Impact of Extraction Methods and Transportation Conditions on Lipid Profiles of Bovine Oocytes.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Lipopolysaccharide-binding protein in follicular fluid is associated with the follicular inflammatory status and granulosa cell steroidogenesis in dairy cows.

Metabolic stress and subsequent hepatic dysfunction in high-producing dairy cows are associated with inflammatory diseases and declining fertility. Lipopolysaccharide (LPS)-binding protein (LBP) is produced by hepatocytes and controls the immune response, suggesting that it is involved in the pathophysiology of inflammation-related attenuation of reproductive functions during metabolic stress. This study investigated the effect of LBP on the inflammatory status, oocyte quality, and steroidogenesis in the follicular microenvironment of dairy cows. Using bovine ovaries obtained from a slaughterhouse, follicular fluid and granulosa cells were collected from large follicles to evaluate the follicular status of metabolism, inflammation, and steroidogenesis. Cumulus-oocyte complexes were aspirated from small follicles and subjected to in vitro embryo production. The results showed that follicular fluid LBP concentrations were significantly higher in cows with fatty livers and hepatitis than in those with healthy livers. Follicular fluid LBP and LPS concentrations were negatively correlated, whereas LPS concentration showed a positive correlation with the concentrations of non-esterified fatty acids (NEFA) and ß-hydroxybutyric acid in follicular fluid. The blastulation rate of oocytes after in vitro fertilization was impaired in cows in which coexisting large follicles had high NEFA levels. Follicular fluid NEFA concentration was negatively correlated with granulosa cell expression of the estradiol (E2) synthesis-related gene (CYP19A1). Follicular fluid LBP concentration was positively correlated with follicular fluid E2 concentration and granulosa cell CYP19A1 expression. In conclusion, follicular fluid LBP may be associated with favorable conditions in the follicular microenvironment, including low LPS levels and high E2 production by granulosa cells.

Exploring gonadotropin dosing effects on MII oocyte retrieval in ovarian stimulation.

PURPOSE: Ovarian stimulation with gonadotropins is crucial for obtaining mature oocytes for in vitro fertilization (IVF). Determining the optimal gonadotropin dosage is essential for maximizing its effectiveness. Our study aimed to develop a machine learning (ML) model to predict oocyte counts in IVF patients and retrospectively analyze whether higher gonadotropin doses improve ovarian stimulation outcomes. METHODS: We analyzed the data from 9598 ovarian stimulations. An ML model was employed to predict the number of mature metaphase II (MII) oocytes based on clinical parameters. These predictions were compared with the actual counts of retrieved MII oocytes at different gonadotropin dosages. RESULTS: The ML model provided precise predictions of MII counts, with the AMH and AFC being the most important, and the previous stimulation outcome and age, the less important features for the prediction. Our findings revealed that increasing gonadotropin dosage did not result in a higher number of retrieved MII oocytes. Specifically, for patients predicted to produce 4-8 MII oocytes, a decline in oocyte count was observed as gonadotropin dosage increased. Patients with low (1-3) and high (9-12) MII predictions achieved the best results when administered a daily dose of 225 IU; lower and higher doses proved to be less effective. CONCLUSIONS: Our study suggests that high gonadotropin doses do not enhance MII oocyte retrieval. Our ML model can offer clinicians a novel tool for the precise prediction of MII to guide gonadotropin dosing.

Fructose improves titanium dioxide nanoparticles induced alterations in developmental competence of mouse oocytes.

AIMS: We investigated the effects of intraperitoneal injections of titanium dioxide nanoparticles (TiO2 NPs, 100 mg/kg) for 5 consecutive days on the developmental competence of murine oocytes. Furthermore, study the effects of TiO2 NPs on antioxidant and oxidative stress biomarkers, as well as their effects on expression of apoptotic and hypoxia inducing factor-1α (HIF1A) protein translation. Moreover, the possible ameliorating effects of intraperitoneal injections of fructose (2.75 mM/ml) was examined. MATERIALS AND METHODS: Thirty sexually mature (8-12 weeks old; ~ 25 g body weight) female mice were used for the current study. The female mice were assigned randomly to three treatment groups: Group1 (G1) mice were injected intraperitoneal (ip) with deionized water for 5 consecutive days; Group 2 (G2) mice were injected ip with TiO2 NPs (100 mg/kg BW) for 5 consecutive days; Group 3 (G3) mice were injected ip with TiO2 NPs (100 mg/kg BW + fructose (2.75 mM) for 5 consecutive days. RESULTS: Nano-titanium significantly decreased expression of GSH, GPx, and NO, expression of MDA and TAC increased. The rates of MI, MII, GVBD and degenerated oocytes were significantly less for nano-titanium treated mice, but the rate of activated oocytes was significantly greater than those in control oocytes. TiO2 NPs significantly increased expression of apoptotic genes (BAX, Caspase 3 and P53) and HIF1A. Intraperitoneal injection of fructose (2.75 mM/kg) significantly alleviated the detrimental effects of TiO2 NPs. Transmission electron microscopy indicated that fructose mitigated adverse effects of TiO2 NPs to alter the cell surface of murine oocytes. CONCLUSION: Results of this study suggest that the i/p infusion of fructose for consecutive 5 days enhances development of murine oocytes and decreases toxic effects of TiO2 NPs through positive effects on oxidative and antioxidant biomarkers in cumulus-oocyte complexes and effects to inhibit TiO2-induced increases in expression of apoptotic and hypoxia inducing factors.

C-Phycocyanin improves the quality of goat oocytes after in vitro maturation and vitrification.

In vitro maturation (IVM) and cryopreservation of goat oocytes are important for establishing a valuable genetic bank for domesticated female animals and improving livestock reproductive efficiency. C-Phycocyanin (PC) is a Spirulina extract with antioxidant, antiinflammatory, and radical scavenging properties. However, whether PC has positive effect on goat oocytes IVM or developmental competence after vitrification is still unknown. In this study, we found that first polar body extrusion (n = 293), cumulus expansion index (n = 269), and parthenogenetic blastocyst formation (n = 281) were facilitated by adding 30 µg/mL PC to the oocyte maturation medium when compared with the control groups and that supplemented with 3, 10, 100 or 300 µg/mL PC (P < 0.05). Although PC supplementation did not affect spindle formation or chromosome alignment (n = 115), it facilitated or improved cortical granules migration (n = 46, P < 0.05), mitochondria distribution (n = 39, P < 0.05), and mitochondrial membrane potential (n = 46, P < 10-4). Meanwhile, supplementation with 30 µg/mL PC in the maturation medium could significantly inhibit the reactive oxygen species accumulation (n = 65, P < 10-4), and cell apoptosis (n = 42, P < 0.05). In addition, PC increased the oocyte mRNA levels of GPX4 (P < 0.01), and decreased the mRNA and protein levels of BAX (P < 0.01). Next, we investigated the effect of PC supplementation in the vitrification solution on oocyte cryopreservation. When compared with the those equilibrate in the vitrification solution without PC, recovered oocytes in the 30 µg/mL PC group showed higher ratios of normal morphology (n = 85, P < 0.05), survival (n = 85, P < 0.05), first polar body extrusion (n = 62, P < 0.05), and parthenogenetic blastocyst formation (n = 107, P < 0.05). Meanwhile, PC supplementation of the vitrification solution increased oocyte mitochondrial membrane potential (n = 53, P < 0.05), decreased the reactive oxygen species accumulation (n = 73, P < 0.05), promoted mitochondria distribution (n = 58, P < 0.05), and inhibited apoptosis (n = 46, P < 10-3). Collectively, our findings suggest that PC improves goat oocyte IVM and vitrification by reducing oxidative stress and early apoptosis, which providing a novel strategy for livestock gamete preservation and utilization.

Frozen autologous and donor oocytes are associated with differences in clinical and neonatal outcomes compared with fresh oocytes: a Society for Assisted Reproductive Technology Clinic Outcome Reporting System Analysis.

Objective: To study the clinical and neonatal outcomes of embryos derived from frozen oocytes relative to fresh oocytes in both autologous and donor oocyte cycles after fresh embryo transfer (ET). Design: This is a retrospective cohort study using the Society for Assisted Reproductive Technology Clinic Outcome Reporting System database between 2014 and 2015. Setting: The Society for Assisted Reproductive Technology Clinic Outcome Reporting System database was used to identify autologous and donor oocyte cycles that resulted in a fresh ET during 2014 and 2015. Patients: There were 154,706 total cycles identified that used embryos derived from fresh or frozen oocytes and resulted in a fresh ET, including 139,734 autologous oocyte cycles and 14,972 donor oocyte cycles. Interventions: Generalized linear regression models were used to compare the clinical and neonatal outcomes of frozen oocytes relative to fresh oocytes. Models were adjusted for maternal age, body mass index, smoking status, parity, infertility diagnosis, number of embryos transferred, and preimplantation genetic testing. An additional sensitivity analysis was performed to examine singleton pregnancies separately. Main Outcome Measures: The live birth (LB) rate was the primary outcome. Secondary outcomes include pregnancy and birthweight outcomes. Results: Differences in clinical and neonatal outcomes between fresh and frozen-thawed oocytes after fresh ET were observed. Specifically, our study found a higher incidence of high-birthweight infants after the use of frozen oocytes relative to fresh oocytes in both autologous oocytes (12.5% [frozen] vs. 4.5% [fresh], adjusted risk ratio [aRR] 2.67, 95% confidence interval [CI] 1.65-4.3) and donor oocyte cycles (6.2% [frozen] vs. 4.6% [fresh], aRR 1.42, 95% CI 1.1-1.83). This finding remained true when the analysis was restricted to singleton gestations only for both groups: autologous (17.3% [frozen] vs. 7.1% [fresh], aRR 2.77, 95% CI 1.74-4.42) and donor oocytes (9.4% [frozen] vs. 7.8% [fresh], aRR 1.38, 95% CI 1.07-1.77). Additionally, we observed a decrease in LB (aRR 0.81, 95% CI 0.77-0.85); clinical pregnancy (aRR 0.83, 95% CI 0.8-0.87); and an increase in biochemical pregnancy loss (aRR 1.22, 95% CI 1.05-1.43) after the use of frozen oocytes in donors, but not autologous cycles. Conclusions: Our findings of an increased incidence of high-birthweight infants after the transfer of embryos derived from frozen oocytes in both autologous and donor oocyte cycles raise questions about oocyte vitrification and deserve further study. Additionally, the finding of a decreased likelihood of LB with frozen-donor oocytes compared with fresh donor oocytes is an important finding, especially because more patients are seeking to use frozen oocytes in their donor egg cycles. Future research should be directed toward these findings to optimize the use of frozen oocytes in clinical practice.

Unravelling the role of HAS2, GREM1, and PTGS2 gene expression in cumulus cells: implications for human oocyte development competency - a systematic review and integrated bioinformatic analysis.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

Women's reproductive choice and (elective) egg freezing: is an extension of the storage limit missing a bigger issue?

Egg freezing can allow women to preserve their eggs to avoid age-related infertility. The UK's recent extension of elective egg freezing storage has been welcomed as a way of enhancing the reproductive choices of young women who wish to delay having children. In this paper, I explore the issue of enhancing women's reproductive choices, questioning whether there is a more significant aspect overlooked in egg freezing. While increasing storage limits expands reproductive choices for some women, focus on this extension alone, I argue, misses a fundamental issue with egg preservation that often remains ignored; the importance of effective information on egg freezing and the effect this has on women's reproductive choices. Ultimately, I highlight the crucial role of balanced information in enhancing women's choices regarding egg freezing and argue that focusing on extending and increasing provision may obscure this real opportunity to empower women and their authentic reproductive choices.

Superior performance of biocomposite nanoparticles PLGA-RES in protecting oocytes against vitrification stimuli.

Irreversible cryogenic damage caused by oocyte vitrification limits its widespread use in female fertility preservation. In recent years, nanoparticles (NPs) have gained great attention as potential alternatives in protecting oocytes against cryoinjuries. In this paper, a novel composite nanoparticle, poly (lactic-co-glycolic acid)-resveratrol (PLGA-RES) was designed to improve the biocompatibility and sustained release properties by encapsulating natural antioxidant RES into PLGA NPs. Firstly, biotoxicity and oxidation resistance of PLGA-RES were determined, and the results showed that PLGA-RES had nontoxic effect on oocyte survival during in vitro maturation (IVM) (97.08% ± 0.24% vs. 98.89% ± 1.11%, p > 0.05). Notably, PLGA-RES even increased maturation (65.10% ± 4.11% vs. 52.85% ± 2.87%, p < 0.05) and blastocyst rate (56.13% ± 1.36% vs. 40.91% ± 5.85%, p < 0.05). Moreover, the reduced reactive oxygen species (ROS) level (13.49 ± 2.30 vs. 34.07 ± 3.30, p < 0.01), increased glutathione (GSH) (44.13 ± 1.57 vs. 37.62 ± 1.79, p < 0.01) and elevated mitochondrial membrane potential (MMP) levels (43.10 ± 1.81 vs. 28.52 ± 1.25, p < 0.01) were observed in oocytes treated with PLGA-RES when compared with that of the control group. Subsequently, the role of PLGA-RES played in oocytes during vitrification was systematically evaluated. The results showed that the addition of PLGA-RES during vitrification and thawing significantly improved the survival rate (80.42% ± 1.97% vs. 75.37% ± 1.3%, p < 0.05). Meanwhile, increased GSH (15.09 ± 0.86 vs. 14.51 ± 0.78, p < 0.01) and mitochondrial membrane potential (22.56 ± 3.15 vs. 6.79 ± 0.60, p < 0.01), decreased reactive oxygen species levels (52.11 ± 2.95 vs. 75.41 ± 7.23, p < 0.05) and reduced mitochondrial abnormality distribution rate (25.00% ± 0.29% vs. 33.33% ± 1.15%, p < 0.01) were assessed in vitrified MII oocytes treated with PLGA-RES. Furthermore, transcriptomic analyses demonstrated that PLGA-RES participated in endocytosis and PI3K/AKT/mTOR pathway regulation, which was verified by the rescued expression of ARRB2 and ULK3 protein after PLGA-RES treatment. In conclusion, PLGA-RES exhibited potent antioxidant activity, and could be used as an efficacious strategy to improve the quality of vitrified oocytes.

Divergent composition and transposon-silencing activity of small RNAs in mammalian oocytes.

BACKGROUND: Small RNAs are essential for germ cell development and fertilization. However, fundamental questions remain, such as the level of conservation in small RNA composition between species and whether small RNAs control transposable elements in mammalian oocytes. RESULTS: Here, we use high-throughput sequencing to profile small RNAs and poly(A)-bearing long RNAs in oocytes of 12 representative vertebrate species (including 11 mammals). The results show that miRNAs are generally expressed in the oocytes of each representative species (although at low levels), whereas endo-siRNAs are specific to mice. Notably, piRNAs are predominant in oocytes of all species (except mice) and vary widely in length. We find PIWIL3-associated piRNAs are widespread in mammals and generally lack 3'-2'-O-methylation. Additionally, sequence identity is low between homologous piRNAs in different species, even among those present in syntenic piRNA clusters. Despite the species-specific divergence, piRNAs retain the capacity to silence younger TE subfamilies in oocytes. CONCLUSIONS: Collectively, our findings illustrate a high level of diversity in the small RNA populations of mammalian oocytes. Furthermore, we identify sequence features related to conserved roles of small RNAs in silencing TEs, providing a large-scale reference for future in-depth study of small RNA functions in oocytes.

Improvement of survivability and developmental ability in vitrified rat oocytes.

Oocyte cryopreservation is useful for human fertility treatment and strain preservation in both experimental and domestic animals. However, the embryonic development of vitrified rat oocytes was lower than that of vitrified embryos. To increase the viability of vitrified oocytes, intracellular ice formation during cooling and warming must be prevented. Rapid warming is important to prevent ice formation. Furthermore, suppressing the spontaneous activation of oocytes is also important because vitrification promotes the spontaneous activation of rat oocytes, and thus compromise developmental competence of the gametes. MG132, a proteasome inhibitor, suppresses the spontaneous activation of rat oocytes. Here, we examined the effects of rapid warming and MG132 treatment on the survival and embryonic development of vitrified rat oocytes. The warming rate was adjusted by changing the vitrification solution volume and warming solution temperature. The survival rate of oocytes vitrified in 10 µL solution and warmed at 50 °C (94%) was significantly higher than that of oocytes vitrified in 100 µL and 10 µL solution and warmed at 37 °C (49% and 81%, respectively). Furthermore, the rate of embryonic development of vitrified oocytes treated with MG132 during vitrification, warming, and intracytoplasmic sperm injection (ICSI) (44%) was significantly higher than that of untreated gametes (10%). Offspring were obtained after transferring embryos derived from MG132-treated vitrified oocytes (14%). Altogether, the survivability of vitrified rat oocytes increased by rapid warming, and MG132 improved embryonic development after ICSI.

Loss of UBE2S causes meiosis I arrest with normal spindle assembly checkpoint dynamics in mouse oocytes.

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.

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.

Fibronectin 1 supports oocyte in vitro maturation in pigs.

Oocyte in vitro maturation (IVM) based on the follicular fluid (FF) environment can exploit untapped resources, however, what FF factors regulate oocyte maturation remains unclear. This work demonstrated that serum and FF significantly promoted oocyte polar body extrusion (PBE) and subsequent embryo development, and FF was especially effective. Fibronectin 1 (FN1) was predicted as one potential candidate to regulate oocyte maturation by proteomics. FN1 transcription obviously decreased, and the protein expression significantly increased and migrated to plasma membrane or even outside during oocyte IVM. Treatment with 10 ng/mL FN1 significantly improved oocyte PBE rate. FN1 significantly upregulated the percentage of regular spindle morphology, downregulated the γ-H2AX level, decreased the levels of ROS and apoptosis, and increased GSH and mitochondrion contents by ameliorating the expression of corresponding genes. Moreover, FN1 significantly increased the p-PI3K level to enhance the activation of PI3K signaling pathway. In conclusion, this study discovers and confirms that FN1 is one factor in FF that significantly enhances oocyte maturation, and the underlying mechanism is that FN1 ameliorates oocyte nuclear and cytoplasmic maturation by promoting the activation of PI3K signaling pathway.

Ursolic acid alleviates meiotic abnormalities induced by 3-nitropropionic acid in mouse oocytes.

3-nitropropionic acid (3-NPA), a toxic metabolite produced by mold, is mainly found in moldy sugarcane. 3-NPA inhibits the activity of succinate dehydrogenase that can induce oxidative stress injury in cells, reduce ATP production and induce oxidative stress in mouse ovaries to cause reproductive disorders. Ursolic acid (UA) has a variety of biological activities and is a pentacyclic triterpene compound found in many plants. This experiment aimed to investigate the cytotoxicity of 3-NPA during mouse oocyte in vitro maturation and the protective effects of UA on oocytes challenged with 3-NPA. The results showed that UA could alleviate 3-NPA-induced oocyte meiotic maturation failure. Specifically, 3-NPA induced a decrease in the first polar body extrusion rate of oocytes, abnormal distribution of cortical granules, and an increase in the proportion of spindle abnormalities. In addition, 3-NPA caused mitochondrial dysfunction and induced oxidative stress, including decreases in the GSH, mitochondrial membrane potential and ATP levels, and increases in the ROS levels, and these effects led to apoptosis and autophagy. The addition of UA could significantly improve the adverse effects caused by 3-NPA. In general, our data show that 3-NPA affects the normal development of oocytes during the in vitro culture, and the addition of UA can effectively repair the damage caused by 3-NPA to oocytes.

XPR1: a regulator of cellular phosphate homeostasis rather than a Pi exporter.

Phosphate (Pi) is an essential nutrient, and its plasma levels are under tight hormonal control. Uphill transport of Pi into cells is mediated by the two Na-dependent Pi transporter families SLC34 and SLC20. The molecular identity of a potential Pi export pathway is controversial, though XPR1 has recently been suggested by Giovannini and coworkers to mediate Pi export. We expressed XPR1 in Xenopus oocytes to determine its functional characteristics. Xenopus isoforms of proteins were used to avoid species incompatibility. Protein tagging confirmed the localization of XPR1 at the plasma membrane. Efflux experiments, however, failed to detect translocation of Pi attributable to XPR1. We tested various counter ions and export medium compositions (pH, plasma) as well as potential protein co-factors that could stimulate the activity of XPR1, though without success. Expression of truncated XPR1 constructs and individual domains of XPR1 (SPX, transmembrane core, C-terminus) demonstrated downregulation of the uptake of Pi mediated by the C-terminal domain of XPR1. Tethering the C-terminus to the transmembrane core changed the kinetics of the inhibition and the presence of the SPX domain blunted the inhibitory effect. Our observations suggest a regulatory role of XPR1 in cellular Pi handling rather than a function as Pi exporter. Accordingly, XPR1 senses intracellular Pi levels via its SPX domain and downregulates cellular Pi uptake via the C-terminal domain. The molecular identity of a potential Pi export protein remains therefore elusive.

Effect of different concentrations of resveratrol on nuclear maturation and in-vitro development competence of oocytes of Nili Ravi buffalo.

Buffaloes are considered animals of the future with the ability to survive under unfavorable conditions. However, the lack of access to superior germplasm poses a significant challenge to increasing buffalo production. Resveratrol has been shown to improve oocyte quality and developmental competence in various animals during in vitro embryo development. However, limited information is available on the use of resveratrol to improve the in vitro maturation and development competence of Nili Ravi buffalo oocytes. Therefore, the current study aimed to investigate the influence of different concentrations of resveratrol on the maturation, fertilization, and development of buffalo oocytes under in vitro conditions. Oocytes were collected from ovaries and subjected to in vitro maturation (IVM) using varying concentrations of resveratrol (0 µM, 0.5 µM, 1 µM, 1.5 µM, and 2 µM), and the maturation process was assessed using a fluorescent staining technique. Results indicated no significant differences in oocyte maturation, morula rate, and blastocyst rate among the various resveratrol concentrations. However, the cleavage rate notably increased with 1 µM and 1.5 µM concentrations of resveratrol (p < 0.05). In conclusion, the study suggests that adding 1 µM of resveratrol into the maturation media may enhance the cleavage and blastocyst hatching of oocytes of Nili Ravi buffaloes. These findings hold promise for advancing buffalo genetics, reproductive performance, and overall productivity, offering potential benefits to the dairy industry, especially in Asian countries.

Blastocyst formation in vitrified-warmed preimplantation embryos derived from vitrified-warmed oocytes in a mouse model.

OBJECTIVE: The purpose of this study was to use a mouse model to investigate the blastocyst formation rate in vitrified-warmed embryos derived from vitrified-warmed oocytes. METHODS: Metaphase II oocytes obtained from BDF1 mice were vitrified and warmed, followed by fertilization with epididymal sperm. On day 3, a total of 176 embryos, at either the eight-cell or the morula stage, were vitrified-warmed (representing group 1). For group 2, 155 embryos at the same developmental stages were not vitrified, but rather were directly cultured until day 5. Finally, group 3 included day-5 blastocysts derived from fresh oocytes, which served as fresh controls. The primary outcome measured was the rate of blastocyst formation per day-3 embryo at the eight-cell or morula stage. RESULTS: The rates of blastocyst formation per day-3 embryo were comparable between groups 1 and 2, at 64.5% and 69.7%, respectively (p>0.05). The formation rates of good-quality blastocysts (expanded, hatching, or hatched) were also similar for groups 1 and 2, at 35.5% and 43.2%, respectively (p>0.05). For the fresh oocytes (group 3), the blastocyst formation rate was 75.5%, which was similar to groups 1 and 2. However, the rate of good-quality blastocyst formation in group 3 was 57.3%, significantly exceeding those of group 1 (p=0.001) and group 2 (p=0.023). CONCLUSION: Regarding developmental potential to the blastocyst stage, vitrified-warmed day-3 embryos originating from vitrified-warmed oocytes demonstrated comparable results to non-vitrified embryos from similar oocytes. These findings indicate that day-3 embryos derived from vitrified-warmed oocytes can be effectively cryopreserved without incurring cellular damage.

In vitro culture of mechanically isolated murine primary follicles in the presence of human platelet lysate PLTMax.

OBJECTIVE: To develop a system for the culture of murine preantral ovarian follicles using Human Serum Albumin (HSA) and Human Platelet Lysate (PLTMax). METHODS: Mechanically isolated preantral follicles (N=146) were obtained from Swiss mice and cultured in DMEM:F12 medium for ten days in a 96-well plate with conical bottom. The medium was supplemented with penicillin, streptomycin, and equine chorionic gonadotropin. Additional proteins were tested in 4 test groups: G1: human serum albumin (HSA), G2: human platelet lysate (PLTM), and G3 and G4: HSA + PLTMax at lower and higher concentrations, respectively. Cellular vitality and oocyte morphology were evaluated on day 11 of culture. RESULTS: The highest follicular growth (3.4 fold) was achieved in HSA (G1), while a significantly lower (1.8 fold) growth was achieved in the presence of PLTM (G2, G4) and even further reduced (1.2 fold) when HSA and PLTM were combined (G3). Cellular vitality was close to 70-80% among the four groups, and the highest number of intact oocytes were found in G1. CONCLUSIONS: PLTM did not improve follicular development and oocyte maturation compared to HSA but preserved cell vitality.

Effects of Leonurine on oocyte maturation and parthenogenetic embryo development in sheep.

Leonurine (LEO), an alkaloid isolated from Leonurus spp., has anti-oxidant, anti-inflammatory and anti-apoptotic effects and can prevent damage caused by reactive oxygen species (ROS). These properties suggest that it can improve the maturation rate of oocytes and developmental ability of embryos, which are key parameters in animal breeding. In this study, the effects of LEO on in vitro maturation and early embryonic development in sheep oocytes were evaluated. Among various doses examined (0, 10, 20 and 40 µM), a dose of 20 µM was optimal with respect to the oocyte maturation rate. Compared with estimates in the control group, GSH levels and mitochondrial membrane potential of sheep oocytes treated with 20 µM LEO were significantly higher, and 40 µM LEO would affect oocyte maturation. Additionally, ROS levels were significantly lower, expression levels of the antioxidant genes CAT and SOD1 were significantly higher, and there was no significant difference in GPX3 expression. The Bax/Bcl-2 ratio and Caspase-3 expression were significantly reduced in the 20 µM LEO group. During early embryonic development in vitro, the cleavage rate and blastocyst rate were significantly higher in the 20 µM LEO treatment group compared to other groups. GSH levels and mitochondrial membrane potential were significantly higher, while ROS levels were significantly lower, and expression levels of the antioxidant genes CAT, GPX3 and SOD1 were significantly higher in eight-cell embryos treated with 20 µM LEO than in the control group. The Bax/Bcl-2 ratio and Caspase-3 levels were significantly decreased. In summary, LEO can reduce the effect of oxidative stress, improve the oocyte maturation rate and enhance embryonic development.