and genetic improvement, using quantitative real time PCR (q RT PCR), light and electron microscopy, and in situ hybridization. Wild brooders of P. indicus (n = 2576) were sampled along the South east coast of India, during 2016 to 2021. Of these ∼ 58 % of the brooders were positive for WSSV, and almost 50 % of infected wild brooders were at the various stages of reproductive maturation. WSSV-PCR positive brooders (n = 200) were analysed for vertical WSSV transmission. The q RT PCR studies of reproductive tissues revealed that 61 % (n = 13) of spermatophore, 54 % (n = 28) of immature ovaries and 48 % (n = 27) of ripe ovaries were infected with WSSV. The lowest level of infection was recorded in females with ripe ovaries (6.84 × 101 ± 9.79 × 100 ng genomic DNA) followed by fertilized eggs (1.59 × 102 ± 3.69 × 101 ng genomic DNA), and larvae (nauplius and zoea). The histology of gravid females with high WSSV copies showed pyknotic and karyorrhectic germinal vesicle with degenerated cortical rods. Conversely, the gravid females with low WSSV copies showed fully developed ovary without characteristic signs of WSSV infection. Transmission electron microscopic studies clearly established the presence of WSSV particles in both ovaries and spermatophores. When subjected to in situ hybridization, WSSV-specific signals were observed in connective tissues of spermatophore, although gravid ovary and fertilized eggs were failed to produce WSSV specific signals. The present study provides the first molecular and histological evidence for trans-ovarian vertical transmission of WSSV. Development of disease-free base population being the cornerstone and first step in establishing the breeding program, the present findings could be a basis for development of such programs.