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Germinal vesicles are alternative targets for female fertility preservation due to their availability and high resilience against non-physiological conditions. Preserved germinal vesicles can then be transferred to fresh cytoplasts to reconstitute viable oocytes. Here, we describe a germinal vesicle preservation method that employs non-ionizing microwave radiations imparting energy to water molecules, which results in rapid and homogeneous drying of the sample. Trehalose is added as a xero-protectant before the radiations, enabling isothermal vitrification of the disaccharide sugar during drying. While the technique is still considered experimental, studies have shown that DNA and structural integrity can be effectively maintained in dried/rehydrated germinal vesicles. Importantly, the dry-preservation approach allows supra-zero temperature storage of the samples, offering a cost-effective and energy-saving alternative to traditional methods relying on ultra-low freezing temperatures. The protocol outlines a comprehensive procedure involving germinal vesicle oocyte collection, trehalose loading, microwave drying, storage, and rehydration. The simplicity of the protocol facilitates the ease of manipulation, making it an accessible method for researchers. While initially developed for domestic cats, the protocol can be adapted for other species with necessary modifications, considering potential species-specific responses to dehydration stress. Summary Sentence This is a detailed protocol for microwave-assisted dehydration of GVs to allow fertility preservation and long-term storage at supra-zero temperatures. Graphical Abstract

The success rate of maturation (IVM) for human oocytes is clinically significant, prompting a focus on optimizing IVM media culture. While various factors have been incorporated to improve outcomes, the role of Ghrelin hormone, despite its multifunctional nature, remains poorly investigated. This study aimed to determine the most effective concentration of the growth hormone releasing protein-6 (GHRP-6), Ghrelin hormone agonist, in the culture medium. In this experimental study, a total of 240 human germinal vesicle (GV) oocytes were collected and cultured in varying concentrations of GHRP-6. Maturation rates were assessed during two days of culture, and compared against a blastocyst media (single-step culture) as control group and another IVM media, human tubal fluid (HTF) 10%, as the sham group. Additionally, the expression levels of two genes associated with nuclear and cytoplasmic maturation were compared in the number of 164 GV oocytes randomly cultured in the most effective concentration of GHRP-6, control and sham groups for 24 hours, using real-time polymerase chain reaction (PCR). The optimal concentration of GHRP-6 for the IVM procedure was determined to be 75 ng/ml, resulting in a maturation rate of 70% on the first day and 80% on the second day. These results surpassed those of other culture media on both days. Real-time PCR data indicated that, despite the early appearance of the first polar body (PB1) on 24h of culture, the Ghrelin agonist did not elevate the expression levels of , genes associated with meiotic progression and membrane proteins, respectively. In summary, while GHRP-6 showed potential in promoting nucleonic maturation by significantly inducing the appearance of PB1 between GVs within 24 hours, it did not exhibit statistically significant improvements in cytoplasmic maturation in metaphase 2 oocytes (MII) during this timeframe.

Although it is well known that maternal ageing causes reduced oocyte quality and fertility, little information is known about its effect on germ cell epigenetics. In the present study, we compared the gene expression and DNA methylation profiles in germinal vesicle oocytes from young (8-week-old) and aged (18-month-old) mice using single-cell RNA-sequencing and single-cell whole-genome DNA methylation sequencing. We found significant differences in the data from the two groups. Oocytes from aged mice showed significant changes in the expression of some metabolism-related genes, such as mitochondria-associated genes, that was in line with our expectations. Expression of some genes associated with reproduction also showed significant differences. DNA methylation levels were also changed in oocytes from aged mice. The two groups had significant gaps in hypermethylation and hypomethylation levels on each chromosome. These data provide useful information for further understanding the mechanisms of oocyte ageing.

Background: Vitrification as the most efficient method of cryopreservation, enables successful storage of oocytes for couples who undergo specific procedures including surgery and chemotherapy. However, the efficacy of in vitro maturation (IVM) methods with vitrified germinal vesicle (GV) oocytes could be improved. Objective: As melatonin and follicular fluid (FF) might enhance IVM conditions, we used these supplements to assess the maturation rate of vitrified GV oocytes and their artificial fertilization rate. Materials and Methods: Four hundred mouse GV oocytes were harvested, vitrified, and assigned into control (C-Vit-GV) and treatment groups of melatonin (M-Vit-GV), human follicular fluid (HFF-Vit-GV), and a combination (M + HFF-Vit-GV). A non-vitrified group of GV oocytes (non-Vit-GV) and a group of in vivo matured metaphase II (Vivo-MII) oocytes served as control groups to evaluate the vitrification and IVM conditions, respectively. Maturation of GV oocytes to MII and further development to two-cell-stage embryos were determined in the different groups. Results: Development to two-cell embryos was comparable between the Vivo-MII and non-Vit-GV groups. IVM and in vitro fertilization (IVF) results in the non-Vit-GV group were also comparable with the C-Vit-GV oocytes. In addition, the IVM and IVF outcomes were similar across the different treatment groups including the M-Vit-GV, HFF-Vit-GV, M + HFF-Vit-GV, and C-Vit-GV oocytes. Conclusion: Employing an appropriate technique of vitrification followed by suitable IVM conditions can lead to reasonable IVF outcomes which may not benefit from extra supplementations. However, whether utilizing other supplementation formulas could improve the outcome requires further investigation. Key words: Vitrification, Germinal vesicle, In vitro oocyte maturation, Melatonin, Follicular fluid.

Background: To increase the results of infertility treatment, many efforts have been made to improve the treatment methods. As assisted reproductive technology is mainly using cell culture methods, one of the approaches to improve this technology is conditioned medium from different sources. It is desirable to apply in vitro maturation (IVM) and use oocytes from normal cycles instead of stimulating ovulation. Objective: To investigate the effect of human cumulus cell condition medium (hCCCM) on the IVM of immature mouse oocytes and morphology. Materials and Methods: In this experimental study, 240 germinal vesile oocytes were collected from four-six wk-old mice after 48 hr of 5IU pregnant mare serum gonadotropin (PMSG) injection and cultured in hCCCM (test group, n = 120) and DMEM + 20% FBS (control group, n = 120). The IVM rates and changes in perivitelline space (PVS) and shape were investigated at 8, 16, and 24 hr following the culture. The mature (MII) oocytes were subjected to in vitro fertilization (IVF) and the fertilization rate was assessed in three days. Results: A significant difference was observed between the maturation rates in the hCCCM and control groups (24.16% vs 0%; p = 0.001), as well as morphologic changes between the two groups (p = 0.04, p = 0.05). The development rate for MII oocytes attained from IVM in the hCCCM group was 27.58% (2-cell) and 6.89% (4-cell). Data displayed that hCCCM is an effective medium for oocytes maturation compared to the control medium. Conclusion: hCCCM supports oocyte in vitro growth and maturation. Moreover, hCCCM changes the oocyte shape and size of perivitelline space. Key words: Germinal vesicle, Cumulus cell, Conditioned medium, In vitro fertilization, In vitro maturation, Oocyte.

Objectives We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos. Results There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes ( P  < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels. Conclusion Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

C-type natriuretic peptide (CNP) is a recently identified meiotic inhibitor in mice. However, it has not been investigated in porcine oocytes to date. This study aimed to demonstrate the inhibitory effect of CNP against germinal vesicle breakdown (GVBD) in porcine oocyte meiotic resumption. Immunohistochemical analysis revealed intense natriuretic peptide receptor 2 (NPR2) immunoreactivity in the oocyte surrounded cumulus cells in the follicles. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) analysis showed the expression of npr2 mRNA only in cumulus cells but not in oocytes, suggesting that cumulus cells are the targets of CNP. When cumulus–oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with various concentrations of CNP (10, 50, 100, 500, and 1,000 nM), inhibitory effect was observed in the COC group, but not in the DO group, confirming that CNP indirectly inhibits GVBD via cumulus cells. This evidence is the first indication that the CNP-NPR2 pathway is involved in meiotic arrest in porcine oocytes. Furthermore, we investigated the effect of oocyte-derived paracrine factor (ODPF) on npr2 mRNA expression level in cumulus cells by evaluating changes in mRNA expression in oocytectomised COCs (OXCs) by real-time PCR. A significant decrease in npr2 mRNA expression level was observed in OXCs, whereas mRNA expression level was restored in OXCs with DOs, indicating that ODPF participates in the regulation of npr2 expression in porcine cumulus cells.

The preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration during IVM may affect the nuclear kinetics, all experiments were performed under two distinct O2 concentrations: 20% and 5% O2. First, we determined the time course of changes in nuclear maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2, respectively, and 67.6% and 47.3% under 5% O2, respectively) were almost double those of late GVBD oocytes (44.8% and 26.0% under 20% O2, respectively, and 40.4% and 17.9% under 5% O2, respectively). With no observable alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method for high developmental competent oocytes.

The present study aimed to investigate the effect of thiamine on growth, hematological, egg thiamine content and oocyte nucleus migration indices in sterlet sturgeon (Acipenser ruthenus). A total of 45 female fish (698.6 ± 8.9 g) were distributed in nine fiberglass tanks with three treatments (each in three replicates). Experimental fish were fed once a day with practical diet supplemented with 1 g kg⁻¹ amprolium hydrochloride (as the antithiamine) for 5 months before spawning. Thiamine hydrochloride was injected to fish with one of three doses at 0, 5 and 50 mg kg⁻¹ body weight (BW) at three different stages. At the end of each month, fish were weighed and growth parameters such as weight gain, specific growth rate, feed efficiency and final weight were determined. At the end of the 5-month period, hematological parameters and egg thiamine content were measured. The results showed no significant differences in terms of growth performance. Hemoglobin, number of red blood cells and white blood cells were not significantly different among the treatments, but hematocrit was significantly higher in fish injected with 5 mg kg⁻¹ BW thiamine. The mean corpuscular volume and mean corpuscular hemoglobin were not significantly different, but lower value of mean corpuscular hemoglobin concentration was observed in fish injected with 5 mg thiamine. Percentage of lymphocytes, neutrophils, eosinophils and monocytes were not significantly different among treatments. The results of the germinal vesicle migration index at different stages showed no significant differences in different groups. Results showed that free thiamine, thiamine pyrophosphate and total thiamine in eggs significantly increased at 50 mg kg⁻¹ injection dose compared with the control, but no significant different was found in thiamine monophosphate. The results revealed that thiamine injection to sterlet broodstocks has positive accumulation in egg thiamine content and some hematological parameters but have no effect on growth and ovarian maturation. We conclude that thiamine can affect some physiological features of broodstock, which needs to be specified in further studies.

For most intracellular structures with larger than molecular dimensions, little is known about the connection between underlying molecular activities and higher order organization such as size and shape. Here, we show that both the size and shape of the amphibian oocyte nucleolus ultimately arise because nucleoli behave as liquid-like droplets of RNA and protein, exhibiting characteristic viscous fluid dynamics even on timescales of < 1 min. We use these dynamics to determine an apparent nucleolar viscosity, and we show that this viscosity is ATP-dependent, suggesting a role for active processes in fluidizing internal contents. Nucleolar surface tension and fluidity cause their restructuring into spherical droplets upon imposed mechanical deformations. Nucleoli exhibit a broad distribution of sizes with a characteristic power law, which we show is a consequence of spontaneous coalescence events. These results have implications for the function of nucleoli in ribosome subunit processing and provide a physical link between activity within a macromolecular assembly and its physical properties on larger length scales.