Effect of Ethanol on Parthenogenetic Activation and α-Tocopherol Supplementation during In Vitro Maturation on Developmental Competence of Summer-Collected Bovine Oocytes.

The use of α-tocopherol during in vitro maturation (IVM) is an alternative to minimize the adverse effects of heat stress on oocyte competence. However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O2, 5% CO2 at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 µM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 µM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 µM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.

Optimal Treatment of 6-Dimethylaminopurine Enhances the In Vivo Development of Canine Embryos by Rapid Initiation of DNA Synthesis.

Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 µM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, p < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle.

In vitro maturation using an agarose matrix with incorporated extracellular matrix proteins improves porcine oocyte developmental competence by enhancing cytoplasmic maturation.

Here, we present a novel in vitro maturation (IVM) system comprising an agarose matrix supplemented with extracellular matrix (ECM) proteins for enhanced maturation of immature oocytes within cumulus-oocyte complexes (COCs) derived from porcine medium antral follicles (MAFs). Immunocytochemical analyses of integrin subunit α2 , α5 , α6 , ß1 , and ß4 expression suggested that integrin α2 ß1 , α5 ß1 , α6 ß1 , and α6 ß4 play pivotal roles in IVM of porcine immature oocytes. Combinatorial supplementation of fibronectin interacting with integrin α5 ß1 , collagen interacting with integrin α2 ß1 , and laminin interacting with integrin α6 ß1 and α6 ß4 to the agarose matrix had no significant effect on nuclear maturation. However, the number of parthenogenetic embryos that developed into blastocysts increased when oocytes were matured using agarose IVM matrices supplemented with fibronectin, collagen, or laminin. Furthermore, significant increases in cytoplasmic maturation-related parameters (BMP15 level, cumulus cell expansion score, intra-oocyte ATP level, and index of cortical granule distribution) were observed in COCs matured in vitro using ECM protein-incorporated agarose matrices. Our data suggest that mature porcine oocytes with enhanced developmental competence and high-quality cytoplasm can be generated via IVM using agarose matrices supplemented with fibronectin, collagen, or laminin.

The antioxidant dieckol reduces damage of oxidative stress-exposed porcine oocytes and enhances subsequent parthenotes embryo development.

This study investigated the effect of the antioxidant dieckol, a component of Ecklonia cava, on maturation and developmental competence of porcine oocytes exposed to oxidative stress in vitro. Oocytes were matured in in vitro maturation (IVM) medium containing various concentrations of dieckol. The blastocyst formation rate was highest in the 0.5 µM dieckol-treated (0.5 DEK) group. The reactive oxygen species level was decreased, and the level of glutathione and expression of antioxidant genes (NFE2L, SOD1, and SOD2) at metaphase II were increased in the 0.5 DEK group. Abnormal spindle organization and chromosome misalignment were prevented in the 0.5 DEK group. Expression of maternal markers (CCNB1 and MOS) and activity of p44/42 mitogen-activated protein kinase were increased in the 0.5 DEK group. After parthenogenetic activation, the total number of cells per blastocyst was increased and the percentage of apoptotic cells was decreased in the 0.5 DEK group. Expression of development-related genes (CX45, CDX2, POU5F1, and NANOG), antiapoptotic genes (BCL2L1 and BIRC5), and a proapoptotic gene (CASP3) were altered in the 0.5 DEK group. These results indicate that the antioxidant dieckol improves IVM and subsequent development of porcine oocytes and can be used to improve the quality of oocytes under peroxidation experimental conditions.

Alpha-lipoic acid improves the maturation and the developmental potential of goat oocytes in vitro.

Oxidative stress inevitably occurs during oocyte maturation in vitro. α-lipoic acid (α-LA) has a strong antioxidant capacity, but the effect of α-LA on parthenogenetic activation of oocytes was rarely reported. This study aims to investigate the effect of supplementing α-LA to in vitro maturation medium on the subsequent developmental ability of goat parthenogenetic embryos during oocytes maturation. In the study, the goat cumulus-oocyte complex was divided into the experimental (with 25 µmol/L α-LA) and the control (without α-LA) groups. Oxidase expression was measured using RT-qPCR. After 18-22 hr of maturation, the oocytes were then parthenogenetic activated. The total antioxidant capacity of embryos was measured after 0, 24, 48, 72 and 96 hr of culture. Rates of oocyte maturation and the rates of development for parthenogenetic embryos in the α-LA group were significantly improved by 7.88% (p < .05) and 5.41% (p < .05) compared with those in the control group, respectively. After 24 hr, the difference in total antioxidant capacity was extremely significant in both groups. An evident decrease in the control group and a minor decrease in the α-LA group were observed (p < .01). The ratio of inner cell mass cells to the total cell number of blastocysts in the α-LA group increased compared with that in the control group (p < .05) on day 8. α-LA significantly promoted the expression of SOD and GPX4 of parthenogenetic blastocysts and maturated oocytes. α-LA (25 µmol/L) improved the maturation rate and the developmental competence of the parthenogenetic activation of oocytes, which might be mediated by maintaining the total antioxidant ability of oocytes during the culture period.

Procyanidin B1 promotes in vitro maturation of pig oocytes by reducing oxidative stress.

Oxidative stress negatively affects the in vitro maturation (IVM) of oocytes. Procyanidin B1 (PB1) is a natural polyphenolic compound that has antioxidant properties. In this study, we investigated the effect of PB1 supplementation during IVM of porcine oocytes. Treatment with 100 µM PB1 significantly increased the MII oocytes rate (p <0.05), the parthenogenetic (PA) blastocyst rate (p <0.01) and the total cell number in the PA blastocyst (p < 0.01) which were cultured in regular in vitro culture (IVC) medium. The PA blastocyst rate of regular MII oocytes activated and cultured in IVC medium supplemented with 100 and 150 µM PB1 significantly increased compared with control (p < 0.01 and p < 0.05). We also evaluated the reactive oxygen species (ROS) levels, mitochondrial membrane potential (Δψm) levels, glutathione (GSH) levels, and apoptotic levels in MII oocytes and cumulus cells following 100 µM PB1 treatment. The results showed that the PB1 supplementation decreased ROS production and apoptotic levels. In addition, PB1 was found to increase Δψm levels and GSH levels. In conclusion, PB1 inhibited apoptosis of oocytes and cumulus cells by reducing oxidative stress. Moreover, PB1 improved the quality of oocytes and promoted PA embryo development. Taken together, our results suggest that PB1 is a promising antioxidant additive for IVM of oocytes.

Ginsenoside Rb1 protects porcine oocytes against methylglyoxal damage thus it improves the quality of parthenogenetic activation and in vitro fertilization embryos.

Panax ginseng, a functional food, has been widely used as an edible nourishment and medicinal supplement. Ginsenoside Rb1 is a major bioactive ingredient of ginseng, which shows very specific anti-apoptosis and anti-oxidant activities. Methylglyoxal (MGO) is one of intermediate products of glucose metabolism, which is absorbed easily from high sugar foods or carbonated beverages. It may involve in a variety of detrimental processes in vivo. However, it has not been fully explored the effects of ginsenoside Rb1 on MGO-induced oocytes damage. This study found that MGO-induced DNA damage and mitochondrial dysfunction result in the failure of porcine oocytes maturation and low in vitro development capacity of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos. Conversely, Rb1 supplementation recovered the rate of maturation, and improved in vitro development capacity of PA and IVF embryos. Rb1 also provided porcine oocytes a lower level of reactive oxygen species production, higher level of ATP content and mitochondrial membrane potential, and stimulated pluripotency gene expression in blastocysts. The findings of this study reveal ginsenoside Rb1 protects porcine oocyte from the cytotoxicity effects of methylglyoxal and provides novel perspectives for the protection of reproduction system by functional food of ginseng.

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.

Melatonin protects against defects induced by malathion during porcine oocyte maturation.

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.

Production of collared peccary (Pecari tajacu Linnaeus, 1758) parthenogenic embryos following different oocyte chemical activation and in vitro maturation conditions.

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ±â€¯1.2 vs. 100% ±â€¯0.0), presence of first polar body (65.9% ±â€¯1.2 vs. 70.5% ±â€¯1.8), nuclear status in second metaphase (62.5% ±â€¯11.6 vs. 68.4% ±â€¯4.9), cytoplasmic maturation (100.0% ±â€¯0.7 vs. 75.0% ±â€¯0.7), reactive oxygen species levels (0.5 ±â€¯0.2 vs. 0.3 ±â€¯0.1), and mitochondrial membrane potential (1.1 ±â€¯0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.

The autophagic inducer and inhibitor display different activities on the meiotic and developmental competencies of porcine oocytes derived from small and medium follicles.

This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3-6 mm in diameter) and small follicles (SF, 1-2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress.

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 µM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.

Effect of single and combined treatments with MPF or MAPK inhibitors on parthenogenetic haploid activation of bovine oocytes.

In bovine, correct oocyte artificial activation is a key step in ICSI and other reproductive biotechnologies, and still needs to be improved. The current study was designed to compare the activating efficiency of ionomycin (Io) followed by: a 4 h time window and ethanol (4h-Et), roscovitine (Rosc), dehydroleucodine (DhL), cycloheximide (CHX) or PD0325901 (PD), each as a single treatment, and then combine them in novel protocols. Parthenogenetic haploid activation was evaluated in terms of pronuclear (PN) formation, second polar body (2PB) extrusion, ploidy of day 2 embryos and in vitro development. Combined treatments with Io-4h-Et-Rosc and Io-Rosc/CHX increased PN formation (92.2% and 96%, respectively) compared with Io-Rosc, Io-CHX or Io-4h-Et, which were equally efficient at inducing PN formation (82-84%) and 2PB extrusion (62.1-70.5%). Oocyte activation with Io-DhL and Io-Rosc/DhL resulted in higher 2PB extrusion rates (90% and 95.9%, respectively) but lower PN formation (49.4-58.8%) and cleavage rates (36-57.9%), as occurred with Io-CHX/DhL (76.4% and 70.4%, respectively). For the first time, results show that Io followed by the MAPK inhibitor PD induces PN formation and 2PB extrusion, but PD combined with Rosc or CHX resulted in low rates of haploid day 2 embryos. In conclusion, DhL strongly induces 2PB extrusion but leads to poor PN formation and embryo development. PD induces bovine oocyte activation but results in low rates of haploid embryos. In contrast, the improved PN formation rates after treatment with combined Io-4h-Et-Rosc and Io-Rosc/CHX suggest they should be further evaluated in ART, aiming to increase success rates in bovine.

NLRP7 is expressed in the ovine ovary and associated with in vitro pre-implantation embryo development.

NLRP (NACHT, LRR and PYD domain-containing proteins) family plays pivotal roles in mammalian reproduction. Mutation of NLRP7 is often associated with human recurrent hydatidiform moles. Few studies regarding the functions of NLRP7 have been performed in other mammalian species rather than humans. In the current study, for the first time, the function of NLRP7 has been explored in ovine ovary. NLRP7 protein was mainly located in ovarian follicles and in in vitro pre-implantation embryos. To identify its origin, 763 bp partial CDS of NLRP7 deriving from sheep cumulus oocyte complexes (COCs) was cloned, it showed a great homology with Homo sapiens. The high levels of mRNA and protein of NLRP7 were steadily expressed in oocytes, parthenogenetic embryos or IVF embryos. NLRP7 knockdown by the combination of siRNA and shRNA jeopardized both the parthenogenetic and IVF embryo development. These results strongly suggest that NLRP7 plays an important role in ovine reproduction. The potential mechanisms of NLRP7 will be fully investigated in the future.

Pioglitazone improves porcine oocyte maturation and subsequent parthenogenetic embryo development in vitro by increasing lipid metabolism.

Optimization of culture conditions is important to improve oocyte maturation and subsequent embryo development. In particular, this study analyzed the effects of increasing concentrations of PIO in the maturation medium on spindle formation and chromosome alignment, glutathione, and intracellular ROS levels and expression of selected genes related to maternal markers, apoptosis, and lipid metabolism. The percentage of oocytes displaying normal spindle formation and chromosome alignment was higher in the 1 µM PIO (1 PIO)-treated group than in the control group. The glutathione level was significantly higher in the 1 PIO-treated group than in the control group, while the reactive oxygen species level did not differ. Expression of maternal marker (MOS and GDF9), antiapoptotic (BIRC5), and lipid metabolism-related (ACADS, CPT2, SREBF1, and PPARG) genes was higher in the 1 PIO-treated group than in the control group, while expression of a proapoptotic gene (CASP3) was lower. The blastocyst formation rate and the percentage of blastocysts that reached at least the hatching stage on Days 6 and 7, and the percentage of blastocysts containing more than 128 cells were significantly higher in the 1 PIO-treated group than in the control group. These results indicate that PIO treatment during in vitro maturation improves porcine oocyte maturation and subsequent parthenogenetic embryo development mainly by enhancing lipid metabolism and antioxidant defense in oocytes.

Allicin protects porcine oocytes against damage during aging in vitro.

Allicin, a chemical component of garlic, has strong antioxidant activity and is thought to exert antiaging effects in vitro. We investigated whether allicin treatment would protect porcine oocytes and embryos from postovulatory aging mediated by apoptosis and autophagy. The rates of oocyte survival and polar body extrusion in samples treated with 1 µM allicin (1 AL) were significantly higher than in untreated samples (0 AL). In addition, 1 AL prevented defects in spindle formation and chromosome alignment, as well as decreases in the expression of maturation markers, during in vitro aging. In this study, we considered allicin to be a regulator of autophagy rather than an antioxidant or antiapoptotic agent. At the embryo level, although the cleavage rate after parthenogenetic activation was similar in all groups, the blastocyst formation rate was higher in the 1 AL group than in the 0 AL group. Our findings demonstrate that allicin effectively prevents the deterioration of porcine oocytes during aging in vitro, and could therefore be used to improve the quality of aged oocytes used in in vitro experiments.

Systematically improved in vitro culture conditions reveal new insights into the reproductive biology of the human parasite Schistosoma mansoni.

Schistosomes infect over 200 million people. The prodigious egg output of these parasites is the sole driver of pathology due to infection, yet our understanding of sexual reproduction by schistosomes is limited because normal egg production is not sustained for more than a few days in vitro. Here, we describe culture conditions that support schistosome sexual development and sustained egg production in vitro. Female schistosomes rely on continuous pairing with male worms to fuel the maturation of their reproductive organs. Exploiting these new culture conditions, we explore the process of male-stimulated female maturation and demonstrate that physical contact with a male worm, and not insemination, is sufficient to induce female development and the production of viable parthenogenetic haploid embryos. We further report the characterization of a nuclear receptor (NR), which we call Vitellogenic Factor 1 (VF1), that is essential for female sexual development following pairing with a male worm. Taken together, these results provide a platform to study the fascinating sexual biology of these parasites on a molecular level, illuminating new strategies to control schistosome egg production.

Baicalin improves IVM of pig oocytes and subsequent preimplantation embryo development by inhibiting apoptosis.

Baicalin, a monomer of flavonoids extracted from dried roots of Scutellaria baicalensis, is used to treat female infertility. However, the effect of baicalin on oocyte maturation is unknown. In this study we investigated the effects of baicalin on the IVM of pig oocytes and subsequent embryo development following parthenogenetic activation (PA). We found that 0.1µgmL-1 baicalin significantly (P<0.05) increased the IVM rate of oocytes compared with the non-treatment (control) group by reducing levels of reactive oxygen species (ROS). In addition, the mRNA expression of genes related to nuclear maturation and cumulus cell expansion, mitochondrial membrane potential and ATP content was significantly (P<0.05) higher in baicalin-treated than control oocytes. To determine whether baicalin treatment during IVM of pig oocytes improves subsequent development of PA embryos, we measured the cleavage and blastocyst formation rates, as well as the number of cells per blastocyst. All these parameters were significantly (P<0.05) higher in the baicalin-treated than control group. In conclusion, this study demonstrates that baicalin improves pig oocyte maturation and subsequent embryo development invitro by inhibiting production of ROS and reducing apoptosis in oocytes.

Calcium ionophore enhanced developmental competence and apoptotic dynamics of goat parthenogenetic embryos produced in vitro.

Parthenogenetically developed embryos are efficient sources of in vitro embryo production, having less ethical issue and being useful for investigating culture conditions/treatments, early developmental, genomic studies, and homonymous source of stem cells. Keeping its advantages in mind, we aimed to study the effects of different activating agents on embryo production and its quality and gene expression. In the present study, 1348 immature oocytes recovered were parthenogenetically developed to embryos. Usable-quality immature oocytes were collected by puncturing the surface follicles and matured in in vitro maturation (IVM) medium for 27 h in a humidified 5% CO2 incubator at 38.5°C. The matured oocytes were parthenogenetically activated by exposure to 5 µM calcium ionophore for 5 min or 7% ethanol for 7 min sequentially followed by 4 h incubation in 2 mM 6-DMAP and then in vitro cultured (IVC) in RVCL/G-2 medium for 8 days. Matured oocytes were activated by calcium ionophore, the cleavage rate observed was 76.67 ± 3.47%, and further they developed into 4-cell, 8-16-cell, morula, blastocyst, and hatched blastocyst with 85.30 ± 1.57%, 70.60 ± 2.00%, 45.05 ± 2.66%, 22.89 ± 2.40%, and 5.70 ± 1.97%, respectively. Whereas ethanol-activated oocytes showed cleavage rate of 87.60 ± 1.70% and further culture developed into 4-cell, 8-16 cell, morula, blastocyst, and hatched blastocyst with 86.14 ± 1.03%, 71.56 ± 2.21%, 40.90 ± 2.45%, 19.02 ± 1.26%, and 2.22 ± 0.38%, respectively. Blastocyst developed from calcium ionophore-activated oocytes showed significantly (P < 0.05) higher total cell number (282.25 ± 27.02 vs 206.00 ± 40.46) and a lower apoptotic index (2.42 ± 0.46 vs 4.07 ± 1.44) than blastocyst developed from ethanol-activated oocytes. The relative expression of anti-apoptotic genes (BCL2, BCL2A1, MCL) at different stages of embryos produced by either calcium ionophore or ethanol activation was found to be increased in earlier stages and decreased in later stages of embryonic development. Similarly, when these embryos were subjected to pro-apoptotic genes (BAX, BAD, BAK), expression was found to be slightly higher in blastocysts than other stages. This study shows that calcium ionophore-activated blastocysts were developmentally more competent than the ethanol-activated blastocysts.

Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification.

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.