NEK2 plays an essential role in porcine embryonic development by maintaining mitotic division and DNA damage response via the Wnt/ß-catenin signalling pathway.

NIMA-related kinase 2 (NEK2) is a serine/threonine protein kinase that regulates mitosis and plays pivotal roles in cell cycle regulation and DNA damage repair. However, its function in porcine embryonic development is unknown. In this study, we used an NEK2-specific inhibitor, JH295 (JH), to investigate the role of NEK2 in embryonic development and the underlying regulatory mechanisms. Inhibition of NEK2 after parthenogenesis activation or in vitro fertilization significantly reduced the rates of cleavage and blastocyst formation, the numbers of trophectoderm and total cells and the cellular survival rate compared with the control condition. NEK2 inhibition delayed cell cycle progression at all stages from interphase to cytokinesis during the first mitotic division; it caused abnormal nuclear morphology in two- and four-cell stage embryos. Additionally, NEK2 inhibition significantly increased DNA damage and apoptosis, and it altered the expression levels of DNA damage repair- and apoptosis-related genes. Intriguingly, NEK2 inhibition downregulated the expression of ß-catenin and its downstream target genes. To validate the relationship between Wnt/ß-catenin signalling and NEK2 during porcine embryonic development, we cultured porcine embryos in JH-treated medium with or without CHIR99021, a Wnt activator. CHIR99021 co-treatment strongly restored the developmental parameters reduced by NEK2 inhibition to control levels. Our findings suggest that NEK2 plays an essential role in porcine embryonic development by regulating DNA damage repair and normal mitotic division via the Wnt/ß-catenin signalling pathway.

Enhancement of porcine in vitro embryonic development through luteolin-mediated activation of the Nrf2/Keap1 signaling pathway.

BACKGROUND: Oxidative stress, caused by an imbalance in the production and elimination of intracellular reactive oxygen species (ROS), has been recognized for its detrimental effects on mammalian embryonic development. Luteolin (Lut) has been documented for its protective effects against oxidative stress in various studies. However, its specific role in embryonic development remains unexplored. This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism. RESULTS: After undergoing parthenogenetic activation (PA) or in vitro fertilization, embryos supplemented with 0.5 µmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates, with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control. Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control. Moreover, Lut supplementation significantly augmented mitochondrial content and membrane potential. Intriguingly, activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut, leading to the upregulation of antioxidant-related gene transcription levels. To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development, we cultured PA embryos in a medium supplemented with brusatol, with or without the inclusion of Lut. The positive effects of Lut on developmental competence were negated by brusatol treatment. CONCLUSIONS: Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence, and offers insight into the mechanisms regulating early embryonic development.

Luteolin supplementation during porcine oocyte maturation improves the developmental competence of parthenogenetic activation and cloned embryos.

Luteolin (Lut), a polyphenolic compound that belongs to the flavone subclass of flavonoids, possesses anti-inflammatory, cytoprotective, and antioxidant activities. However, little is known regarding its role in mammalian oocyte maturation. This study examined the effect of Lut supplementation during in vitro maturation (IVM) on oocyte maturation and subsequent developmental competence after somatic cell nuclear transfer (SCNT) in pigs. Lut supplementation significantly increased the proportions of complete cumulus cell expansion and metaphase II (MII) oocytes, compared with control oocytes. After parthenogenetic activation or SCNT, the developmental competence of Lut-supplemented MII oocytes was significantly enhanced, as indicated by higher rates of cleavage, blastocyst formation, expanded or hatching blastocysts, and cell survival, as well as increased cell numbers. Lut-supplemented MII oocytes exhibited significantly lower levels of reactive oxygen species and higher levels of glutathione than control MII oocytes. Lut supplementation also activated lipid metabolism, assessed according to the levels of lipid droplets, fatty acids, and ATP. The active mitochondria content and mitochondrial membrane potential were significantly increased, whereas cytochrome c and cleaved caspase-3 levels were significantly decreased, by Lut supplementation. These results suggest that Lut supplementation during IVM improves porcine oocyte maturation through the reduction of oxidative stress and mitochondria-mediated apoptosis.

Cadmium exposure impairs oocyte meiotic maturation by inducing endoplasmic reticulum stress in vitro maturation of porcine oocytes.

Cadmium (Cd) is toxic metal that can induce various diseases, such as cardiovascular, nervous, and reproductive systems. This study investigated the effect of Cd exposure on porcine oocyte maturation and the underlying mechanism. Porcine cumulus-oocyte complexes were exposed various Cd concentration and tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum (ER) stress during in vitro maturation (IVM). After IVM, we evaluated meiotic maturation, ER stress, and oocyte quality by Cd exposure. Cd exposure inhibited cumulus cell expansion and meiotic maturation, increased oocyte degeneration, and induced ER stress. The levels of spliced XBP1 and ER stress-associated transcripts, markers of ER stress, were elevated in Cd-treated cumulus-oocyte complexes and denuded oocytes during IVM. Moreover, Cd-induced ER stress impaired oocyte quality by disrupting mitochondrial function and elevating intracellular reactive oxygen species levels while decreasing ER function. Interestingly, TUDCA supplementation significantly decreased the expression of ER stress-related genes and increased the quantity of ER compared with the Cd treatment. Additionally, TUDCA was also able to rescue excessive levels of ROS and restore normal mitochondrial function. Moreover, the addition of TUDCA under Cd exposure greatly ameliorated Cd-mediated detrimental effects on meiotic maturation and oocyte quality, including cumulus cell expansion and MII rate. These findings suggest that Cd exposure during IVM impairs the meiotic maturation of oocytes by inducing of ER stress.

Restoration of Developmental Competence in Low-Quality Porcine Cumulus-Oocyte Complexes through the Supplementation of Sonic Hedgehog Protein during In Vitro Maturation.

The sonic hedgehog (SHH) pathway is an important signaling pathway for mammalian ovarian folliculogenesis and oocyte maturation. A previous study demonstrated that low-quality porcine cumulus-oocyte complexes (COCs) have low developmental competence, with lower SHH signaling protein expression before and after in vitro maturation (IVM) than high-quality COCs. However, there is no reported evidence on the restorative effects of SHH protein supplementation during the IVM of low-quality porcine COCs. Therefore, this study investigated the effects of SHH protein supplementation on the IVM of low-quality porcine COCs, as assessed by brilliant cresyl blue (BCB) staining. To examine this, we designed four groups: (i) BCB- (low-quality), (ii) BCB- + SHH, (iii) BCB+ (high-quality), and (iv) BCB+ + SHH. While the supplementation of SHH protein with high-quality COCs had no effect, supplementation with low-quality COCs significantly improved cumulus cell expansion, metaphase II rate, and subsequent embryo development following parthenogenetic activation. Our results provide the first evidence that the low developmental competence of low-quality porcine COCs can be improved by supplementation with the SHH protein. These results indicate that an active SHH signaling pathway is required for the acquisition of developmental competence in porcine COCs.

Expression of the melatonergic system during meiotic maturation of cynomolgus monkey cumulus-oocyte complexes.

BACKGROUND: Melatonin is a multifunctional hormone synthesized in the pineal gland and peripheral reproductive tissues that regulates many biological processes. There is increasing evidence for a role of melatonin in oocyte maturation and embryonic development in various mammals. However, no study has reported evidence for the existence of melatonergic system, such as melatonin synthesis enzymes, melatonin membrane receptors, or melatonin binding sites in non-human primate cumulus-oocyte complexes (COCs). METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were performed to detect transcripts and proteins of the rate-limiting enzyme in melatonin synthesis (arylalkylamine N-acetyltransferase, AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NRH: quinone oxidoreductase 2, NQO2) in cynomolgus monkey COCs. RESULTS: RT-PCR analyses revealed the presence of AANAT, MT1, MT2, and NQO2 transcripts in granulosa cells, germinal vesicle (GV)- and metaphase II (MII)-stage cumulus cells, and oocytes. Immunocytochemistry revealed the presence of AANAT, MT1, MT2, and NQO2 proteins in GV- and MII-stage COCs. CONCLUSIONS: Our results provide the first evidence for the existence of the rate-limiting enzyme required for melatonin synthesis, melatonin membrane receptors, and a melatonin binding site in non-human primate COCs.

Anethole improves the developmental competence of porcine embryos by reducing oxidative stress via the sonic hedgehog signaling pathway.

BACKGROUND: Anethole (AN) is an organic antioxidant compound with a benzene ring and is expected to have a positive impact on early embryogenesis in mammals. However, no study has examined the effect of AN on porcine embryonic development. Therefore, we investigated the effect of AN on the development of porcine embryos and the underlying mechanism. RESULTS: We cultured porcine in vitro-fertilized embryos in medium with AN (0, 0.3, 0.5, and 1 mg/mL) for 6 d. AN at 0.5 mg/mL significantly increased the blastocyst formation rate, trophectoderm cell number, and cellular survival rate compared to the control. AN-supplemented embryos exhibited significantly lower reactive oxygen species levels and higher glutathione levels than the control. Moreover, AN significantly improved the quantity of mitochondria and mitochondrial membrane potential, and increased the lipid droplet, fatty acid, and ATP levels. Interestingly, the levels of proteins and genes related to the sonic hedgehog (SHH) signaling pathway were significantly increased by AN. CONCLUSIONS: These results revealed that AN improved the developmental competence of porcine preimplantation embryos by activating SHH signaling against oxidative stress and could be used for large-scale production of high-quality porcine embryos.

Arsenic exposure during porcine oocyte maturation negatively affects embryonic development by triggering oxidative stress-induced mitochondrial dysfunction and apoptosis.

Arsenic (AS), an environmental contaminant, is a known human carcinogen that can cause cancer of the lung, liver, and skin. Furthermore, AS induces oxidative stress and mitochondrial impairments in mammalian cells. However, limited information is available on the effect of AS exposure on oocyte maturation of porcine, whose anatomy, physiology, and metabolism are similar to those of human. Therefore, we examined the effect of AS exposure on the in vitro maturation (IVM) of porcine oocytes and the possible underlying mechanisms. Cumulus-cell enclosed oocytes were cultured with or without AS for maturation, and then were used for analyses. This study indicated that AS under a concentration of 1 µM significantly increased the abnormal expansion of cumulus cells and the number of oocytes maintained in meiotic arrest. In addition, AS exposure significantly reduced subsequent development of embryos and increased the rate of apoptosis of blastocysts following parthenogenetic activation (PA) and in vitro fertilization (IVF). Moreover, AS exposure induced oxidative stress with increased reactive oxygen species (ROS), and decreased glutathione (GSH), leading to reduced mitochondrial membrane potential, mitochondrial quantity, DNA damage, excessive autophagy activity, and early apoptosis in porcine oocytes. Taken together, the results demonstrated that AS exposure exerts several negative effects, such as meiotic defects and embryo developmental arrest by causing mitochondrial dysfunction and apoptosis via inducing oxidative stress.

Hyaluronidase 6 Does Not Affect Cumulus-Oocyte Complex Dispersal and Male Mice Fertility.

Glycosylphosphatidylinositol-anchored sperm hyaluronidases (HYAL) assist sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we demonstrated that sperm from HYAL 5 and 7 double-knockout (dKO) mice produced significantly less offspring than sperm from wild-type mice due to defective COC dispersal. However, the HYAL6 gene remained active in the sperm from the dKO mice, indicating that they were not entirely infertile. This study explored the role of HYAL6 in fertilization by analyzing HYAL6-mutant mice. In this mouse model, HYAL5 and HYAL7 were present in the HYAL6-knockout sperm, and they could disperse hyaluronic acid. We found that HYAL6 was present on the surface of sperm. However, male mice lacking the HYAL6 gene had normal fertility, testicular integrity, and sperm characteristics. Furthermore, in vitro fertilization assays demonstrated that HYAL6-deficient epididymal sperm functioned normally. Therefore, HYAL6 is dispensable for fertilization.

Combined Chaetocin/Trichostatin A Treatment Improves the Epigenetic Modification and Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos.

Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.

Luteolin Orchestrates Porcine Oocyte Meiotic Progression by Maintaining Organelle Dynamics Under Oxidative Stress.

Increasing evidence has demonstrated that oxidative stress impairs oocyte maturation, but the underlying mechanisms remain largely unknown. Here, for the first time, we examined the antioxidant role of luteolin in meiotic progression and the underlying mechanisms. Supplementation of 5 µM luteolin increased the rates of first polar body extrusion and blastocyst formation after parthenogenetic activation, and the expression levels of oocyte competence (BMP15 and GDF9)-, mitogen-activated protein kinase (MOS)-, and maturation promoting factor (CDK1 and Cyclin B)-related genes were also improved. Luteolin supplementation decreased intracellular reactive oxygen species levels and increased the expression levels of oxidative stress-related genes (SOD1, SOD2, and CAT). Interestingly, luteolin alleviated defects in cell organelles, including actin filaments, the spindle, mitochondria, the endoplasmic reticulum, and cortical granules, caused by H2O2 exposure. Moreover, luteolin significantly improved the developmental competence of in vitro-fertilized embryos in terms of the cleavage rate, blastocyst formation rate, cell number, cellular survival rate, and gene expression and markedly restored the competencies decreased by H2O2 treatment. These findings revealed that luteolin supplementation during in vitro maturation improves porcine meiotic progression and subsequent embryonic development by protecting various organelle dynamics against oxidative stress, potentially increasing our understanding of the underlying mechanisms governing the relationship between oxidative stress and the meiotic events required for successful oocyte maturation.

Lycopene Improves In Vitro Development of Porcine Embryos by Reducing Oxidative Stress and Apoptosis.

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 µM lycopene. The results indicate that 0.1 µM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 µM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 µM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 µM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0-2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2-4 days) and late phases (4-6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

Heat stress impairs oocyte maturation through ceramide-mediated apoptosis in pigs.

Heat stress (HS) is an emerging issue that greatly impairs the reproductive performance of animals and humans. In particular, disruption of oocyte maturation due to HS is considered a major cause of impaired reproductive performance. HS is known to induce ceramide generation, which causes reactive oxygen species (ROS) production and mitochondrial dysfunction, thereby inducing apoptosis. Therefore, we investigated whether inhibition of ceramide generation ameliorates HS-induced apoptosis in porcine cumulus-oocyte complexes (COCs) using specific inhibitors of the de novo (fumonisin B1, FB1) and hydrolytic pathways (desipramine, Des) of ceramide formation. We investigated the effects of FB1 and Des supplementation under HS conditions (41.5 °C for 44 h) on in vitro maturation (IVM) of porcine COCs. After IVM, HS significantly reduced proportion of COCs exhibiting fully expanded cumulus cells and the rate of metaphase II in oocytes. After parthenogenetic activation (PA), HS significantly reduced the rates of cleavage and blastocyst formation with a lower total cell number and a higher percentage of apoptosis in blastocysts. However, FB1 or Des supplementation under HS avoided detrimental effects of HS on expansion of cumulus cells, nuclear maturation of oocytes, and embryonic development after PA including the rates of cleavage and blastocyst formation, total cell number, and the percentage of apoptosis in blastocysts. Furthermore, FB1 or Des addition under HS, compared with HS alone, significantly decreased ceramide generation, ROS production, cytochrome C expression, and apoptosis and increased mitochondrial membrane potential in COCs, reaching levels comparable with those of the control. Taken together, our results indicate that HS impaired oocyte maturation through ceramide-mediated apoptosis.

Effect of Triclosan Exposure on Developmental Competence in Parthenogenetic Porcine Embryo during Preimplantation.

Triclosan (TCS) is included in various healthcare products because of its antimicrobial activity; therefore, many humans are exposed to TCS daily. While detrimental effects of TCS exposure have been reported in various species and cell types, the effects of TCS exposure on early embryonic development are largely unknown. The aim of this study was to determine if TCS exerts toxic effects during early embryonic development using porcine parthenogenetic embryos in vitro. Porcine parthenogenetic embryos were cultured in in vitro culture medium with 50 or 100 µM TCS for 6 days. Developmental parameters including cleavage and blastocyst formation rates, developmental kinetics, and the number of blastomeres were assessed. To determine the toxic effects of TCS, apoptosis, oxidative stress, and mitochondrial dysfunction were assessed. TCS exposure resulted in a significant decrease in 2-cell rate and blastocyst formation rate, as well as number of blastomeres, but not in the cleavage rate. TCS also increased the number of apoptotic blastomeres and the production of reactive oxygen species. Finally, TCS treatment resulted in a diffuse distribution of mitochondria and decreased the mitochondrial membrane potential. Our results showed that TCS exposure impaired porcine early embryonic development by inducing DNA damage, oxidative stress, and mitochondrial dysfunction.

Chaetocin Improves Pig Cloning Efficiency by Enhancing Epigenetic Reprogramming and Autophagic Activity.

Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (suv39h1 and suv39h2) and DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.

Effect of Oocyte Quality Assessed by Brilliant Cresyl Blue (BCB) Staining on Cumulus Cell Expansion and Sonic Hedgehog Signaling in Porcine during In Vitro Maturation.

Brilliant cresyl blue (BCB) staining is used to select developmentally competent cumulus-oocyte complexes (COCs) for in vitro maturation (IVM). However, limited attention has been paid to what drives the higher developmental competence of BCB+ COCs. Sonic hedgehog signaling (SHH) is an important signaling pathway for ovarian follicular development and oocyte maturation. Therefore, this study investigated the effect of oocyte quality assessed by BCB staining on cumulus cell expansion, oocyte nuclear maturation, subsequent embryo development, apoptosis levels, and SHH signaling protein expression, in porcine COCs. After IVM, BCB+ COCs exhibited a significantly higher proportion of complete cumulus cell expansion and metaphase II rate in oocytes than BCB- COCs. After in vitro fertilization, the BCB+ group showed a significantly higher monospermy rate, fertilization efficiency, percentage of cleavage and blastocyst formation, with a higher total cell number and a lower apoptosis in blastocysts as compared with the BCB- group. Furthermore, significantly lower apoptosis levels and a higher expression of SHH-signaling proteins in COCs were observed, before and after IVM. In conclusion, high-quality oocytes had a greater potential to expand their surrounding cumulus cells with active SHH signaling and a lower apoptosis. This could provide COCs with a proper environment for maturation, thereby leading to a better subsequent embryo development.

Butylparaben Is Toxic to Porcine Oocyte Maturation and Subsequent Embryonic Development Following In Vitro Fertilization.

Parabens are widely used in personal care products due to their antimicrobial effects. Although the toxicity of parabens has been reported, little information is available on the toxicity of butylparaben (BP) on oocyte maturation. Therefore, we investigated the effects of various concentrations of BP (0 µM, 100 µM, 200 µM, 300 µM, 400 µM, and 500 µM) on the in vitro maturation of porcine oocytes. BP supplementation at a concentration greater than 300 µM significantly reduced the proportion of complete cumulus cell expansion and metaphase II oocytes compared to the control. The 300 µM BP significantly decreased fertilization, cleavage, and blastocyst formation rates with lower total cell numbers and a higher rate of apoptosis in blastocysts compared to the control. The BP-treated oocytes showed significantly higher reactive oxygen species (ROS) levels, and lower glutathione (GSH) levels than the control. BP significantly increased the aberrant mitochondrial distribution and decreased mitochondrial function compared to the control. BP-treated oocytes exhibited significantly higher percentage of γ-H2AX, annexin V-positive oocytes and expression of LC3 than the control. In conclusion, we demonstrated that BP impaired oocyte maturation and subsequent embryonic development, by inducing ROS generation and reducing GSH levels. Furthermore, BP disrupted mitochondrial function and triggered DNA damage, early apoptosis, and autophagy in oocytes.

Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs.

Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.

SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus-oocyte complex penetration defect.

The glycosylphosphatidylinositol-anchored sperm hyaluronidases (Hyals), sperm adhesion molecule 1 (SPAM1) and HYAL5, have long been believed to assist in sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we have shown that mouse sperm devoid of either Spam1 or Hyal5 are still capable of penetrating the COC and that the loss of either Spam1 or Hyal5 alone does not cause male infertility in mice. In the present study, we found that Spam1/Hyal5 double knockout (dKO) mice produced significantly fewer offspring compared with wild-type (WT) mice, and this was due to defective COC dispersal. A comparative analysis between WT and Spam1/Hyal5 dKO epididymal sperm revealed that the absence of these 2 sperm Hyals resulted in a marked accumulation of sperm on the outside of the COC. This impaired sperm activity is likely due to the deficiency in the sperm Hyals, even though other somatic Hyals are expressed normally in the dKO mice. The fertilization ability of the Spam1/Hyal5 dKO sperm was restored by adding purified human sperm Hyal to the in vitro fertilization medium. Our results suggest that Hyal deficiency in sperm may be a significant risk factor for male sterility.-Park, S., Kim, Y.-H., Jeong, P.-S., Park, C., Lee, J.-W., Kim, J.-S., Wee, G., Song, B.-S., Park, B.-J., Kim, S.-H., Sim, B.-W., Kim, S.-U., Triggs-Raine, B., Baba, T., Lee, S.-R., Kim, E. SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus-oocyte complex penetration defect.