Visualization of intracellular ice formation and growth in mouse oocytes.

BACKGROUND: Characterization of intracellular ice formation (IIF) in oocytes during the freezing and thawing processes will contribute to optimizing their cryopreservation. However, the observation of the ice formation process in oocytes is limited by the spatiotemporal resolution of the cryomicroscope systems. OBJECTIVE: To observe the intracellular icing of oocytes during cooling and rewarming, and to study the mechanism of formation and growth of intracellular ice in oocytes. MATERIALS AND METHODS: Mouse oocytes were frozen at different cooling rates to induce intracellular ice formation using a cryomicroscopy system consisting of a microscope equipped with a cryogenic cold stage, an automatic cooling system, a temperature control system, and a high-speed camera. The growth patterns of intracellular ice in oocytes were analyzed from the images recorded. Finally, the growth rate of intracellular ice formation in oocytes was calculated using an automatic intracellular ice tracking method. RESULTS: The IIF temperature decreased gradually with the increase in cooling rate. Initiation sites of IIF could be classified into three categories: marginal type, internal type and coexisting type. There was a strong predominance for ice crystal initiation site in the oocytes, with up to 80% of the initiation sites located in the marginal region. The intracellular ice growth modes of darkening and twitching cells were characterized by "spreading" and "clustering", respectively. In addition, twitching cells started to recrystallize during rewarming, while darkening cells did not. The instantaneous maximal growth rate of ice crystals in twitching cells was about 10 times higher than that in darkening cells. CONCLUSION: By visualising the growth of ice crystals in mouse oocytes during cooling and rewarming, we obtained valuable information on the kinetics of ice formation and melting in these cells. This information can help us understand how ice formation and melting affect the viability and quality of oocytes after cryopreservation. Doi.org/10.54680/fr24310110412.

Development and evaluation of a usable blastocyst predictive model using the biomechanical properties of human oocytes.

PURPOSE: The purposes of this study were to determine whether biomechanical properties of mature oocytes could predict usable blastocyst formation better than morphological information or maternal factors, and to demonstrate the safety of the aspiration measurement procedure used to determine the biomechanical properties of oocytes. METHODS: A prospective split cohort study was conducted with patients from two IVF clinics who underwent in vitro fertilization. Each patient's oocytes were randomly divided into a measurement group and a control group. The aspiration depth into a micropipette was measured, and the biomechanical properties were derived. Oocyte fertilization, day 3 morphology, and blastocyst development were observed and compared between measured and unmeasured cohorts. A predictive classifier was trained to predict usable blastocyst formation and compared to the predictions of four experienced embryologists. RESULTS: 68 patients and their corresponding 1252 oocytes were included in the study. In the safety analyses, there was no significant difference between the cohorts for fertilization, while the day 3 and 5 embryo development were not negatively affected. Four embryologists predicted usable blastocyst development based on oocyte morphology with an average accuracy of 44% while the predictive classifier achieved an accuracy of 71%. Retaining the variables necessary for normal fertilization, only data from successfully fertilized oocytes were used, resulting in a classifier an accuracy of 81%. CONCLUSIONS: To date, there is no standard guideline or technique to aid in the selection of oocytes that have a higher likelihood of developing into usable blastocysts, which are chosen for transfer or vitrification. This study provides a comprehensive workflow of extracting biomechanical properties and building a predictive classifier using these properties to predict mature oocytes' developmental potential. The classifier has greater accuracy in predicting the formation of usable blastocysts than the predictions provided by morphological information or maternal factors. The measurement procedure did not negatively affect embryo culture outcomes. While further analysis is necessary, this study shows the potential of using biomechanical properties of oocytes to predict embryo developmental outcomes.

The impact of maternal age on aneuploidy in oocytes: Reproductive consequences, molecular mechanisms, and future directions.

Age-related aneuploidy in human oocytes is a major factor contributing to decreased fertility and adverse reproductive outcomes. As females age, their oocytes are more prone to meiotic chromosome segregation errors, leading primarily to aneuploidy. Elevated aneuploidy rates have also been observed in oocytes from very young, prepubertal conceptions. A key barrier to developing effective treatments for age-related oocyte aneuploidy is our incomplete understanding of the molecular mechanisms involved. The challenge is becoming increasingly critical as more people choose to delay childbearing, a trend that has significant societal implications. In this review, we summarize current knowledge regarding the process of oocyte meiosis and folliculogenesis, highlighting the relationship between age and chromosomal aberrations in oocytes and embryos, and integrate proposed mechanisms of age-related meiotic disturbances across structural, protein, and genomic levels. Our goal is to spur new research directions and therapeutic avenues.

Treatment of mares with the non-steroidal anti-inflammatory drug (NSAID) phenylbutazone transiently affects in vitro maturation of equine oocytes and blastocyst development after Intracytoplasmic Sperm Injection (ICSI).

Mares enrolled in assisted reproductive technologies (ARTs) programs are often treated with non-steroidal anti-inflammatory drugs (NSAIDs), particularly phenylbutazone (Bute), due to chronic lameness. The current study was performed to determine the effect of Bute administration on the developmental competence of in vitro-matured equine oocytes subjected to Intracytoplasmic Sperm Injection (ICSI). In a Preliminary Study, immature cumulus-oocyte complexes (COCs) recovered by post-mortem ovary harvested from two healthy mares (n = 2) treated for 10 days with Bute (4.4 mg/kg, PO, BID), and four non-treated healthy mares (n = 4), were matured in vitro and subjected to Piezo-driven ICSI. Lower oocyte in vitro maturation [Bute: 25% (3/12) vs. Control: 61% (28/46)] and blastocyst rates [Bute: 0% (0/12) vs. Control: 18% (5/28)] were observed in the Bute-treated when compared to the Control mares (P < 0.05). In the Main Experiment, a group of healthy mares (n = 9) received a daily dose of Bute (4.4 mg/kg, orally, SID) for 10 days. A control group of mares (n = 10) was treated with an equal volume of placebo. Mares in both groups were subjected to ultrasound-guided transvaginal oocyte aspiration (TVA) on days 3, 33, and 77 following the last dose of Bute (PT). Recovered COCs from both mare groups were matured in vitro and subjected to Piezo-driven ICSI. By day-3 PT, oocyte in vitro maturation rate was similar between mare groups [Bute: 65% (36/55) vs. Control: 67% (78/116); P > 0.05], while oocyte recovery [Bute: 53% (55/103) vs. Control: 70% (116/166)], cleavage [Bute: 31% (11/36) vs. Control: 62% (48/78)] and blastocyst rates [Bute: [0%] (0/36) vs. Control: 28% (22/78)] were significantly different (P < 0.05). By day 33 PT and 77 PT, differences on oocyte recovery, in vitro maturation, cleavage, and blastocyst rates were not observed between mare groups. In summary, the administration of Bute for 10 consecutive days (4.4 mg/kg, PO, SID, or BID) is associated with a decrease in the ability of immature equine oocytes to undergo in vitro-maturation (Preliminary Study) and develop to the blastocyst stage following ICSI (Preliminary Study and Main Experiment). This negative effect appeared to be transient, as 30- and 77-days post-treatment, no differences on in vitro maturation, cleavage or blastocyst rates were observed.

Live birth derived from a markedly large polar body oocyte: a rare case report.

Oocytes with excessively large first polar bodies (PB1) often occur in assisted reproductive procedures. Many times these oocytes are discarded without insemination and, as a result, the application of this portion of oocytes has scarcely been reported to date. Few studies have examined large PB1 oocytes in infertile women and have virtually entirely studied genetic variations for large PB1 oocyte abnormalities. Here, we describe an unusual case of a live birth from a remarkably large PB1 oocyte in a frozen embryo transfer (FET) cycle. This is the first instance of a successful live birth resulting from a PB1 oocyte with an extremely large polar body measuring 80 µM × 40 µM in size. The large PB1 oocyte was performed by an early rescue intracytoplasmic sperm injection (r-ICSI) and was formed into a blastocyst on day 5. Following FET, a healthy boy baby weighing 3100 g was finally delivered by caesarean section at 37 weeks and 5 days after conception. Additionally, there were no complications throughout the antenatal period or the perinatal phase of this following full-term delivery. In this study, it is revealed for the first time that a huge PB1 oocyte can be fertilized, resulting in the growth of a blastocyst, a subsequent pregnancy, and a live birth. This new information prompts us to reconsider the use of large PB1 oocytes. More insightful talks should be given attention to prevent the waste of embryos because not all oocytes with aberrant morphology are unavailable.

Role and action mechanisms of miR-149 and miR-31 in regulating function of pig cumulus cells and oocytes.

Understanding the mechanisms for oocyte maturation and optimizing the protocols for in vitro maturation (IVM) are greatly important for improving developmental potential of IVM oocytes. The miRNAs expressed in cumulus cells (CCs) play important roles in oocyte maturation and may be used as markers for selection of competent oocytes/embryos. Although a recent study from our group identified several new CCs-expressed miRNAs that regulate cumulus expansion (CE) and CC apoptosis (CCA) in mouse oocytes, validation of these findings and further investigation of mechanisms of action in other model species was essential before wider applications. By using both in vitro and in vivo pig oocyte models with significant differences in CE, CCA and developmental potential, the present study validated that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes. We demonstrated that miR-149 and miR-31 targeted SMAD family member 6 (SMAD6) and transforming growth factor ß2 (TGFB2), respectively, in the transforming growth factor-ß (TGF-ß) signaling. Furthermore, both miR-149 and miR-31 increased CE and decreased CCA via activating SMAD family member 2 (SMAD2) and increasing the expression of SMAD2 and SMAD family member 4. In conclusion, the present results show that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes by activating the TGF-ß signaling, suggesting that they might be used as markers for pig oocyte quality.

Melatonin protects oogenesis from hypobaric hypoxia-induced fertility damage in mice.

Environmental hypoxia adversely affects reproductive health in humans and animals at high altitudes. Therefore, how to alleviate the follicle development disorder caused by hypoxia exposure and to improve the competence of fertility in plateau non-habituated female animals are important problems to be solved urgently. In this study, a hypobaric hypoxic chamber was used for 4 weeks to simulate hypoxic conditions in female mice, and the effects of hypoxia on follicle development, proliferation and apoptosis of granulosa cells, reactive oxygen species (ROS) levels in MII oocyte and 2-cell rate were evaluated. At the same time, the alleviating effect of melatonin on hypoxic exposure-induced oogenesis damage was evaluated by feeding appropriate amounts of melatonin daily under hypoxia for 4 weeks. The results showed that hypoxia exposure significantly increased the proportion of antral follicles in the ovary, the number of proliferation and apoptosis granulosa cells in the follicle, and the level of ROS in MII oocytes, eventually led to the decline of oocyte quality. However, these defects were alleviated when melatonin was fed under hypoxia conditions. Together, these findings suggest that hypoxia exposure impaired follicular development and reduced oocyte quality, and that melatonin supplementation alleviated the fertility reduction induced by hypoxia exposure.

A rare germline BMP15 missense mutation causes hereditary ovarian immature teratoma in human.

Ovarian immature teratomas (OITs) are malignant tumors originating from the ovarian germ cells that mainly occur during the first 30 y of a female's life. Early age of onset strongly suggests the presence of susceptibility gene mutations for the disease yet to be discovered. Whole exon sequencing was used to screen pathogenic mutations from pedigrees with OITs. A rare missense germline mutation (C262T) in the first exon of the BMP15 gene was identified. In silico calculation suggested that the mutation could impair the formation of mature peptides. In vitro experiments on cell lines confirmed that the mutation caused an 84.7% reduction in the secretion of mature BMP15. Clinical samples from OIT patients also showed a similar pattern of decrease in the BMP15 expression. In the transgenic mouse model, the spontaneous parthenogenetic activation significantly increased in oocytes carrying the T allele. Remarkably, a mouse carrying the T allele developed the phenotype of OIT. Oocyte-specific RNA sequencing revealed that abnormal activation of the H-Ras/MAPK pathway might contribute to the development of OIT. BMP15 was identified as a pathogenic gene for OIT which improved our understanding of the etiology of OIT and provided a potential biomarker for genetic screening of this disorder.

Vitamin D and follicular recruitment in the in vitro fertilization cycle.

OBJECTIVE: Vitamin D (VD) is a fat-soluble steroid hormone, synthesized by the skin, most known for its role in bone mineral balance. Vitamin D receptors (VDR) are also found in the female reproductive system, but their role remains unclear. The objective of this study was to analyze the relationship between serum vitamin D levels and the number of oocytes retrieved after ovarian stimulation. METHODS: This is a retrospective study involving 267 patients undergoing in vitro fertilization (IVF) carried out in the Fertipraxis clinic, a private practice facility. The patients were initially divided into two groups according to their VD levels. Group 1 included 152 patients with VD levels < 30 ng/mL and group 2 had 115 patients with VD levels > 30 ng/mL. They were further analyzed and separated considering their age, anthropometric data, ovarian reserve, amount of gonadotropin used, and follicles obtained until trigger day. RESULTS: In our analysis, there were no difference in the number of follicles and oocytes retrieved, nor in the number of mature oocytes obtained from patients with both vitamin D deficiency and sufficiency. CONCLUSIONS: The results of our study show no difference among number of follicles, oocytes retrieved and mature oocytes obtained after ovarian stimulation according to their vitamin D serum levels. Further higher-quality studies are needed to evaluate the possible roles of serum vitamin D levels in other stages of human fertilization process.

Effect of chemical activators after intracytoplasmic sperm injection (ICSI) on embryo development in alpacas.

Low motility and low sperm concentration are characteristics of alpaca semen. Thus, the intracytoplasmic sperm injection (ICSI) technique represents an alternative to improve the reproductive capacity of the male. However, the effect of post-ICSI activation in alpaca is not yet known. The aim of the present study was to compare the effect of chemical activators on alpaca embryo development after ICSI. Alpaca ovaries were collected from a local slaughterhouse and transported to the laboratory. Category I, II and III oocytes were matured for 30 h at 38.5 °C. After ICSI, injected oocytes were randomly divided and activated as follows: i) 5 µM ionomycin for 5 min, ii) 7% ethanol for 4 min, iii) 5 µM ionomycin for 5 min, window period 3 h plus 7% ethanol for 4 min, iv) 5 µM ionomycin for 5 min, window period 3 h, a second ionomycin treatment for 5 min, followed by 1.9 mM 6-DMAP for 3 h, v) 10 mM SrCl2 for 3 h. Culture was carried out for 5 days in SOFaa at 38.5 °C. The cleavage rate was the lowest in the SrCl2 group, morula development was the lowest in the SrCl2 and without activation groups, and blastocyst stage was not different between groups (P<0.05). The rates with SrCl2 were lower in total embryos produced, whereas in transferable embryos they were lower with 2Io/6-DMAP and with SrCl2 (P<0.05). In conclusion, alpaca oocyte activation is more efficient with ionomycin and ethanol to produce transferable embryos.

Histological and Cytological Techniques to Study Perinatal Mouse Ovaries and Oocytes.

The regulation of female fertility in mammals depends on critical processes during oocyte development and maturation. Therefore, it is crucial to use specific approaches when studying mammalian female fertility to preserve ovary and oocyte structures effectively. The methods of collecting and culturing ovaries and oocytes play an essential role in the study of mammalian follicle development and oocyte quality. This chapter presents a collection of protocols that focus on various methods for studying mammalian ovaries and oocytes, providing researchers with a variety of approaches to choose from.

Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells.

Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor ß (Esr2/ERß) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERß and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERß and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERß and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERß and GPER in cumulus cells.

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

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

Identification and molecular characterization of genes modulating progression of an oocyte from M-I to M-II in rat ovary.

BACKGROUND: To achieve oocyte competence for successful fertilization, bidirectional communication between oocyte and granulosa cells is crucial. The acquisition of meiotic competency in oocyte is facilitated by various regulatory genes however, expression pattern of these genes is not well documented during meiotic transition from Metaphase-I to Metaphase-II stage. Therefore, the present research analyzed the expression pattern of regulatory genes that are involved in the transition from M-I to M-II stages in rat oocyte. METHODS: The analysis of the data was conducted by applying an array of bioinformatic tools. The investigation of gene group interactions was carried out by employing the STRING database, which relies on co-expression information. The gene ontology (GO) analysis was performed utilizing the comparative GO database. Functional annotation for GO and pathway enrichment analysis were performed for genes involved in networking. The GO obtained through computational simulations was subsequently validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. RESULTS: The findings of our study suggest that there is a distinct gene expression pattern in both the oocyte and granulosa cells. This pattern indicates that oocyte-secreted factors, such as BMP15 and GDF9, play a crucial role in regulating the progression of the meiotic cell cycle from the M-I to M-II stages. We have also examined the level of mRNA expression of genes including CYP11A1, CYP19A1, and STAR, which are crucial for the steroidogenesis. CONCLUSIONS: It is fascinating to observe that the oscillatory pattern of specific key genes may hold significance in the process of in vitro oocyte maturation, specifically during the transition from the M-I to M-II stage. It might be useful for determining biomarker genes and potential pathways that play a role in attaining oocyte competency, thereby aiding in the assessment of oocyte quality for the purpose of achieving successful fertilization.

Apigenin delays postovulatory oocyte aging by reducing oxidative stress through SIRT1 upregulation.

After ovulation, senescent oocytes inevitably experience reduced quality and defects in embryonic development. Apigenin (API) is a flavonoid with a wide range of pharmacological effects. Therefore, this study examined the protective effects of API on the quality of porcine oocytes during in-vitro ageing and the underlying mechanisms. The results showed that API treatment could reduce the activation rate after aging for 48 h. In addition, API significantly reduced reactive oxygen species, abnormal distribution of mitochondria, early apoptosis in ageing oocytes, increased glutathione, and mitochondrial adenosine triphosphate levels in ageing oocytes. Importantly, API increased the embryonic development rate in aged oocytes. We also examined molecular changes, finding decreased sirtuin 1 expression in in-vitro postovulatory oocytes, but API reversed this effect. Our results suggest that API attenuates the deterioration of oocyte quality during in-vitro ageing, possibly by reducing oxidative stress through the upregulation of sirtuin 1.

Transcriptome analysis of porcine embryos derived from oocytes vitrified at the germinal vesicle stage.

Vitrification of porcine immature oocytes at the germinal vesicle (GV) stage reduces subsequent embryo yield and changes at the molecular level may occur during embryonic development. Therefore, the present study used porcine parthenogenetic embryos as a model to investigate the effect of GV oocyte vitrification on the transcriptional profiles of the resultant embryos at the 4-cell and blastocyst stages using the Smart-seq2 RNA-seq technique. We identified 743 (420 up-regulated and 323 down-regulated) and 994 (554 up-regulated and 440 down-regulated) differentially expressed genes (DEGs) from 4-cell embryos and blastocysts derived from vitrified GV oocytes, respectively. Functional enrichment analysis of DEGs in 4-cell embryos showed that vitrification of GV oocytes influenced regulatory mechanisms related to transcription regulation, apoptotic process, metabolism and key pathways such as the MAPK signaling pathway. Moreover, DEGs in blastocysts produced from vitrified GV oocytes were enriched in critical biological functions including cell adhesion, cell migration, AMPK signaling pathway, GnRH signaling pathway and so on. In addition, the transcriptomic analysis and quantitative real-time PCR results were consistent. In summary, the present study revealed that the vitrification of porcine GV oocytes could alter gene expression patterns during subsequent embryonic developmental stages, potentially affecting their developmental competence.

Optimizing oocyte electroporation for genetic modification of porcine embryos: Evaluation of the parthenogenetic activation.

In reproductive biology, understanding the effects of novel techniques on early embryo development is of paramount importance. To date, the effects of electrical activation on oocytes prior to in vitro fertilization (IVF) are not well understood. The aim of this study was to investigate the effects of oocyte electroporation prior to IVF on embryo development and to differentiate between true embryos and parthenotes by using a TPCN2 knock-out (KO) male to evaluate the presence of the KO allele in the resulting blastocysts. The study consisted of three experiments. The first one examined oocyte electroporation with and without subsequent IVF and found that electroporated oocytes had higher activation rates, increased occurrence of a single pronucleus, and no effect on sperm penetration. Cleavage rates improved in electroporated oocytes, but blastocyst rates remained constant. Genotype analysis revealed a significant increase in the proportion of parthenotes in the electroporated groups compared to the IVF control (30.2 % vs. 6.8 %). The second experiment compared two electroporation media, Opti-MEM and Nuclease-Free Duplex Buffer (DB). DB induced higher oocyte degeneration rates, and lower cleavage and blastocyst rates than Opti-MEM, while parthenogenetic formation remained consistent (60.0 and 48.5 %). In the third experiment, the timing of electroporation relative to IVF was evaluated (1 h before IVF, immediately before IVF and 7 h after IVF). Electroporation immediately before IVF resulted in higher activation rates and different pronuclear proportions compared to the other timing groups. The penetration rate was higher in the immediate electroporation group, and cleavage rate improved in all electroporated groups compared to the control. Blastocyst rates remained constant. Genotyping revealed no significant differences in parthenote proportions among the timing groups, but these were higher than the control (56.25 %, 63.89 %, 51.61 %, 2.44 %, respectively), and showed higher mutation rates when electroporation was performed 7 h after IVF. Overall, this comprehensive study sheds light on the potential of electroporation for creating genetically modified embryos and the importance of media selection and timing in the process, the best media being the Opti-MEM and the more efficient timing regarding mutation rate, 7 h post-IVF, even when the parthenote formation did not differ among electroporated groups. Further studies are needed to reduce the parthenogenetic activation while maintaining high mutation rates to optimize the use of this procedure for the generation of gene-edited pig embryos by oocyte/zygote electroporation.

SIRT2 regulates apoptosis by inducing mitophagy in sheep cumulus cells.

Cumulus cells surrounding oocytes furnish nutritional support crucial for oocyte maturation in vitro, and thereby enhance oocyte quality significantly. Our previous studies affirmed the role of SIRT2 in regulation of mitochondrial function in sheep granulosa cells. However, the effect of SIRT2 action on mitophagy in these cells remain unclear. Here, RNA-seq was used to scrutinize pathways where differentially expressed genes (DEGs) are enriched following SIRT2 knockdown in cumulus cells. Prior to SIRT2 knock down, cumulus cells were treated with the mitophagy inhibitor Mdivi-1. Potential mechanisms by which SIRT2 affects apoptosis via mitophagy were explored. Results indicated that DEGs after SIRT2 knockdown were enriched in various pathways including mitochondria, mitophagy, and apoptosis. The expression levels of CASP3/CASP9 were significantly increased after mitophagy activation (P < 0.01), whereas inhibition of mitophagy had no effect on apoptosis (P > 0.05). Pretreatment of cumulus cells with Mdivi-1 prior to SIRT2 knockdown significantly reduced the expression of mitophagy-related genes, the number of autolysosomes, the expression of CASP3/CASP9, and the levels of Ca2+ and cytochrome C (P < 0.05). In addition, an improvement in mitochondrial morphology and increases in ATP levels and mitochondrial DNA (mtDNA) copy numbers were observed. Interestingly, double knockdown of SIRT2 and MAPK15 was found to reverse increased mitophagy and apoptosis activity caused by SIRT2 knockdown. Our findings indicate that SIRT2 modulate apoptosis in cumulus cells by regulating mitophagy, with MAPK15 likely playing a pivotal role in this process.

The ability of donkey sperm to induce oocyte activation and mule embryo development after ICSI.

Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey sperm's fertilization capability in vitro to further improve donkey ICSI. In this work, we initially studied the localization of donkey sperm Phospholipase C zeta (PLCζ) and assessed the sperm's capacity to induce pronuclear formation and maternal SMARCA4 recruitment upon injection into pig oocytes through ICSI. Subsequently, we investigated the injection of donkey sperm into horse oocytes, evaluating in vitro production up to the blastocyst stage using sperm from different jacks, including frozen and refrigerated samples. Distinct patterns of PLCζ localization were observed for donkey sperm cells compared to their horse counterparts. Additionally, donkey sperm exhibits a reduced ability to induce porcine oocyte activation. However, when injected into horse oocytes, donkey sperm demonstrated sufficient capability to induce oocyte activation as no discernible differences in cleavage or blastocyst rates are observed between in vitro produced mules and horse ICSI embryos. Our study not only delineates PLCζ localization in donkey sperm but also suggests potential differences in the ability to induce oocyte activation in pigs compared to horses while observing no distinctions in pronuclear recruitment of SMARCA4. Interestingly, donkey sperm remains sufficiently capable of inducing horse oocyte activation for in vitro mule blastocyst production.

Oocyte competence and gene expression in parthenogenetic produced embryos from repeat breeder and normally fertile buffaloes (Bubalus bubalis) raised in sub-humid tropical climate.

The reproductive management of the buffalo species still faces several unresolved problems, which directly affect the productivity of the herd, one of them being the presence of repeat breeder females. Given this scenario, this study aimed to verify the developmental competence of oocytes obtained from repeat breeder females and submitted to parthenogenetic activation. In addition, embryo gene expression was compared to normally fertile females. Murrah buffaloes were divided into two groups: repeat breeder (RB, n = 8) and normally fertile or control (CR, n = 7). Cumulus-oocyte complexes (COCs) were aspirated by transvaginal ovum pick-up from estrus synchronized females. The COCs were submitted to IVM for 24 h, and subsequently, the oocytes were activated using ionomycin, followed by 6-DMAP. Afterwards, the presumptive parthenotes were cultured for six or seven days in a microenvironment of 5 % CO2, 5 % O2, and 90 % N2 at 38.5 °C. The expression of OCT4, GLUT1, BCL2 and TFAM genes from blastocysts was evaluated. The overall COCs recovery rate was 70.9 % (190/268). The maturation (57.8 vs 71.1), cleavage (45.2 vs 62.2) and blastocyst (30.1 vs 45.9) rates did not differ (P > 0.05) between RB and CR females, respectively. Similarly, no significant difference (P > 0.05) was observed for the expression of studied genes in both RB and CR females. In conclusion, oocytes obtained from RB were as developmentally competent as those collected from CR females, with similar energy metabolism and in vitro development capacity. Thus, the low fertility rate of repeat breeder buffaloes, when compared to normal cyclic females, must be due to subsequent events to the blastocyst stage.