Vitro-matured Porcine Oocytes Research Articles

In Vivo-Matured Oocyte Resists Post-Ovulatory Aging through the Hub Genes DDX18 and DNAJC7 in Pigs.

Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.

Bezafibrate prevents aging in in vitro-matured porcine oocytes.

Bezafibrate, a fibrate drug used as a lipid-lowering agent to treat hyperlipidemia, is a pan-agonist of peroxisome proliferator-activated receptor alpha. It can enhance mitochondrial fatty acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis. After ovulation, oocytes may get arrested at the metaphase II (MII) stage until fertilization beyond optimal timing, which is termed as post-ovulatory aging. Post-ovulatory aging is a disease that degrades DNA, mitochondria, and oxidative system, and has a negative impact on embryo development and quality; however, the impact of bezafibrate during post-ovulatory aging has not been fully defined. In the present study, we assessed the ability of bezafibrate to prevent the progression of aging in in vitro conditions as well as the underlying mechanisms in pigs. An appropriate concentration of this drug (50 μM) was added, and then oxidative stress, reactive oxygen species downstream, mitochondrial biogenesis, and mitochondrial function were analyzed via immunofluorescence staining and real-time polymerase chain reaction. Bezafibrate significantly alleviated reactive oxygen species and ameliorated glutathione production simultaneously in oocytes and embryos. Moreover, it diminished H2A.X and attenuated CASPASE 3 expression produced by oxidative stress in oocytes and embryos. Furthermore, bezafibrate remarkably improved the mitochondrial function and blastocyst quality as well as markedly reduced the mitochondria/TOM20 ratio and mtDNA copy number. The elevated PARKIN level indicated that mitophagy was induced by bezafibrate treatment after post-ovulatory aging. Collectively, these results suggest that bezafibrate beneficially affects against porcine post-ovulatory oocyte aging in porcine by its antioxidant property and mitochondrial protection.

MEMS cytometer for porcine oocyte deformation measurement

Mechanical properties of cells are indicators of biological potential, quality and presumptive pathological status. Mammalian oocytes are particularly important cells from the point of view of testing their fertilizing potential and performing in vitro fertilization. However, only occasionally are their mechanical parameters measured.A new method for deformation measurement of in vitro-matured porcine oocytes has been developed and presented in this study. The method was used for multiparametric characterization of gametes using a MEMS-type microcytometer and a new methodology for determining cell deformability. The novel chip design, principle of operation, and fabrication processes are described in the study. Using original analysis of microscopic images with the usage of dedicated software and data analysis algorithms, deformation parameters were determined. Results indicate the relationships between parameters under the influence of cell compression and show that the behaviour of oocytes during measurements depends on the morphological quality of the cumulus-oocyte complexes they originated from.

Effect of porcine uterus as ex vivo model of fertilizing ability and gene expression pattern on blastocysts

The success of in vitro embryo production demonstrates that the oviduct can be bypassed during early embryonic development. Using an ex vivo model of porcine uterus is one of the strategies used to investigate fertilization within the oviductal environment. In this study, in vitro-matured porcine oocytes (MII) were fertilized with 7.5 × 107, 15 × 107, or 30 × 107 sperm cells for 20 min in the oviduct of a porcine uterine ex vivo model. MII oocytes used for in vitro fertilization (IVF) served as control 1; those cultured in the oviduct of the ex vivo model for 20 min before IVF served as control 2. In present study, the penetration rate, polyspermy, and fertilization efficiency, and accumulated reactive oxygen species (ROS) levels in the treatment groups were significantly decreased compared to those in the control 1 group. During embryonic development, the cleavage rates in the treatment groups were significantly lower than those in the control groups. The cleavage rate in the 30 × 107 sperm cell-treated group was higher than that in the 7.5 × 107 sperm cell-treated group. The blastocyst formation rate in control 1 and 2, and 30 × 107 sperm cell-treated groups increased compared to that in the 7.5 and 15 × 107 sperm cell-treated groups. PCNA, HSP70.2, and GLUT1 were upregulated in the treatment groups and POU5F1, BAX, GPX1 were upregulated in the treatment and control 2 groups, compared to the control 1 group. These results suggest that an ex vivo model may decrease the penetration rate and fertilization efficiency by increasing the accumulated ROS levels and inducing the expression of apoptosis- and stress-related genes. However, the model improved the monospermy rate and expression of embryo developmental competence genes. This is the first study that evaluates the effect of an ex vivo model of porcine uterus on fertilization parameters, and the development of porcine embryos.

Effects of recombinant OVGP1 protein on <i>in vitro</i> bovine embryo development

Previously, our group demonstrated that recombinant porcine oviductin (pOVGP1) binds to the zona pellucida (ZP) of in vitro-matured (IVM) porcine oocytes with a positive effect on in vitro fertilization (IVF). The fact that pOVGP1 was detected inside IVM oocytes suggested that this protein had a biological role during embryo development. The aim of this study was to evaluate the effects of pOVGP1 on bovine in vitro embryo development. We applied 10 or 50 µg/ml of pOVGP1 during IVF, embryonic in vitro culture (IVC), or both, to evaluate cleavage and embryo development. Blastocyst quality was assessed by analyzing the expression of important developmental genes and the survival rates after vitrification/warming. pOVGP1 was detected in the ZP, perivitelline space, and plasma membrane of blastocysts. No significant differences (P > 0.05) were found in cleavage or blastocyst yield when 10 or 50 µg/ml of pOVGP1 was used during IVF or IVC. However, when 50 µg/ml pOVGP1 was used during IVF + IVC, the number of blastocysts obtained was half that obtained with the control and 10 µg/ml pOVGP1 groups. The survival rates after vitrification/warming of expanded blastocysts cultured with pOVGP1 showed no significant differences between groups (P > 0.05). The use of pOVGP1 during IVF, IVC, or both, increased the relative abundance of mRNA of DSC2, ATF4, AQP3, and DNMT3A, the marker-genes of embryo quality. In conclusion, the use of pOVGP1 during bovine embryo in vitro culture does not affect embryo developmental rates but produces embryos of better quality in terms of the relative abundance of specific genes.

46 SPINDLE CONFIGURATION OF IN VITRO-MATURED BOVINE OOCYTES EXPOSED TO SODIUM CHLORIDE OR SUCROSE PRIOR TO CRYOTOP VITRIFICATION

It has been previously described that a simple treatment with medium containing elevated NaCl or sucrose concentrations increases the cryotolerance and developmental competence of in vitro-matured porcine oocytes after vitrification and parthenogenetic activation (Lin et al. 2009 Reprod. Fertil. Dev. 21, 338–344). This work was designed to study whether the exposure to increased concentrations of NaCl or sucrose before vitrification improves cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, in vitro-matured oocytes were exposed to different NaCl and sucrose concentrations (from 375 to 808 mOsm) for 1 h. In Experiment 2, and according to the results obtained in the first experiment, oocytes were exposed to 375 mOsm NaCl or sucrose solution, vitrified, and warmed. Nontreated oocytes were used as controls. In both experiments, oocytes were fixed after treatment and microtubule, and chromosome distribution was analysed by immunocitochemistry. All statistical analyses were conducted with the IBM SPSS 19 for Windows (IBM corp., Chicago, IL). ANOVA was performed to analyse differences in meiotic spindle. Statistical significance was set at P < 0.05. After exposure to 375 mOsm of NaCl or sucrose, similar percentages of oocytes showing normal chromosome distribution were obtained compared to the control group (83.4, 71.8, and 85.0%, respectively). Groups treated with higher concentrations (443 to 808 mOsm) triggered significantly lower proportions of normal spindles. After vitrification/warming, no significant differences were observed between nonvitrified oocytes (71.3%) and those treated with NaCl before vitrification/warming procedure (41.9%) when normal chromosome organisation was analysed. Significantly higher percentages of normal chromosome configuration were observed when oocytes were exposed to sucrose before vitrification (34.2%) compared with control-vitrified oocytes (23.3%). However, pretreatment with NaCl or sucrose before vitrification did not trigger significant differences in terms of percentages of normal microtubule configuration (41.9 and 32.9%, respectively) compared with control-vitrified oocytes (40.2 and 24.4%, respectively), although both treatments differed significantly from control (79.1 and 81.7%, respectively). In conclusion, this study showed that a 375-mOsm NaCl or sucrose pretreatment of bovine oocytes before vitrification did not have a deleterious effect on the organisation of the meiotic spindle of vitrified/warmed bovine oocytes. Further experiments are required to investigate whether in vitro-matured oocytes subjected to this osmotic treatment could improve their development competence after being vitrified/warmed.

301 ZINC IS INVOLVED IN PORCINE OOCYTE MEIOTIC MATURATION

Zinc is an extremely important trace element that plays important roles in several biological processes. In this study, we investigated the role of zinc during meiotic resumption and metaphase arrest in in vitro-matured porcine oocytes. Oocytes at either germinal vesicle (GV) or MII stage were treated with TPEN, a Zn2+ chelator. Meiotic resumption and activation were assayed. Effect of PMA, a PKC activator, on GV breakdown (GVBD) and oocytes activation after TPEN treatment were checked. Results showed that depletion of zinc with 3 µM TPEN-blocked oocytes at GV stage (60.85 ± 5.15 v. 15.60 ± 0.20%; P < 0.05) after 25 h of maturation. The 10-µM TPEN treatment at MII stage significantly (P < 0.05) increased pronucleus formation (90.61 ± 9.10 v. 5.56 ± 9.62%; P < 0.05) and the second polar body extrusion (93.64 ± 5.53 v. 8.59 ± 8.34%; P < 0.05). The p34cdc2 activity was decreased in both MII and GVBD oocytes that were treated with TPEN as compared to control. Phosphorylated MAPK measured by Western blot was also decreased in GVBD oocytes when zinc was depleted. This might be explained by the low expression of C-mos Cyclin B1 and Cdc2 at this stage. Treatment of the oocytes with PKC agonist PMA (100 nM) rescued the meiotic resumption arrest observed after TPEN treatment (GV stage: 26.91 ± 3.10 v. 83.89 ± 11.94%; P < 0.05). The level of phosphorylation of MAPK and p34cdc2 activity were rescued when PMA were used. Treatment oocytes with 100 nM PMA in the GV stage also increased the signal of zinc indicator, fluozin-3-a.m., by about 4-fold in cytoplasm (P < 0.05). These results showed that zinc regulates meiotic resumption probably through PKC. However, although the TPEN treatment reduced phosphorylation of PKC substrates in both meiotic resumption and the MII stage, rescue of PKC substrates phosphorylation with PMA did not prevent the activation of oocytes caused by zinc depletion. These data demonstrate that zinc regulates meiotic resumption via a PKC-dependent pathway, but independent of that in maintaining of metaphase arrest in porcine oocytes.This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ009601 and PJ009098), Rural Development Administration, Republic of Korea.

Timing of oviductal fluid collection, steroid concentrations, and sperm preservation method affect porcine in vitro fertilization efficiency

To determine optimal conditions for the inclusion of oviductal fluid (OF) in IVF protocols. Experimental prospective study. Mammalian reproduction research laboratory. Oviducts and ovaries from porcine females were collected at a slaughterhouse. A total of 30 oviducts and 1,285 oocytes were used. Boar-ejaculated spermatozoa were also used. In vitro-matured porcine oocytes were preincubated with OF collected from animals before or after ovulation and later fertilized in vitro. Zona pellucida digestion time in oocytes after preincubation in OF was assessed. Concentrations of E2 and P4 in OF were measured. IVF was performed, including within the culture media the E2 and P4 concentrations found in the preovulatory OF. The effect of preovulatory OF on IVF efficiency was compared between fresh and frozen-thawed spermatozoa. E2 and P4 concentrations in OF; penetration and monospermy rates; number of spermatozoa within the ooplasm and on the zona pellucida after IVF under different experimental conditions; zona pellucida resistance to protease digestion. Preincubation of oocytes in OF collected before ovulation enhances IVF efficiency in the pig compared with OF collected after ovulation (29.58 ± 3.84 vs. 11.03 ± 2.69). When frozen-thawed spermatozoa are used for the IVF of these OF-treated oocytes, their fertilization ability increases compared with fresh semen. OF collected before and after ovulation shows significantly different concentrations of E2 (99.00 ± 8.72 vs. <10 pg/mL) and P4 (2.53 ± 0.66 vs. 12.27 ± 2.33 ng/mL), respectively. Addition of E2 and P4 at concentrations similar to those in the OF before ovulation partially simulates the effect of the fluid on IVF outcome. Preincubation of oocytes in OF collected before ovulation is a suitable protocol for increasing the efficiency of IVF with fresh semen in the pig model and could be a useful tool to increase the fertilization ability of frozen-thawed spermatozoa in other species. E2 concentrations in preovulatory OF are higher than those reported in blood serum at the same phase of the estrous cycle.

The effects of glial cell line‐derived neurotrophic factor on the in vitro matured porcine oocyte transcriptome

It is well documented that oocytes from small antral follicles are less competent than those derived from large follicles, and we have previously shown that glial cell line-derived neurotrophic factor (GDNF) enhances developmental competence in oocytes from antral follicles. Exactly how GDNF effects this change and if it depends on the stage of oocyte development is currently unknown. The objective of this study was to examine the transcriptomic effects of follicle size and GDNF on the in vitro maturation of porcine oocytes. Microarray analysis uncovered differentially expressed transcripts among in vitro-matured porcine oocytes from different-size antral follicles, in the absence or presence of GDNF. Oocytes isolated from small follicles showed a lower state of maturation than those from large follicles, with several transcripts associated with meiotic arrest. Addition of GDNF to the culture media had effects that depended on the stage of the follicle from which the oocyte was isolated, with those from small follicles showing decreased expression of genes associated with acetyltransferase activity while those from large follicles showed decreased metabolic activity. In summary, our results revealed considerable differences between the transcriptomes of small- and large-follicle-derived oocytes. Furthermore, GDNF affects the developmental competence of oocytes in follicle-stage dependent manner. Thus, improving our understanding of the requirements for successful in vitro maturation of porcine oocytes will inform current reproductive technologies, with implications for the future of animal and human health.

Transgenic pigs with pancreas-specific expression of green fluorescent protein.

The development and regeneration of the pancreas is of considerable interest because of the role of these processes in pancreatic diseases, such as diabetes. Here, we sought to develop a large animal model in which the pancreatic cell lineage could be tracked. The pancreatic and duodenal homeobox-1 (Pdx1) gene promoter was conjugated to Venus, a green fluorescent protein, and introduced into 370 in vitro-matured porcine oocytes by intracytoplasmic sperm injection-mediated gene transfer. These oocytes were transferred into four recipient gilts, all of which became pregnant. Three gilts were sacrificed at 47–65 days of gestation, and the fourth was allowed to farrow. Seven of 16 fetuses obtained were transgenic (Tg) and exhibited pancreas-specific green fluorescence. The fourth recipient gilt produced a litter of six piglets, two of which were Tg. The founder Tg offspring matured normally and produced healthy first-generation (G1) progeny. A postweaning autopsy of four 27-day-old G1 Tg piglets confirmed the pancreas-specific Venus expression. Immunostaining of the pancreatic tissue indicated the transgene was expressed in β-cells. Pancreatic islets from Tg pigs were transplanted under the renal capsules of NOD/SCID mice and expressed fluorescence up to one month after transplantation. Tg G1 pigs developed normally and had blood glucose levels within the normal range. Insulin levels before and after sexual maturity were within normal ranges, as were other blood biochemistry parameters, indicating that pancreatic function was normal. We conclude that Pdx1-Venus Tg pigs represent a large animal model suitable for research on pancreatic development/regeneration and diabetes.

266 COMPARISON OF OOCYTES FROM LARGE-SIZED (≥8-mm) and MEDIUM-SIZED (3- to 7-mm) FOLLICLES FOR IN VITRO EMBRYO PRODUCTION IN PIGS

Despite recent efforts to improve culture systems, the developmental competence of in vitro-matured (IVM) porcine oocytes is still inferior compared with those that have been in vivo matured. In pigs, cumulus–oocyte complexes (COC) are usually aspirated from 3- to 7-mm follicles and matured for 42 to 44 h in vitro. In this study, we compared oocytes obtained from large-sized (≥8-mm) and medium-sized (3- to 7-mm) follicles in terms of nuclear maturation, intracellular reduced glutathione levels, gene expression, and embryo development after IVM. In the control group, COC (n = 521) were aspirated from 3- to 7-mm follicles and matured for 22 h with hormones (eCG/hCG) and subsequently matured in vitro for 20 to 22 h without hormones at 39°C, 5% CO2. In the large follicle (LF) group, COC (n = 256) were obtained from follicles larger than 7 mm and were subjected to IVM reduced for 18 h. The maturation medium was TCM-199 supplemented with 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng mL–1 of epidermal growth factor, 75 µg mL–1 of kanamycin, 1 µg mL–1 of insulin, and 10% (vol/vol) porcine follicular fluid without hormones. Nuclear status and reduced glutathione content in oocytes were investigated by Hoechst 33342 staining and CellTracker Blue (CMF2HC; Invitrogen, Carlsbad, CA, USA), respectively. The abundance of messenger RNA of genes reflecting the developmental competence of oocytes was analysed in cumulus cells by real-time PCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as the reference. In addition, oocytes were subjected to parthenogenetic activation to assess in vitro embryo developmental competence. Data were analysed by Student’s t-test using SPSS version 17.0 (IBM Corporation, Armonk, NY, USA) and presented as means. All experiments were replicated at least three times. The average frequency of ovaries having ≥8-mm follicles was 2.3% (44/1953 in 11 replicates). Before IVM, the nuclear-stage oocytes from ≥8-mm follicles were as follows: germinal vesicle stage = 15.2%; metaphase I (MI) stage = 55.4%; anaphase I and telophase I (AI + TI) stages = 15.8%; and metaphase II (MII) stage = 13.6%. After 6 h of IVM, 4.2% of oocytes were at the germinal vesicle stage and frequencies of the MI, AI + TI, and MII stages were 43.6, 9.4, and 42.8%, respectively. After 18 h, IVM frequencies of the MI and MII stages were 13.0 and 87.0%, respectively. Oocytes of the LF group showed a significant (P &lt; 0.001) increase in intracellular reduced glutathione level (1.41 v. 1.00) compared with the control (42- to 44-h matured oocytes). Cumulus cells in the LF group showed lower (P &lt; 0.1) messenger RNA expression of COX-2 (cyclooxygenase-2) and TNFAIP6 (tumor necrosis factor, α-induced protein 6), and higher (P &lt; 0.1) expression of PCNA (proliferating cell nuclear antigen) and Nrf2 (NF-E2-related factor 2) compared with the control. After parthenogenetic activation, the oocytes from the LF group had significantly (P &lt; 0.05) higher blastocyst rates and total cell numbers in blastocysts than did the control group (90.1% and 73.6 v. 50.5% and 55.3, respectively). In conclusion, oocytes from preovulatory LF require only 18 h to complete maturation in vitro, and their developmental competence is higher than those obtained from MF. Although limited, oocytes from ≥8-mm follicles offer an alternative source of material for the production of transgenic pigs by somatic cell nuclear transfer. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

Effects of Hoechst 33342 staining and ultraviolet irradiation on mitochondrial distribution and DNA copy number in porcine oocytes and preimplantation embryos

Hoechst 33342 (H342), in combination with ultraviolet (UV) irradiation, is frequently used to aid or confirm the enucleation of porcine oocytes in somatic cell nuclear transfer programs. The exposure of oocytes to H342 and UV irradiation has a deleterious effect on the development of in vitro-fertilized porcine oocytes, with increasing exposure to UV irradiation (up to 30 sec) having more drastic effects. It has been hypothesized that this decrease in embryonic development could be due to damage to the mitochondrial DNA (mtDNA). To investigate this hypothesis, we analyzed the mitochondrial distribution and DNA copy number of in vitro-matured porcine oocytes exposed to H342/UV and the subsequent embryonic development compared with the mitochondrial distribution and DNA copy number of in vivo-derived oocytes and embryos. Using quantitative, real-time polymerase chain reaction (qPCR) protocols to analyze mtDNA and confocal laser scanning microscopy with MitoTracker Deep Red to determine mitochondrial distribution, we demonstrated that the simultaneous exposure of in vitro-matured porcine oocytes to H342 staining and UV irradiation is associated with reduced oocyte developmental competence and abnormal mitochondrial distribution in the resulting cleaved embryos. In addition, 2- to 4-cell embryos derived from oocytes exposed to H342/UV showed a significant decrease in mtDNA copy number. These results should be considered when H342/UV procedure is used during nuclear transfer in recipient porcine oocytes.

Exposure of in vitro-matured porcine oocytes to SYBR-14 and fluorescence impairs their developmental capacity

Staining with Hoechst 33342 followed by ultraviolet irradiation is frequently used to aid or confirm the enucleation of recipient oocytes in porcine somatic cell nuclear transfer programs. However, the procedure has a clearly deleterious effect on the developmental ability of oocytes. This study evaluated the effectiveness of a longer-wavelength fluorochrome (SYBR-14) for visualizing maternal chromosomes in in vitro-matured porcine oocytes and the effects of this dye in combination with fluorescence excitation on the subsequent in vitro fertilization and embryo development of the oocytes. In the first experiment, the oocytes were exposed to different concentrations (1, 3, 5 and 7μg/mL) of SYBR-14 at different incubation times (5, 10 and 30min) in a 4×3 factorial design. The optimal condition for proper metaphase-II plate and first polar body visualization was a 10-min incubation with 5μg/mL of SYBR-14. In the second experiment, the degeneration rate of the oocytes 18h after exposure to SYBR-14 (5μg/mL for 10min) and fluorescence excitation for 5 or 30s was significantly higher (p<0.002) than that obtained for non-exposed oocytes. The fertilization parameters were not influenced by the treatments. The cleavage and blastocyst rates during culture were lower (p<0.001) for the oocytes exposed to SYBR-14 and fluorescence than for those in the non-exposed group. These results indicate that the exposure of mature oocytes to SYBR-14 and fluorescence for periods as short as 5s increased the rate of oocyte degeneration and limited their subsequent developmental competence.

195 ALTERED mRNA TRANSCRIPT EXPRESSION OF IN VITRO- VERSUS IN VIVO-MATURED PORCINE OOCYTES

In contrast to oocytes matured in vitro, porcine embryos that result from in vivo maturation and fertilization have a high developmental competence and readily make the transition from oocyte to blastocyst. This observation led us to investigate the transcript profile differences between in vivo- and in vitro-matured porcine oocytes. For the in vivo-matured group, oviducts of 3 gilts of similar genetic background were flushed 2 days after detection of standing oestrus. MII oocytes were collected in pools of 10 and snap frozen in liquid nitrogen for RNA isolation. The in vitro-matured oocytes were obtained by euthanizing 3 gilts, again with a similar genetic background and recovering the ovaries. Follicles (2 to 8 mm in size) were aspirated and oocytes with multiple layers of cumulus cells and uniform cytoplasm were placed in M-199 supplemented with LH, FSH and epidermal growth factor for 42 h. Upon maturation, cumulus cells were stripped and the healthy MII oocytes were collected in pools of 10 and snap frozen. Total RNA was extracted from 3 pools of 10 oocytes for both treatments using an All prep DNA/RNA micro isolation kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized using oligo (dT′) primed reverse transcriptase with superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was synthesized using DNA polymerase I and sequenced using Illumina Genome Analyzer II. All reads were aligned to a custom-built porcine transcriptome. There were over 18 million reads in the 2 maturation groups that tiled to the 34 433-member transcriptome: 1317 transcripts were detected with a P ≤ 0.1 (Students t-test), a minimum of 7 reads in at least 1 of the treatments and ≥2-fold difference. Real-time PCR was used on selected transcripts. Comparative CT Method was used on an IQ real-time PCR system with the Bio–Rad SYBR green mix. Statistical differences were determined using the Proc general linear model procedure of SAS (SAS Institute Inc., Cary, NC) and means separated with a l.s.d. (P ≤ 0.05). The misrepresented transcripts from the sequencing data were also characterized using the functional annotation tool DAVID. Twelve pathways were overrepresented in the in vitro-matured oocytes (the top 4 are pathways to cancer, spliceosome, cell cycle and ubiquitin-mediated proteolysis). Eight pathways were underrepresented in the in vitro-matured oocytes (the top 4 are cytoskeleton regulation, T-cell receptor signaling pathway, ubiquitin-mediated proteolysis and cell cycle). Eight transcripts were selected for real-time PCR. ZP2 was higher in the in vitro-matured oocytes as determined by both sequencing and real time. ATG4, HSP90, UBAP2 and SOX4 were not different, regardless of assay. SLC7A3, MRPS36 and PDHX2 were not different based on sequencing, but based on real-time MRPS36 and PDHX2, were higher in the in vivo group and SLC7A3 was higher in the in vitro group. In conclusion, there is an abundance of misregulated transcripts and altered pathways in in vitro-matured oocytes. This dataset is a tool that may provide clues to improve the in vitro maturation process so that in vitro-matured oocytes will be more like their in vivo-matured counterparts, thus improving developmental competence. Funded by Food for the 21st Century.

Effects of Hoechst 33342 staining and ultraviolet irradiation on the developmental competence of in vitro-matured porcine oocytes

Hoechst 33342 (H342) in combination with ultraviolet (UV) irradiation is frequently used to assist the enucleation of porcine oocytes in somatic cell nuclear transfer programs. This work evaluated the effects of H342 (5 μg/mL for 12 min) staining and/or exposure to UV irradiation on fertilisability and developmental capacity of porcine oocytes matured in vitro. In Experiment 1, a total of 1388 mature oocytes were distributed in the following groups: Group 1: oocytes without treatment (Control), Group 2: oocytes stained with H342, Group 3: oocytes stained with H342 and UV irradiated for 30 sec, and Group 4: oocytes UV irradiated for 30 sec. Oocytes from each group were exposed to thawed spermatozoa and cultured for 18 h to assess fertilization parameters or for 7 d to evaluate embryo development. Sperm penetration (P < 0.001) and monospermy (P < 0.04) were lower in oocytes exposed to H342/UV (80.7 ± 4.5% and 30.7 ± 5.4%, respectively) than in oocytes from the control group (94.9 ± 4.3 and 50.0 ± 4.9, respectively). The oocytes exposed to H342/UV showed lower (P < 0.001) cleavage (49.8 ± 2.9%) and blastocyst (7.7 ± 2.9%) rates than oocytes from the other groups (range: 73.8 ± 2.9% to 77.7 ± 2.9% and 22.3 ± 2.9% to 30.9 ± 3.0%, respectively). Experiment 2 was designed to evaluate the effect of shorter UV irradiation (5 sec). A total of 1835 mature oocytes were separated into the same groups as those of Experiment 1. The fertilization parameters and the cleavage rates were not influenced by the different treatments. However, the oocytes exposed to H342 and UV irradiation for 5 sec showed a lower (P < 0.02) rate of blastocyst formation (15.2 ± 4.5%) than the oocytes from other groups (range: 26.1 ± 4.5% to 30.7 ± 4.5%). In conclusion, our results demonstrate that the combination of H342 staining with UV irradiation has a clear deleterious effect on the developmental ability of oocytes, with the effects being more intense with increased exposure to UV irradiation.

Use of polarized light microscopy in porcine reproductive technologies

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) could be useful for a non-invasive evaluation of the meiotic spindle and may allow removal of nuclear structures without fluorochrome staining and ultraviolet exposure. In this study, PLM was used to assess its potential application in porcine reproductive technologies. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein in in vitro-matured porcine oocytes; to examine its effects on the oocyte developmental competence; to select oocytes based on the presence of the meiotic spindle detected by PLM; and to assess the efficiency oocyte enucleation assisted with PLM. In the first experiment, the presence of microtubule-polymerized protein was assessed and confirmed in oocytes (n = 117) by immunostaining and chromatin detection. In the second experiment, oocytes (n = 160) were exposed or not (controls) to PLM for 10 minutes, and then parthenogenetically activated and cultured in vitro. In the third experiment, development competence of oocytes with a positive or negative signal to PLM was analyzed after in vitro fertilization. Finally, oocytes (n = 54) were enucleated using PLM as a tool to remove the meiotic spindle. A positive PLM signal was detected in 98.2 % of the oocytes, which strongly correlated (r = 1; p < 0.0001) with the presence of microtubule-polymerized protein as confirmed by immunostaining. Oocytes exposed to PLM did not differ significantly from controls on cleavage, total blastocyst, expanded blastocyst rates and total cell numbers. The percentage of oocytes at the MII stage and blastocyst formation rate in the negative PLM group significantly differed from control and PLM positive groups. Overall efficiency of spindle removal using the PLM-Oosight system was 92.6%. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured porcine oocytes and does not exert detrimental effects on porcine oocyte developmental competence. Selecting oocytes by the presence of a PLM signal provides limited improvement on IVF results. Finally, PLM appears as an efficient method to enucleate porcine oocytes.

245 ANALYSIS OF CORTICAL GRANULE EXUDATE OBTAINED BY CHEMICAL OOCYTE ACTIVATION IN PIGS

Cortical granules (CG) are clue organelles in the mammalian oocyte because once released, their content modifies the zona pellucida (ZP) and oolema, thus preventing polyspermy. However, research on putative CG proteins has progressed slowly because of the picogram amount of proteins contained in CG. Isolation and identification of CG contents in porcine oocytes would help to elucidate the molecular mechanism involved in blocking polyspermic fertilization. Our objective was to study the contents of CG from in vitro-matured (IVM) porcine oocytes, and to achieve this objective, CG exudate was collected after its release from chemically activated oocytes. Oocytes were subjected to IVM in porcine oocyte medium supplemented with 50 μM β-mercaptoethanol for 44 h. After the IVM period, the ZP was removed by protease treatment (0.5% pronase in PBS), and the ZP-free oocytes were activated with calcium ionophore A23187 (6.5 μM, 2 min) in a medium consisting of 114.06 mM NaCl, 3.20 mM KCl, 0.50 mM MgCl2·6H2O, 10.00 mM sodium lactate, 0.35 mM NaH2PO4, 5.00 mM glucose, 25.07 mM NaHCO3, and 8.00 mM calcium lactate·5H2O. After activation, oocytes were transferred to fresh medium without calcium ionophore and kept for 30 min to allow release of the CG content. After this time, medium containing the CG exudate was collected, as well as the activated oocytes, and both samples were stored at –80°C until analysis. Samples were thawed and the CG proteins were concentrated by centrifugation in 10-kDa centrifugal devices (Microcon, Millipore, Billerica, MA) following the manufacturer’s instructions. The CG exudates from activated oocytes (n = 300) and activated oocytes (n = 125) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In brief, 4% stacking and 12% separating gel was used and run using 25 mM Tris–0.2 M glycine buffer, pH 8.6, containing 0.1% SDS for 1.5 h at 150 V and room temperature. After electrophoresis, the gel was silver stained. Thirteen strong bands were identified in the CG exudate lane, with an approximate molecular mass from approximately 45 to 105 kDa. However, the lane for activated oocytes showed faint protein bands. The presence of well-defined bands in the CG exudate lane might correspond to different CG-derived proteins. These preliminary results show a new approach for studying CG content. Further proteomic analysis of the bands will help to describe specific proteins contained in these organelles, shedding light on the role of the cortical reaction in pigs. Supported by MEC and FEDER (AGL2009-12512-C02-01) and Okayama Universit R. R. was granted funding by JSPS (Ref. S-09210).

Positive effects of Forskolin (stimulator of lipolysis) treatment on cryosurvival of in vitro matured porcine oocytes

In order to examine its effect on oocyte lipid content and cryosurvival, Forskolin was added to the medium for in vitro maturation of porcine oocytes. Treatments were control (IVM without Forskolin during the 42 h incubation period), addition of 10 μM Forskolin for the entire 42 h (0–42) and addition of 10 μM Forskolin between 24 and 42 h only (24–42). In Experiment 1, treatments did not differ significantly in cleavage rate, but the blastocyst formation rate was lower in the 0–42 group than for control and 24–42 group oocytes (17, 32 and 40%, respectively; P < 0.05). It was shown in Experiment 2 that Forskolin treatment from 0–42 h and from 24–42 h significantly reduced lipid content of oocytes compared to that of control cells (65 and 99 vs. 140 μm 2 intensity of fluorescence, respectively; P < 0.05). In Experiment 3, the percentage of oocyte survival after cryopreservation and thawing was significantly higher in both Forskolin treatment groups than in control oocytes (72% for 0–42, 65% for 24–42 and 52% for control; P < 0.05). However, Forskolin treatment did not increase cleavage rates of vitrified in vitro matured porcine oocytes (Control group 28%, 0–42 h group 0%, 24–42 h group 26.67%). Addition of Forskolin affected the nuclear maturation of porcine oocytes. The percentage of PBE (polar body extrusion) were significantly reduced in the 0–42 h group (0–42 h group 42.00 ± 2.08 vs. Control group 79.70 ± 2.82 and 24–42 h group 70.60 ± 2.83; P < 0.05). The 24–42 h group showed similar nuclear status to that of the Control group. We propose that delipation engendered by incubation with 10 μM Forskolin during 24–42 hours of maturation increased cryosurvival of in vitro-maturated porcine oocytes and that attendant chemical lipolysis did not impair their further development as it may have done in oocytes incubated with Forskolin for the full 42 h.

351 POLARIZED LIGHT MICROSCOPY: DETECTION OF MICROTUBULES AND ITS EFFECTS ON THE VIABILITY OF IN VITRO-MATURED PORCINE OOCYTES

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) is used as a tool in human and, recently, in farm animals assisted reproductive technologies. PLM could be useful for a non-invasive evaluation of the meiotic spindle. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured porcine oocytes and to examine the effects of PLM on the oocyte developmental competence. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in vitro for 42 h as described by Gil et al. (2004 Theriogenology 62, 544-552). In the first experiment, a total of 97 oocytes from 6 replicates were placed individually in 10-μL drops of TCM-199-Hepes-FCS in a glass Petri dish. PLM was used to detect the presence of polymerized protein which could be forming a meiotic spindle. The presence of polymerized protein and a meiotic spindle was confirmed in individual oocytes by inmunostaining and chromatin detection as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191-201). In the second experiment, a total of 160 oocytes from 4 replicates were exposed or not (controls) to PLM for 10 minutes. Thereafter, the oocytes were parthenogenetically activated and cultured in vitro. Cleavage rate, total blastocyst rate, expanded blastocyst rate on Day 7 and total cell numbers in expanded blastocysts were assessed. Data were analyzed by GLM procedure of SAS. There was a positive correlation (r = 1; P &lt; 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by inmunostaining. A positive PLM signal was detected in 98.9% of the oocytes. A barrel-shape spindle was observed in 94.8% of the individual samples by inmunostaining and all of these oocytes were positive to PLM. Moreover, oocytes exposed to PLM did not differ significantly from controls on cleavage rate (83.7 ± 1.5 v. 84.4 ± 1.5), total blastocyst rate (36.9 ± 3.6 v. 41.2 ± 3.6) and expanded blastocyst rate on Day 7 (21.9 ± 1.7 v. 26.2 ± 1.7), respectively. There were also no differences in total cell numbers counted in expanded blastocysts (32.8 ± 2.6 v. 35.6 ± 2.5). These results indicate that polarized light microscopy did not exert detrimental effects on porcine oocyte developmental competence and it seems an efficient system to detect polymerized protein in in vitro-matured porcine oocytes. Grant support: INIA: RZ2007-00013-00-00. I. Molina, M. Muñoz, B. Trigal and D. Martín are sponsored by INIA, RYC08-03454, Cajastur and PTA2007-0268-I, respectively.

Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs

We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence of in vitro matured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence of in vitro matured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP alphaT). No differences were observed in COMP alphaT values among GSH-treated and control groups. COMP alphaT values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.