Pig Oocytes Research Articles

In vitro Production of Porcine Embryos: Current Status and Possibilities – A Review

Abstract This paper presents the current possibilities, state of knowledge and prospects of in vitro production (IVP) of pig embryos, which consists of in vitro oocyte maturation, in vitro fertilization and in vitro embryo culture. In pigs, oocyte maturation is one of the most important stages in the embryo IVP process. It determines the oocyte’s fertilization ability as well as its embryonic development. Through many research studies of the proper selection of oocytes and appropriate maturation medium composition (especially the addition of various supplements), the in vitro maturation of pig oocytes has been significantly improved. Recent studies have demonstrated that modifications of the diluents and in vitro fertilization media can reduce polyspermy. Furthermore, several adjustments of the porcine culture media with the addition of some supplements have enhanced the embryo quality and developmental competence. These updates show the progress of IVP in pigs that has been achieved; however, many problems remain unsolved.

Assessing the effect of interspecies oocyte nucleolar material dosage on embryonic development

Sequence differences are considered to be the basic cause of developmental failure in interspecies embryos when more distant species are combined. However, other phenomena, such as insufficient or excessive quantity of specific cellular factors, might also influence the outcome. These effects are usually not considered. One of the organelles shown to contain different amount of proteins is the oocyte nucleolus-like body. Here we show that upon interspecies transfer, a single porcine nucleolus-like body is unable to support the development of a mouse parthenogenetic embryo derived from an enucleolated oocyte. However, when the amount of the porcine nucleolar material is increased to equalize the amount of mouse nucleolar material by transferring two nucleolus-like bodies, mouse embryos are able to pass the developmental block elicited by enucleolation. These embryos progress to the blastocyst stage at rates comparable to controls. Thus, using the model of an interspecies nucleolus-like body transplantation between mouse and pig oocytes, we show that an inadequate amount of nucleolar factors, rather than the species origin, affects the development. In a wider context of interspecies nuclear transfer schemes, the observed incompatibility between more distant species might not stem simply from sequence differences but also from improper dosage of key cellular factors.

Fatty acid induced lipolysis influences embryo development, gene expression and lipid droplet formation in the porcine cumulus cells†.

The pig oocyte maturation protocol differs from other mammalian species due to dependence on follicular fluid (FF) supplementation. One of the most abundant components of the porcine follicular fluid are fatty acids (FAs). Although evidence from other mammalian models revealed a negative impact of saturated fatty acids (SFA) on developmental competence of oocytes, pig has not yet been widely analyzed. Therefore, we aimed to investigate whether supplementation of IVM medium with 150 μM of stearic acid (SA) and oleic acid (OA) affects lipid content and expression of genes related to fatty acid metabolism in porcine cumulus–oocyte complexes and parthenogenetic embryo development. We found significant influence of fatty acids on lipid metabolism in cumulus cells without affecting the oocyte proper. The expression of ACACA, SCD, PLIN2, FADS1, and FADS2 genes was upregulated (P < 0.01) in cumulus cells, while their expression in oocytes did not change. The increase in gene expression was more pronounced in the case of OA (e.g., up to 30-fold increase in PLIN2 transcript level compared to the control). The number of lipid droplets and occupied area increased significantly in the cumulus cells and did not change in oocytes after SA treatment. Oleic acid improved the blastocyst rate (48 vs 32% in control), whereas stearic acid did not affect this parameter (27%). Additionally, we have discovered a phenotypic diversity of LD in cumulus cells in response to FA supplementation, suggesting extensive lipolysis in response to SA. Stearic acid excess in maturation media led to the formation of multiple micro lipid droplets in cumulus cells.

Long noncoding RNA 2193 regulates meiosis through global epigenetic modification and cytoskeleton organization in pig oocytes.

Long noncoding RNAs (lncRNAs) regulate a variety of physiological and pathological processes. However, the biological function of lncRNAs in mammalian germ cells remains largely unexplored. Here we identified one novel lncRNA (lncRNA2193) from single-cell RNA sequencing performed on porcine oocytes and investigated its function in oocyte meiosis. During in vitro maturation (IVM), from germinal vesicle (GV, 0 hr), GV breakdown (GVBD, 24 hr), to metaphase II stage (MII, 44 hr), the transcriptional abundance of lncRNA2193 remained stable and high. LncRNA2193 interference by small interfering RNA microinjection into porcine GV oocytes could significantly inhibit rates of GVBD and the first polar body extrusion, but enhance the rates of oocytes with a nuclear abnormality. Moreover, lncRNA2193 knockdown disturbed cytoskeletal organization (F-actin and spindle), and decreased DNA 5-methylcytosine (5mC) and histone trimethylation (H3K4me3, H3K9me3, H3K27me3, and H3K36me3) levels. The lncRNA2193 downregulation induced a decrease of 5mC level could be partially due to the reduction of DNA methyltransferase 3A and 3B, and the elevation of 5mC-hydroxylase ten-11 translocation 2 (TET2). After parthenogenetic activation of MII oocytes, parthenotes exhibited higher fragmentation but lower cleavage rates in the lncRNA2193 downregulated group. However, lncRNA2193 interference performed on mature MII oocytes and parthenotes at 1-cell stage did not affect the cleavage and blasctocyst rates of pathenotes. Taken together, lncRNA2193 plays an important role in porcine oocyte maturation, providing more insights for relevant investigations on mammalian germ cells.

Meiotic block with roscovitine improves competence of porcine oocytes by fine-tuning activities of different cyclin-dependent kinases.

Successful use of oocytes from small follicles (SFs) is of great importance for animal embryo production and human in vitro fertilization with reduced hormone-related side effects. How in vitro meiotic arrest maintenance (MAM) increases the competence of oocytes is not clear. In this study, pig oocytes recovered from SF of 1-2 mm and medium-follicles (MF) of 3-6 mm in diameter from abattoir ovaries were treated by various MAM treatments to improve their competence. The results showed that 25 µM roscovitine or 1 mM db-cAMP efficiently blocked germinal vesicle breakdown in both SF and MF oocytes suggesting a similar cyclin-dependent kinase (CDK) 1 level between the two oocyte groups. MAM with 15- and 25-µM roscovitine alone or with 1-mM db-cAMP improved competence of SF and MF oocytes, respectively, with a promoted chromatin configuration transition from surrounded nucleoli (SN) to re-decondensation (RDC) pattern that supported substantial gene transcription. However, MAM with db-cAMP alone or with higher concentrations of roscovitine did not improve oocyte competence, could not support an SN-to-RDC transition, and/or evoked a premature chromatin condensation (PMC) that suppressed gene transcription. Both CDK2 and CDK5 contents were higher (p < .05) in MF than in SF oocytes. It is concluded that the competence of pig oocytes, particularly that of SF oocytes can be improved by MAM using a proper roscovitine concentration that promotes gene transcription by inhibiting CDK5 while letting CDK2 off to prevent PMC.

Effects of glucose metabolism pathways on nuclear and cytoplasmic maturation of pig oocytes

The developmental competence of IVM porcine oocytes is still low compared with that in their in vivo counterparts. Although many studies reported effects of glucose metabolism (GM) on oocyte nuclear maturation, few reported on cytoplasmic maturation. Previous studies could not differentiate whether GM of cumulus cells (CCs) or that of cumulus-denuded oocytes (DOs) supported oocyte maturation. Furthermore, species differences in oocyte GM are largely unknown. Our aim was to address these issues by using enzyme activity inhibitors, RNAi gene silencing and special media that could support nuclear but not cytoplasmic maturation when GM was inhibited. The results showed that GM in CCs promoted pig oocyte maturation by releasing metabolites from both pentose phosphate pathway and glycolysis. Both pyruvate and lactate were transferred into pig DOs by monocarboxylate transporter and pyruvate was further delivered into mitochondria by mitochondrial pyruvate carrier in both pig DOs and CCs. In both pig DOs and CCs, pyruvate and lactate were utilized through mitochondrial electron transport and LDH-catalyzed oxidation to pyruvate, respectively. Pig and mouse DOs differed in their CC dependency for glucose, pyruvate and lactate utilization. While mouse DOs could not, pig DOs could use the lactate-derived pyruvate.

The key long non-coding RNA screening and validation between germinal vesicle and metaphase II of porcine oocyte in vitro maturation.

Oocyte maturation plays a vitally important role in the reproduction of pigs. However, the roles of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in the developmental process of porcine oocyte maturation are still largely unclear. In this study, a transcriptome analysis of germinal vesicle (GV) and metaphase II (MII) of oocytes from Chinese Duroc pigs was performed. A total of 1,753,030 and 2,486 differentially expressed (DE) mRNAs, 22,811 and 9,868 DE lncRNAs were identified between GV and MII stages, respectively. Furthermore, functional enrichment analysis showed that the common DE mRNAs and DE lncRNAs during the process of maturation were mainly involved in biological process and cellular components. Our study provides new insights of the expression changes of mRNAs and lncRNAs during GV and MII stages, which might contribute to the maturation of oocytes. These results greatly improve our understanding of the molecular mechanisms regulating the maturation of oocytes in pigs.

Optimization of in vitro embryo production and zygote vitrification for the indigenous Vietnamese Ban pig: The effects of different in vitro oocyte maturation systems.

The Vietnamese Ban pig is a precious genetic resource that needs to be preserved. In vitro embryo production from in vitro matured (IVM) oocytes is an important tool for the utilization of cryopreserved porcine sperm. The aim of this study was to compare two media for the IVM of Ban pig oocytes. Immature oocytes were subjected to IVM either in a non-defined (TCM-199+pig follicular fluid) or in a defined base medium (POM+epidermal growth factor). At the end of IVM, the oocytes were in vitro fertilized (IVF) with frozen Ban sperm. Ten hours after IVF, the oocytes were either subjected to orcein staining to check fertilization and maturation status or cultured in vitro for 7days. There was no difference between the two IVM media in terms of percentages of oocyte maturation and blastocyst production. However, the percentage of male pronuclear formation after IVF and the total cell numbers in blastocysts were higher with the defined system. Zygotes obtained by the two IVM systems survived vitrification at similar rates. In conclusion, the two IVM systems were both effective for the production of Ban pig embryos; however, better embryo quality was achieved with the defined one.

Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes.

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

Piglets produced by transfer of embryos obtained by in vitro fertilization of oocytes matured in vitro with thymosin: A case report

The aim of the study was to examine the in vivo viability of in vitro-produced (IVP) porcine embryos obtained from oocytes matured with thymosin. The research material for this study consisted of immature pig oocytes obtained from ovaries after slaughter and ejaculated semen obtained from one boar. The immature oocytes were cultured in vitro until the metaphase II stage in a medium supplemented with thymosin (TMS). The presumptive zygotes obtained were cultured in vitro for 4-40 hours. The presumptive zygotes and 2-4-cell embryos were evaluated in vivo after transferring them to synchronized recipients. After the transfer of embryos from the experimental group into 2 recipients (50 embryos into each gilt) and the transfer of 50 embryos from the control group into 1 recipient, both gilts that had received embryos obtained by in vitro fertilization of oocytes matured with TMS became pregnant and delivered a total of 16 live piglets. After the transfer of embryos from the control group, no pregnancy was achieved. In conclusion, the results of our preliminary study suggest that the maturation of pig oocytes with thymosin supports the in vivo survival of in vitro produced embryos. It is important to note, that this was the first birth of piglets obtained after transfer of IVP embryos in Poland.

181 The effects of aging in the oocyte induced by trichostatin A

Successful fertilization and embryonic development decreases for an aged oocyte that is not fertilized during the optimal window of time after ovulation. If fertilization of an aged oocyte does occur, the developing embryo is prone to have a delay through meiosis and a decrease in cleavage and blastocyst formation. Trichostatin A (TSA) prohibits the breakdown of the germinal vesicle during meiosis. The objective of this study was to create an invitro model to study the effects of aging in pig oocytes. Oocytes (n=1489) were matured with or without TSA (100ngmL−1) for 24 or 48h followed by an additional 16h of maturation without TSA. A portion of the oocytes were evaluated for stage of meiosis (MI and MII) before maturation (n=95), after the first part of maturation (OMI; 24 or 48h, n=363), or after completing maturation (OMII; 16h, n=230). The remaining oocytes (n=801) were fertilized for 6 to 8h and potential embryos developed. A portion of the embryos were evaluated for fertilization characteristics 12h after IVF (n=400). Remaining embryos (n=401) were evaluated for cleavage by 48h and blastocyst formation by 144h, after IVF. Data were analysed using ANOVA and Tukey's test. Oocytes matured without TSA for 64h had a significantly higher (P&amp;lt;0.05) percentage of oocytes at the MI stage of meiosis compared with all other treatment groups (16.00±6.80), and oocytes matured without TSA for 40h had a significantly higher (P&amp;lt;0.05) percentage of oocytes at the MII stage of meiosis compared with all other treatment groups (14.7±11.30). The results (Table 1) indicate that supplementation of TSA to OMI significantly decreased fertilization penetration rates compared with not supplementing TSA for 24h. However, the percent of embryos cleaved by 48h after IVF was significantly higher in oocytes matured for 40h compared with those matured for 64h. The percent of embryos reaching the blastocyst stage of development by 144h after IVF was significantly lower in oocytes matured for 64h compared with those matured for 40h without TSA. In conclusion, these results suggest that supplementation of TSA during early maturation delays meiosis and decreases fertilization, and increasing maturation time causes a decrease in early embryonic development. By supplementing TSA during early maturation and increasing the length of maturation by 24h, we have created an invitro model to study the effects of aging in pig oocytes. Table 1.Fertilization and developmental characteristics of oocytes1 Amount of TSA added during OMI (ngmL−1) Length of OMI (h) Oocytes penetrated (%) Polyspermicoocytes2 (%) Oocytes with MPN2 (%) Cleavage (%) Day 7 blastocyst (%) 0 24 83.00±3.80a 15.70±4.00 75.90±4.70 77.20±4.20a 27.70±4.20a 0 48 72.00±4.50ab 20.80±4.80 76.40±5.00 58.00±5.00bc 12.00±5.00b 100 24 65.00±4.80b 30.80±5.80 73.80±5.50 68.00±4.70ab 21.00±4.70ab 100 48 56.00±5.00b 30.40±6.20 62.50±6.50 46.00±5.00c 8.00±5.00b a,bMeans with different superscripts in the same column differ (P&amp;lt;0.05). 1TSA, trichostatin A; OMI, oocyte maturation 1. 2Percent of the number of oocytes penetrated.

184 Effects of cyanidin supplementation on invitro maturation of pig oocytes

Oxidative stress can have a negative effect on oocyte maturation during invitro production of pig embryos. Imbalance of reactive oxygen species and antioxidant levels can affect the progression of oocyte maturation up to the point of fertilization. Antioxidants are effective in maintaining more ideal reactive oxygen species levels, which help to protect oocytes from potential harmful effects of oxidative stress. Berries from the elder plant (Sambucus sp.) contain high levels of a broad spectrum of antioxidants. One of these antioxidants, cyanidin, when supplemented to maturation medium at 100μM concentrations, reduces reactive oxygen species formation and improves IVF and early embryonic development in pigs. However, changes in the enzyme mechanisms of action during oocyte maturation due to cyanidin supplementation are unknown. Therefore, the objective of this study was to characterise the intracellular oocyte enzyme mechanisms between oocytes supplemented with 100μM cyanidin during 40 to 44h of maturation (n=600) and oocytes without supplementation of cyanidin during maturation (n=558). At the end of maturation, oocytes were evaluated for either glutathione peroxidase (n=300), catalase (n=564), or superoxide dismutase (n=294) activities. Glutathione peroxidase activity was determined by following the rate of NADPH oxidation, catalase activity was determined by following the rate of hydrogen peroxide decomposition, and superoxide dismutase activity was determined by following the reduction rate of cytochrome c, utilising the xanthine-xanthine oxidase system. Data were analysed using ANOVA and Tukey's test. There were no significant differences between oocytes matured with 100μM cyanidin and those that were not when comparing glutathione peroxidase and superoxide dismutase activities. Supplementation of 100μM cyanidin to maturation medium increased (P&amp;lt;0.05) catalase activity in oocytes (0.78±0.15 units/oocyte) compared with no cyanidin supplementation (0.14±0.11 units/oocyte). These results indicate that supplementing 100μM cyanidin to the maturation medium of pig oocytes could reduce the negative effects of oxidative stress by increasing intracellular catalase activity during oocyte maturation.

Mechanism Of Immature Pig Oocyte Injury At A Low Temperature

Mechanism Of Immature Pig Oocyte Injury At A Low Temperature

Supplementation with exogenous coenzyme Q10 to media for in vitro maturation and embryo culture fails to promote the developmental competence of porcine embryos.

The coenzyme Q10 (CoQ10) is a potent antioxidant with critical protection role against cell oxidative stress, caused by the mitochondrial dysfunction. This study evaluated the effects of CoQ10 supplementation to in vitro maturation (IVM) or embryo culture media on the maturation, fertilization and subsequent embryonic development of pig oocytes and embryos. Maturation (Experiment 1) or embryo culture (Experiment 2) media were supplemented with 0 (control), 10, 25, 50 and 100μM CoQ10. The addition of 10-50μM CoQ10 to the IVM medium did not affect the percentage of MII oocytes nor the fertilization or the parameters of subsequent embryonic development. Exogenous CoQ10 in the culture medium neither did affect the development to the 2-4-cell stage nor rates of blastocyst formation. Moreover, the highest concentration of CoQ10 (100μM) in the maturation medium negatively affected blastocyst rates. In conclusion, exogenous CoQ10 supplementation of maturation or embryo culture media failed to improve the outcomes of our in vitro embryo production system and its use as an exogenous antioxidant should not be encouraged.

Biotechnological bases of the development of cloned pig embryos

The term ‘clone’ in animal biotechnology refers to an organism derived from non-sexual reproduction, which is both a direct offspring and a genetic copy of the parent organism. To date, the pig appears to be the most interesting object in cloning research. Somatic cell nuclear transfer in pigs has a wide range of potential applications in various fields of human scientific and economic activities. However, the efficiency of producing cloned embryos in swine is still lower than that of other livestock species, in particular horses and cattle. Somatic cell nuclear transfer is a technically complex multi-stage technology, at each stage of which the pig oocytes, which are more susceptible to changes of surrounding conditions, are affected by various factors (mechanical, physical, chemical). At the stage of oocyte maturation, changes in the cell ultrastructures of the ooplasm occur, which play an important role in the subsequent nuclear reprogramming of the transferred donor cell. Before transfer to the oocyte donor somatic cells are synchronized in the G0/G1 stage of the cell cycle to ensure the normal ploidy of the cloned embryo. When removing the nucleus of pig oocytes maturated in vitro, it is necessary to pay attention to the problem of preserving the viability of cells, which were devoid of their own nuclear material. To perform the reconstruction, a somatic cell is placed, using micro-tools, in the perivitelline space, where the first polar body was previously located, or in the cytoplasm of an enucleated oocyte. The method of manual cloning involves the removal of the oocyte nucleus with subsequent fusion with the donor cell without the use of micromanipulation techniques. The increased sensitivity of oocytes to the environmental conditions causes special requirements for the choice of the system for in vitro culture of cloned pig embryos. In this work, we have reviewed the modern methods used for the production of cloned embryos and identified the technological issues that prevent improving the efficiency of somatic cloning of pigs.

Intranuclear characteristics of pig oocytes stained with brilliant cresyl blue and nucleologenesis of resulting embryos.

Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus-oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB-) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB- oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB- embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB- embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB- oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.

The dynamics of gene expression, lipid composition and DNA methylation reprogramming are different during in vitro maturation of pig oocytes obtained from prepubertal gilts and cycling sows.

This study aimed to characterize the gene expression, lipid composition and DNA methylation reprogramming during in vitro maturation (IVM) of pig oocytes with different developmental competencies. We used prepubertal gilts and cycling sows as a model to obtain oocytes with different levels of competency. We found that genes involved in lipid metabolism, SLC27A4, CPT2 and PLIN2, and DNA methylation, DNMT3A, TET1 and TET3, possessed altered transcript expression levels during IVM. Specifically, SLC27A4 mRNA (p=0.05) increased in oocytes from cycling females, whereas CPT2 (p=0.05), PLIN2 (p=0.02) and DNMT3A (p=0.02) increased in oocytes from prepubertal females during IVM. Additionally, TET3 mRNA increased during IVM in oocytes from prepubertal (p=0.0005) and cycling females (p=0.02). The TET1 transcript decreased (p=0.05) during IVM in oocytes from cycling sows. Regarding lipid composition, mass spectrometry revealed a cluster of ions, with molecular masses higher than m/z 700, which comprises a group of complex phospholipids, was identified in all groups of oocytes, except in those from prepubertal gilts. With respect to DNA methylation reprogramming, it was noted that the less competent oocytes were not able to reprogramme the XIST gene during IVM. We conclude that the maternal mRNA store, lipid composition and epigenetic reprogramming are still being established during maturation and are related to oocyte competence. In addition, we propose that the methylation pattern of the XIST may be used as molecular marker for oocyte competence in pigs.

5-Azacytidine improves the meiotic maturation and subsequent in vitro development of pig oocytes

Treatment of donor cells and/or cloned embryos with cytidine analogues, having an Aza group at its 5th carbon (5-Aza), such as 5-Azacytidine (5-Aza-C) or 5-Aza-2′-deoxycytidine (5-Aza-dC) improves the in vitro development of cloned embryos produced by somatic cell nuclear transfer (SCNT). In vitro maturation (IVM) of immature pig oocytes treated with 5-Aza-C not only results in greater (P < 0.05) meiotic maturation to the MII stage but also enhances the capacity of 5-Aza-C treated oocytes for early embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF) or SCNT in a dose-dependent manner (0–10 μM). Cloned embryos generated from 5-Aza-C (0.01 μM) treated oocytes had an increased capacity to develop to the blastocyst stage (14.1 ± 1.5% compared with 9.6 ± 1.8%), greater probability of hatching (61.8 ± 1.5% compared with 45.0 ± 3.9%) and contained a greater number of cells per blastocyst (38.5 ± 4.4 compared with 30.5 ± 3.4) than those produced from non-treated control oocytes (P < 0.05). Data from the present study indicate that treatment of oocytes with 5-Aza-C may be an important approach to enhance the meiotic maturation and subsequent in vitro development of pig embryos. Future studies should be conducted to determine the underlying mechanism of improved early embryonic development of 5-Aza-C treated oocytes.

Various macromolecules in in vitro growth medium influence growth, maturation, and parthenogenetic development of pig oocytes derived from small antral follicles

Various macromolecules in in vitro growth medium influence growth, maturation, and parthenogenetic development of pig oocytes derived from small antral follicles

Endo-siRNAs repress expression of SINE1B during in vitro maturation of porcine oocyte

Approximately 40% of mammalian genome is made of transposable elements (TEs), and during specific biological processes, such as gametogenesis, they may be activated by global demethylation, so strict silencing mechanism is indispensable for genomic stability. Here, we performed small RNA-seq on Dicer1 knockdown (KD) oocytes in pig, and observed short interspersed nuclear elements 1B (SINE1B) derived endogenous small interfering RNAs (endo-siRNAs), termed SINE1B-siRNAs, were significantly decreased and their biogenesis was dependent on Dicer1 and transcript of SINE1B. Furthermore, by injection of mimics and inhibitors of the SINE1B-siRNAs into germinal vesicle-stage (GV-stage) oocytes, we found the maturation rate was significantly decreased by SINE1B-siRNAs, indicating the SINE1B-siRNAs are indispensible for in vitro maturation (IVM) of porcine oocyte. To figure out the mechanism, we checked the expression pattern and DNA methylation status of SINE1B during IVM of porcine oocytes, and demonstrated the SINE1B-siRNAs could repress SINE1B expression induced by hypomethylation at a post-transcriptional level. Our results suggest that during gametogenesis when the erasure of DNA methylation occurs, endo-siRNAs act as a chronic response to limit retrotransposon activation.