Differing molecular response of young and advanced maternal age human oocytes to IVM.

STUDY QUESTION: What effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation? SUMMARY ANSWER: Although polyadenylated transcript abundance is similar between young and advanced maternal age (AMA) germinal vesicle (GV) oocytes, metaphase II (MII) oocytes exhibit a divergent transcriptome resulting from a differential response to in vitro oocyte maturation. WHAT IS KNOWN ALREADY: Microarray studies considering maternal age or maturation stage have shown that either of these factors will affect oocyte polyadenylated transcript abundance in human oocytes. However, studies considering both human oocyte age and multiple stages simultaneously are limited to a single study that examined transcript levels for two genes by qPCR. Thus, polyadenylated RNA sequencing (RNA-Seq) could provide novel insight into age-associated aberrations in gene expression in GV and MII oocytes. STUDY DESIGN, SIZE, DURATION: The effect of maternal age (longitudinal analysis) on polyadenylated transcript abundance at different stages was analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (YNG; < 30 years; average age 26.8; range 20-29) and five advanced maternal age (AMA; ≥40 years; average age 41.6 years; range 40-43 years) patients. Thus, a total of 10 YNG (5 GV and 5 MII) and 10 AMA (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Patients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided oocyte retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 h incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads on HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, weighted gene correlation network analysis (WGCNA), Ingenuity Pathway Analysis) were performed. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 12 770 genes were determined to be expressed in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least three of five replicates for a minimum of one sample type). Differential gene expression analysis between YNG and AMA oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted P < 0.1 and log2 fold change >1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5 and PRDX1, which have been reported to affect oocyte developmental potential. Despite the similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were correlated (P < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation. LARGE SCALE DATA: Raw data from this study can be accessed through GSE95477. LIMITATIONS, REASONS FOR CAUTION: The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by IVM of patient oocytes. Although the approach has the benefit of identifying intrinsic differences between samples, it may not be completely representative of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Transcriptome profiles of YNG and AMA oocytes, particularly at the MII stage, suggest that aberrant transcript abundance may contribute to the age-associated decline in fertility. STUDY FUNDING/COMPETING INTEREST(S): J.M.R. was supported by an Austin Eugene Lyons Fellowship awarded by the University of California, Davis. The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (awarded to P.J.R.; R01HD070044) and the Fertility Laboratories of Colorado partly supported the research presented in this manuscript.

Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment.

Due to reduced fertility, cryopreserved semen is seldom used for commercial porcine artificial insemination (AI). Predicting the fertility of individual frozen ejaculates for selection of higher quality semen prior to AI would increase overall success. Our objective was to test novel and traditional laboratory analyses to identify characteristics of cryopreserved spermatozoa that are related to boar fertility. Traditional post-thaw analyses of motility, viability, and acrosome integrity were performed on each ejaculate. In vitro fertilization, cleavage, and blastocyst development were also determined. Finally, spermatozoa-oviduct binding and competitive zona-binding assays were applied to assess sperm adhesion to these two matrices. Fertility of the same ejaculates subjected to laboratory assays was determined for each boar by multi-sire AI and defined as (i) the mean percentage of the litter sired and (ii) the mean number of piglets sired in each litter. Means of each laboratory evaluation were calculated for each boar and those values were applied to multiple linear regression analyses to determine which sperm traits could collectively estimate fertility in the simplest model. The regression model to predict the percent of litter sired by each boar was highly effective (p < 0.001, r(2) = 0.87) and included five traits; acrosome-compromised spermatozoa, percent live spermatozoa (0 and 60 min post-thaw), percent total motility, and the number of zona-bound spermatozoa. A second model to predict the number of piglets sired by boar was also effective (p < 0.05, r(2) = 0.57). These models indicate that the fertility of cryopreserved boar spermatozoa can be predicted effectively by including traditional and novel laboratory assays that consider functions of spermatozoa.

Dysregulation of methylation and expression of imprinted genes in oocytes and reproductive tissues in mice of advanced maternal age.

PURPOSE: To evaluate reproductive outcomes in aged compared to young female mice, and determine associated methylation and expression of imprinted genes in reproductive tissues. METHODS: Fetal, placental, and ovarian tissue were collected on d16.5 of pregnancy from young (4-5 weeks) and aged (15 months) mice. Uterine tissue and in vivo matured oocytes were collected from non-pregnant females. Methylation of imprinted genes was determined by restriction enzyme based assays, and transcript abundance of imprinted and nutrient supply genes were analyzed by quantitative PCR (qPCR). RESULTS: Maternal age was associated with fetal growth restriction and placental overgrowth. In maternally aged mice, methylation was minimally dysregulated in fetal tissue, while placental tissue showed aberrant methylation and transcript abundance of imprinted genes. Ovarian methylation and gene expression was severely dysregulated, although oocyte gene expression was only minimally altered. Abundance of Kcnq1 transcripts was significantly (P < 0.05) increased in oocytes obtained from aged females compared to young females. Gene expression was also severely dysregulated in the uterus, including nutrient transport genes. CONCLUSION: Fetal and placental growth abnormalities correspond to aberrant methylation and gene expression in reproductive tissues from maternally aged mice. Significant alterations in gene expression and methylation in the aged ovary suggests that the follicular environment may be compromised. Aberrant methylation and expression of imprinted genes in the aged uterus may contribute to reduced implantation. Maternal age negatively affects imprinted gene methylation and expression in both germ cells and somatic cells of the reproductive tract, contributing to the reduced fertility observed with advanced maternal age.

Fatty acid metabolism during maturation affects glucose uptake and is essential to oocyte competence.

Fatty acid ß-oxidation (FAO) is essential for oocyte maturation in mice. The objective of this study was to determine the effect of etomoxir (a FAO inhibitor; 100 µM), carnitine (1 mM), and palmitic acid (1 or 100 µM) during maturation on metabolism and gene expression of the oocyte and cumulus cells, and subsequent embryo development in the mouse. Carnitine significantly increased embryo development, while there was a decrease in development following maturation with 100 µM palmitic acid or etomoxir (P<0.05) treatment. Glucose consumption per cumulus-oocyte complex (COC) was decreased after treatment with carnitine and increased following etomoxir treatment (P<0.05). Intracellular oocyte lipid content was decreased after carnitine or etomoxir exposure (P<0.05). Abundance of Slc2a1 (Glut1) was increased after etomoxir treatment in the oocyte and cumulus cells (P<0.05), suggesting stimulation of glucose transport and potentially the glycolytic pathway for energy production when FAO is inhibited. Abundance of carnitine palmitoyltransferase 2 (Cpt2) tended to increase in oocytes (P=0.1) after treatment with 100 µM palmitic acid and in cumulus cells after exposure to 1 µM palmitic acid (P=0.07). Combined with carnitine, 1 µM palmitic acid increased the abundance of Acsl3 (P<0.05) and Cpt2 tended to increase (P=0.07) in cumulus cells, suggesting FAO was increased during maturation in response to stimulators and fatty acids. In conclusion, fatty acid and glucose metabolism are related to the mouse COC, as inhibition of FAO increases glucose consumption. Stimulation of FAO decreases glucose consumption and lipid stores, positively affecting subsequent embryo development, while an overabundance of fatty acid or reduced FAO negatively affects oocyte quality.

Alterations in the transcriptome of porcine oocytes derived from prepubertal and cyclic females is associated with developmental potential.

The developmental competence of oocytes is progressively attained as females approach puberty. The poor quality of prepubertally derived oocytes suggests that essential processes during cytoplasmic maturation have not been completed. The objective of this experiment was to identify genes in oocytes that are associated with good (cyclic females) and poor (prepubertal females) developmental competence. Development to the blastocyst stage in vitro was significantly decreased in oocytes derived from prepubertal females compared with cyclic females (5.26 and 12.86%, respectively). Approximately 10% of the oocyte transcriptome was differentially expressed between in vitro-matured oocytes derived from cyclic and prepubertal females (P < 0.05); 58% of differentially expressed genes had increased transcript abundance in oocytes derived from cyclic females. Genes involved in the metabolism and regulation of biological processes had increased transcript abundance in oocytes derived from cyclic females, whereas genes involved in translation were increased in prepubertally derived oocytes. Quantitative PCR confirmed differential expression (P < 0.05) for 6 out of 11 selected genes [DPYD (dihydropyrimidine dehydrogenase), RDH11 (retinol dehydrogenase 11), SFRS4 (serine/arginine-rich splicing factor 4), SFRS7 (serine/arginine-rich splicing factor 7), TL4 (transcribed loci 4), and TOP2B (topoisomerase II ß)] that were differentially expressed with greater than a 2-fold change by microarray, although 3 of these genes, DPYD, TL4, and TOP2B, were in opposing directions by the 2 methods. In conclusion, expression of multiple genes involved in metabolism and translation was significantly altered in oocytes from prepubertal females compared with cyclic females, which was associated with reduced in vitro development to the blastocyst stage. These genes may represent important cellular mechanisms that regulate oocyte quality.

Towards the use of microfluidics for individual embryo culture.

Mammalian embryo development is still relatively inefficient in vitro. Much research has been conducted on the chemical environment, or culture medium, surrounding the embryo, but little attention has been given to the actual physical culture environment, which has changed very little over the years. The application of microfluidics to embryo production in vitro is a tantalising approach that may alleviate some of the limits that traditional microdrop culture places on embryo development and research into gamete and embryo physiology. These devices may lead to enhanced in vitro embryo development and quality by more closely mimicking the in vivo environment. Initial work in this area is promising and gives us proof-of-principle that these unique microfluidic systems may indeed be applicable to in vitro culture of gametes and embryos. The present paper reviews the advantages of microfluidics for in vitro embryo production: how the platforms are manufactured, the current uses of microfluidics in assisted reproduction, static v. dynamic culture environments, individual gamete and embryo culture and the future directions of microfluidic application to in vitro embryo production and manipulation. Finally, preliminary data from our laboratory using a new microfluidic well insert for porcine, bovine and murine embryo culture is discussed.

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs.

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus-oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0mM, 0.02mM, 0.2mM, 2mM, or 20mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM+pFF; positive control) at 38.7 degrees C in 7% CO(2) in air for 40-44h. No significant difference (P>0.05) in nuclear maturation was found between oocytes matured in TCM+pFF or PPM with 0mM, 0.02mM and 0.2mM ammonium chloride. However, nuclear maturation was decreased (P<0.05) in oocytes matured in PPM with 2mM or 20mM ammonium. After IVF, oocytes matured in PPM with 20mM ammonium resulted in embryos with reduced (P<0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20mM ammonium resulted in embryos with lesser (P<0.05) embryonic cleavage compared to TCM+pFF. However, PA embryos derived from oocytes matured in PPM with both 2mM and 20mM ammonium had reduced (P<0.05) blastocyst development compared with TCM+pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.

Detection of anovulation by heatmount detectors and transrectal ultrasonography before treatment with progesterone in a timed insemination protocol.

Our objective was to determine the accuracy of identifying noncycling lactating dairy cows before the application of a timed artificial insemination (AI) protocol [with or without progesterone supplementation via a controlled internal drug-release (CIDR) insert and 2 different timings of AI] by using heatmount detectors and a single ovarian ultrasound examination. At 6 locations in the Midwest, 1,072 cows were enrolled in a Presynch protocol (2 injections of PGF(2alpha) 14 d apart), with the second injection administered 14 d before initiating the Ovsynch protocol (injection of GnRH 7 d before and 48 h after PGF(2alpha) injection, with timed AI at 0 or 24 h after the second GnRH injection). Heatmount detectors were applied to cows just before the first Presynch injection, assessed 14 d later at the second Presynch injection (replaced when activated or missing), and reassessed at initiation of the Ovsynch protocol. Ovaries were examined for the presence of a corpus luteum (CL) by ultrasound before the initiation of treatment. Treatments were assigned to cows based on the presence or absence of a CL detected by ultrasound: 1) no CL + no CIDR; 2) no CL + CIDR insert for 7 d; and 3) CL present. Further, alternate cows within the 3 treatments were assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0 h) or 24 h later. Pregnancy was diagnosed at 33 and 61 d after the second GnRH injection. By using low (<1 ng/mL) concentrations of progesterone in serum as the standard for noncycling status, heatmount detectors were activated on a large percentage of noncycling cows (>60%), whereas the single ultrasound examination incorrectly classified noncycling cows only 21% of the time. Conversely, cycling cows (progesterone > or =1 ng/mL) were correctly identified 70 to 78% of the time by heatmount detectors, but 85 to 92% were correctly identified by ultrasound. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced the incidence of ovulation but increased the proportions of pregnancies per AI at d 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Percentages of cows pregnant and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL and treated with progesterone. Compared with no response, when a follicle ovulated in response to the first GnRH injection, percentage of cows becoming pregnant after the timed AI increased from 33.3 to 41.6%. Timing of AI at 0 or 24 h after the second GnRH injection did not alter pregnancies per AI, but cows having luteal activity before treatment had improved pregnancies per AI compared with noncycling cows. We conclude that identifying noncycling cows by ultrasound was more accurate than by heatmount detectors. Subsequent progesterone treatment of previously cycling cows not having a CL at the onset of Ovsynch increased the proportion of pregnant cows, equal to that of cows having a CL but not treated with progesterone.

A comparative analysis of metabolism and viability in porcine oocytes during in vitro maturation.

The importance of oocyte quality cannot be overstated, because it impacts all subsequent events during development of the embryo, the fetus and even the resulting offspring. Oocyte metabolism plays a critical role in supporting developmental competence via multiple mechanisms. It is beginning to be understood that metabolic pathways not only affect cytoplasmic maturation but may control nuclear maturation as well. A complete understanding of the precise roles that metabolism plays in determining oocyte quality is crucial for developing efficient in vitro maturation systems to support acquisition of oocyte competence. To date, this pursuit has not been entirely successful. Work in our laboratory on porcine oocyte metabolism has elucidated some of the intricate control mechanisms at work within the oocyte, not only for energy production, but also encompassing progression of nuclear maturation, mitochondrial activity and distribution, and oxidative and ionic stresses. We hypothesize that by utilizing oocyte metabolic data, we can develop more appropriate in vitro maturation systems that result in increased oocyte and embryo developmental competence.

The effect of oocyte quality on development.

Oocyte quality affects early embryonic survival, the establishment and maintenance of pregnancy, fetal development, and even adult disease. Quality, or developmental competence, is acquired during folliculogenesis as the oocyte grows, and during the period of oocyte maturation. Assisted reproductive technologies involving ovarian hyperstimulation, or collection of immature oocytes followed by maturation in vitro, perturb this process and result in oocytes with reduced quality. In domestic livestock species, offspring have been produced using in vitro oocyte maturation, although only a small percentage of the original pool of immature oocytes is capable of developing to the blastocyst stage and subsequently resulting in pregnancy. In vitro maturation, as it is currently undertaken, does not support the correct development of oocyte competence. Follicle size affects oocyte quality, potentially implicating messenger RNA or protein stores as factors involved in oocyte competence. Oocytes from preantral follicles grown in vitro are competent to resume meiosis, although development to the blastocyst stage is decreased. An offspring from oocytes produced using this technique was normal at birth but experienced delayed onset health issues, highlighting the importance of oocyte quality long after embryogenesis. Metabolism may play a critical role in oocyte quality because glycolytic activity in mature oocytes is correlated with increased embryonic development. Communication between the oocyte and its surrounding cumulus cells is also important for the development of a competent oocyte. Ovarian stimulation causes delayed embryonic development, increased abnormal blastocyst formation, fetal growth retardation, and increased fetal loss. Thus, although meiosis and even early development may be completed successfully, there are a variety of other processes occurring within the cytoplasm of the oocyte that are required for complete developmental competence. However, the cellular mechanisms that impart oocyte quality are unclear. Until the mechanisms involved in oocyte quality are elucidated, any effort to use assisted reproductive technologies in animals for production or biomedical purposes will be inefficient at best.

Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential.

In vitro maturation (IVM) of goat oocytes with serum-supplemented media results in oocytes with reduced developmental potential. The objective of this study was to develop a defined medium for IVM of goat oocytes that better supports subsequent embryonic development. Cumulus oocyte complexes (COC) were matured for 18-20 hr in: Experiment (1), tissue culture medium 199 (TCM199) with 10% (v/v) goat serum or modified synthetic oviduct fluid maturation medium (mSOFmat) with 2.5, 8.0, or 20.0 mg/ml bovine serum albumin (BSA); Experiment (2), mSOFmat with 4.0, 8.0, 12.0, or 16.0 mg/ml BSA; or Experiment (3), 1.0 mg/ml polyvinyl alcohol (PVA; control), 4.0 mg/ml BSA, 0.5 mg/ml hyaluronate plus 0.5 mM citrate, or hyaluronate, citrate, and BSA. Mature COC were coincubated for 20-22 hr with 12-15 x 10(6) sperm/ml in modified Brackett and Oliphant (mBO) medium. Embryos were cultured for a total of 7 days in G1/2, and evaluated for cleavage, and blastocyst development, hatching, and total cell numbers. In the first experiment, more (P < 0.05) blastocysts developed per cleaved embryo following maturation in mSOFmat with 2.5 or 8.0 mg/ml BSA than with 20.0 mg/ml BSA or TCM199 with 10% goat serum. The various concentrations of BSA used in the second experiment did not affect (P > 0.05) any of the developmental endpoints examined. In the third experiment, developmental potential of oocytes matured with PVA or hyaluronate with citrate was not different (P > 0.05) from oocytes matured in the presence of BSA. These results demonstrate that developmentally competent goat oocytes can be matured under defined conditions.

Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system.

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL(-1) hyaluronan, 0.6 mM cysteine, 10 ng mL(-1) epidermal growth factor (EGF), 0.1 U mL(-1) porcine LH and FSH, and 1 x Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 x 10(5) sperm mL(-1)) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mM bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1% v. 14.3%). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.

Intracellular adenosine triphosphate and glutathione concentrations in oocytes from first estrous, multi-estrous, and testosterone-treated gilts.

Cytoplasmic maturation refers to a variety of cellular changes that must occur in the oocyte in order to progress through subsequent fertilization and embryonic development. Intracellular concentrations of ATP (ATPi) or glutathione (GSHi), indicative of metabolic activity or the ability of the oocyte to form a male pronucleus and cope with cellular stress, respectively, have been used as markers of cytoplasmic maturation in vitro. In the current study, our objective was to determine if concentrations of ATPi and GSHi in oocytes recovered from three groups of gilts were associated with known differences in developmental competence within these populations. In vivo matured oocytes were surgically recovered 36-38 h after the onset of estrus from first estrous gilts, multi-estrous gilts, and multi-estrous gilts receiving testosterone (1mg/2 ml per day; day 13 to estrus, day 0=day of estrus). Concentrations of ATPi and GSHi were determined using a bioluminescent somatic cell assay kit (luciferin-luciferase reaction) and the dithiobisnitrobenzoic acid (DTNB)-glutathione reductase recycling reaction, respectively. There were no differences (P>0.05) between ATPi concentrations in oocytes from the three groups (1.52 +/- 0.10, 1.51 +/- 0.11, 1.56 +/- 0.11pmol per oocyte). In contrast, oocytes from multi-estrous gilts had higher (P<0.05) concentrations of GSHi (31.53 +/- 1.66 to 33.67 +/- 2.30 pmol per oocyte) than oocytes from first estrous gilts (25.07 +/- 0.82). Administration of testosterone did not affect (P>0.05) GSHi concentrations in oocytes from multi-estrous gilts. Differences in developmental potential between the three groups of gilts were apparently not due to different concentrations of ATPi. However, GSHi concentrations were higher in oocytes from multi-estrous gilts, suggesting that reduced developmental potential of oocytes from first-estrus gilts may be related to insufficient amounts of GSHi. The beneficial effect of exogenous testosterone on the percentage of embryos surviving early gestation does not appear to be due to increased GSHi. Of the numerous potential markers of developmental potential, two were examined in the current study, and GSHi appeared to be useful for assessing porcine oocytes.

Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes.

Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at -80 degrees C in groups of 10-20 (GSH) or 5-10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P < 0.05) of GSH (n = 207, 9.82 +/- 0.71 pmol/oocyte; n = 104, 9.73 +/- 0.81 pmol/oocyte; n = 108, 7.89 +/- 0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n = 217, 36.26 +/- 11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n = 70, 0.97 +/- 0.07 pmol/oocyte; TCM-PVA, n = 117, 0.81 +/- 0.13 pmol/oocyte; TCM-MAP, n = 107, 1.02 +/- 0.18 pmol/oocyte; NCSU-pFF, n = 134, 0.71 +/- 0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes.

Use of energy substrates by various stage preimplantation pig embryos produced in vivo and in vitro.

The aim of in vitro embryo systems is to produce embryos of comparable quality to those derived in vivo. Comparison of embryo metabolism as an indicator of viability may be useful in optimization of culture conditions. The aim of the present study was to determine glucose, glutamine and pyruvate use by various stage pig embryos produced in vitro and in vivo. The results indicate that pig embryos use glucose via glycolysis in significant amounts at all stages examined, regardless of embryo origin. In vitro-derived embryos have significantly increased glycolytic activity after the eight-cell stage, whereas in vivo-derived embryos have increased glycolysis at the blastocyst stage. In vivo-derived embryos have higher rates of glycolysis compared with in vitro-derived embryos. Glucose usage through the Krebs cycle for in vitro- and in vivo-derived embryos increased significantly at the blastocyst stage. Pig embryos produced in vitro used constant amounts of glutamine throughout development, whereas in vivo-derived embryos increased glutamine usage after the eight-cell stage. Pyruvate use was minimal at all stages examined for both in vitro- and in vivo-derived pig embryos, showing significant increases at the blastocyst stage. Krebs cycle metabolism of pyruvate, glutamine and glucose by in vivo-derived embryos was higher than that by in vitro-derived embryos. Current in vitro culture conditions produce pig embryos with altered metabolic activity, which may compromise embryo viability.

Development and viability of in vitro derived porcine blastocysts cultured in NCSU23 and G1.2/G2.2 sequential medium.

Porcine embryo development in vitro is relatively inefficient compared to other domestic species. Currently, a single culture medium (NCSU23) is the standard for porcine in vitro systems. However, the G1.2/G2.2 sequential culture system has been beneficial for embryo development in other species. The objective of this study was to compare porcine preimplantation embryo development in vitro and subsequent blastocyst viability and metabolic activity using NCSU23 and G1.2/G2.2 culture media. Oocytes were matured in defined TCM199 base medium for 45 to 47 h and fertilized in mTBM for 4 h. Embryos were cultured in either NCSU23 for 146 h or G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 74 h. Blastocyst substrate use was measured using a modification of the hanging drop technique. Culture in NCSU23 resulted in a higher percentage (P < 0.05) of embryo cleavage (74.0%) and blastocyst development (14.6%) than culture in G1.2/G2.2 (67.8% and 7.8%, respectively). Both NCSU23 and G1.2/G2.2 produced blastocysts with similar mean cell numbers (51.5 +/- 4.3 and 47.1 +/- 4.3, respectively), similar glucose use (10.81 +/- 1.39 and 10.12 +/- 1.72 pmol/embryo/3 h, respectively) and pyruvate use (1.08 +/- 0.056 and 0.88 +/- 0.048 pmol/embryo/3 h, respectively). These data indicate that a sequential culture system can support porcine embryo development in vitro without compromising embryo viability. However, the G1.2/G2.2 system was not as effective as NCSU23 in supporting blastocyst development. Sequential media should be formulated specifically for porcine embryos to improve embryonic cleavage and blastocyst development.