Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.     

Developmental capacity of bovine oocytes cryopreserved after maturation in vitro and of frozen-thawed bovine embryos derived from frozen mature oocytes

The present study was conducted 1) to investigate the post-thaw developmental capacity of in vitro mature bovine oocytes (Metaphase II) frozen by 1.6 M of 1,2-propanediol and 2) to confirm the viability of frozen bovine embryos derived from frozen mature oocytes. The cleavage and developmental rates to the blastocyst stage of frozen-thawed mature oocytes were significantly lower (P<0.01) than that of nonfrozen oocytes. When mature oocytes were treated with hyaluronidase, trypsin, or base solution (solution control) before processing to remove the cumulus cells, the developmental rates to the blastocyst stage of frozen-thawed oocytes were 2.8% (5 180 ), 3.1% (9 295 ) and 1.1% (1 89 ), respectively. The viability and developmental capacity of frozen-thawed bovine embryos derived from frozen mature oocytes were not different from those of frozen-thawed bovine embryos derived from nonfrozen mature oocytes (control). Furthermore, nonfrozen and frozen-thawed embryos derived from frozen-thawed mature oocytes were nonsurgically transferred to recipient cows. One of the four and one of the two recipient cows became pregnant, respectively. The results of this study demonstrated the viability of embryos obtained from frozen-thawed bovine oocytes at Metaphase II followed by in vitro fertilization and culture to the blastocyst stage in vitro.     

Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes

Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.     

<Emphasis Type="Italic">In vitro</Emphasis> fertilization and preimplantation development in the primate

Nonhuman primates represent a strong model for examining the chromosomal, biochemical, and temporal normality of embryos produced byin vitro fertilization. Morein vitro fertilized embryos from the squirrel monkey(Saimiri sciureus) have been produced and examined than with all other primate species combined. In studies over a 13 year period a fertilization rate approximating 60 % has been developed in this species with 30% of these embryos proceeding to the two cell stage and 50% of these to the three-four cell stage. Chromosomal abnormalities (primarily missing or extra chromosomes) at a level of nine to 16% have been found, a value corresponding to that found inin vivo mating andin vitro fertilization in other species. An incidence of triploidy of 16.7% was observed. RNA and protein synthetic rates appear comparable with those of laboratory species subjected toin vitro fertilization and indicate the initial stages of metabolic activity of the newly formed embryo. Similarly, increases in estrogen incorporation appear after fertilization but no effect is observed in progesterone incorporation. Utilizing 2-deoxy-glucose and insulin, it was determined that the glucose requirement as an energy source for early preimplantationin vitro fertilized primate embryos is very low.Of very great importance is the temporal relationship of the development ofin vitro fertilized squirrel monkey embryos compared with similar development in other primates (including humans) afterin vivo andin vitro fertilization. An analysis of over a decade of work with the squirrel monkey embryos demonstrates a pattern of temporal development that is comparable with all other primate species that have been examined (including the human) and comparable with development afterin vivo fertilization.     

Animal oocyte and embryo cryopreservation

Cryopreservation of oocytes and embryos is a crucial step for the widespread and conservation of animal genetic resources. However, oocytes and early embryos are very sensitive to chilling and cryopreservation and although new advances have been achieved in the past few years the perfect protocol has not yet been established. All oocytes and embryos suffer considerable morphological and functional damage during cryopreservation but the extent of the injury as well as differences in survival and developmental rates may be highly variable depending on the species, developmental stage and origin (for example, in vitro produced or in vivo derived, micromanipulated or not). Currently, there are two methods for gamete and embryos cryopreservation: slow freezing and vitrification. We have experienced both techniques but vitrification has become a viable and promising alternative to traditional approaches especially when dealing with in vitro produced or micromanipulated embryos and oocytes. Recently new strategies based on emerging studies in the field of lipid research have been used to reduce intracellular lipid content in bovine in vitro produced embryos and therefore increase their tolerance to micromanipulation and cryopreservation. The addition of a conjugated isomer of linoleic acid, the trans-10, cis-12 octadecadienoic acid to embryo culture medium more than twice improved embryo post-thawing viability after micromanipulation and vitrification. Vitrification was also used for the cryopreservation of embryos belonging to the Portuguese Animal Germplasm Bank project presently running at our facilities. Presented at the International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 2007 at Vale de Santarém, Portugal.     

Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes

In order to gain further insight into IGF-1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF-1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52(ShcA). Western blot analysis using homologous antibodies evidenced a 60-kDa protein, p60(Xl)(Shc), that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60(Xl)(Shc), Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant-negative form of Grb2 specifically inhibits insulin-induced resumption of meiosis. We finally show that Grb2 binds to p60(Shc) in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2-Sos are implicated in ras-dependent Xenopus oocyte maturation induced by insulin/IGF-1; they also indicate that inability of insulin/IGF-1 to activate the Ras-MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.     

Growth hormone and gene expression of in vitro‐matured rhesus macaque oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010. © 2009 Wiley‐Liss, Inc.     

The influence of insulin on the in vitro development of mouse and bovine embryos

To further investigate the role of insulin during preimplantation embryo development, we compared the effects of insulin on the development of mouse and bovine preimplantation embryos and on cell proliferation during culture in vitro in simplex media. The influence of insulin on the development of mouse zygotes was determined during cultivation in mSOF medium, alone or supplemented with glucose. Similarly, the effects of insulin on the bovine preimplantation embryo development were studied in mSOF medium. The addition of insulin into mSOF medium enhanced significantly the number of cells per mouse blastocyst. Moreover, when mSOF medium was supplemented with insulin and 0.2 mmol x l(-1) glucose, the percentage of hatched blastocysts and the mean cell number of mouse blastocysts were significantly higher. Insulin had no significant effect on the development of bovine embryos, produced by in vitro fertilization of in vitro matured oocytes. Neither the rates of developing embryos nor the mean number of cells in blastocysts were different in comparison with control embryos. Our results suggest that the in vitro development of mouse embryos could be enhanced by the addition of insulin to the culture medium and is further improved by the addition of glucose. In contrast to this our results indicate that insulin has no detectable beneficial effect on the preimplantation development of bovine embryos in mSOF medium.     

The characterization of bovine embryos obtained from prepubertal calf oocytes and their viability after non surgical embryo transfer

We have previously shown that the addition of epidermal growth factor (EGF) during in vitro maturation was capable of stimulating the cytoplasmic maturation of cow and calf oocytes. The aim of the present study was to compare calf and cow blastocysts produced in the presence of EGF in terms of total cell number and cell distribution between trophectoderm (TE) and inner cell mass (ICM), pattern of protein synthesis, and ability to establish pregnancy after embryo transfer to recipients. For all experiment, embryos at Day 7 were obtained from IVM/IVF/IVC oocytes. No significant differences were noted in total cell number (cow= 138±46 vs CALF= 142±59; mean±SD) or ICM and TE cell number between calf (ICM= 35 ± 19, TE= 107± 52) and cow (ICM= 38± 21, TE= 99 ± 32) blastocysts, nor in the ICM/total cell number ratio (cow= 0.27± 11, CALF= 0.25 ± 12). No differences were noted in the constitutive and the neosynthetic protein profiles between cow and calf embryos obtained in vitro. The results of embryo transfer, showed that there was higher pregnancy loss following transfer of calf compared with cow embryos. After Day 35, the rate of pregnancy decreases, with only 22% of calf embryos maintaining pregnancy until calving compared with 39% for cow embryos. In conclusion, it would seem that embryos originating from calf oocytes are less capable of establishing pregnancies than embryos obtained from adult oocytes, althrough this difference was not significant. This low viability cannot be explained by differences in cell number or by the protein profiles identifed between these 2 groups of embryos.     

Functional characterization of an insulin-responsive glucose transporter (GLUT4) from fish adipose tissue

Glucose transport across the plasma membrane is mediated by a family of glucose transporter proteins (GLUTs), several of which have been identified in mammalian, avian, and, more recently, in fish species. Here, we report on the cloning of a salmon GLUT from adipose tissue with a high sequence homology to mammalian GLUT4 that has been named okGLUT4. Kinetic analysis of glucose transport following expression in Xenopus laevis oocytes demonstrated a 7.6 +/- 1.4 mM K(m) for 2-deoxyglucose (2-DG) transport measured under zero-trans conditions and 14.4 +/- 1.5 mM by equilibrium exchange of 3-O-methylglucose. Transport of 2-DG by okGLUT4-injected oocytes was stereospecific and was competed by D-glucose, D-mannose, and, to a lesser extent, D-galactose and D-fructose. In addition, 2-DG uptake was inhibited by cytochalasin B and ethylidene glucose. Moreover, insulin stimulated glucose uptake in Xenopus oocytes expressing okGLUT4 and in isolated trout adipocytes, which contain the native form of okGLUT4. Despite differences in protein motifs important for insulin-stimulated translocation of mammalian GLUT4, okGLUT4 was able to translocate to the plasma membrane from intracellular localization sites in response to insulin when expressed in 3T3-L1 adipocytes. These data demonstrate that okGLUT4 is a structural and functional fish homolog of mammalian GLUT4 but with a lower affinity for glucose, which could in part explain the lower ability of fish to clear a glucose load.     

Effects of fetal calf serum, amino acids, vitamins and insulin on blastocoel formation and hatching of in vivo and IVM/IVF-derived porcine embryos developing in vitro

The objective of this study was to determine the effects of fetal calf serum (FCS), non-essential MEM amino acids, MEM vitamins and insulin on blastocoel formation, expansion and hatching in porcine embryos developing in vitro. Addition of 20% FCS to the NCSU 23 medium significantly (P < 0.05) decreased by the compaction and blastocoel formation of 1- to 2-cell embryos developing in vitro. In contrast, more 1- to 2-cell embryos commenced hatching in the media containing amino acids than in control medium (25.7 vs 2.6%, P < 0.01). Amino acids and insulin synergistically enhanced the incidence of blastocoel formation and hatching of porcine embryos developing in vitro (P < 0.05). When early compacted embryos which developed in vitro in NCSU 23 medium were cultured in BSA-free NCSU 23 medium supplemented with 20% FCS, the incidence of hatching was significantly increased compared with that of the control groups (35.7 vs 4.1%, P < 0.01). However, addition of amino acids, vitamins or insulin to the NCSU 23 medium did not enhance the development of early morulae to the hatched embryos (P > 0.1). When either in vivo or IVM/IVF-derived 1- to 2-cell stage embryos were cultured 4 d in the modified NCSU 23 and an additional 4 days in the modified NCSU 23 supplemented in the FCS, the percentages (61.8 and 17.8%, in vivo- and IVM/TVF-derived, respectively) of hatched blastocysts were significantly higher (P < 0.01) than in the control groups (2.9 and 0%, in vivo and IVM/IVF-derived, respectively). These results suggested that dual culture conditions are required to optimize an in vitro culture system for the development of the porcine embryo in vitro.     

Blastocysts derived from adult fibroblasts of a rhesus monkey ( Macaca mulatta) using interspecies somatic cell nuclear transfer

In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus-oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5-5.5% CO2 under various conditions (37-39 °C and 5-20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.     

Pregnancy rate after the transfer of sheep embryos originated from randomly chosen oocytes matured and fertilized in vitro

Randomly chosen sheep oocytes isolated from 2- to 5-mm follicles of hormonally nonstimulated slaughtered females were matured and fertilized in vitro. Using heparin for the induction of ram sperm capacitation, a fertilization rate close to 80% was recorded. After the transfer of 29 embryos cultured to the 2- to 4-cell stage to 4 recipients, each delivered 1 lamb. In another experiment, 34 2-cell embryos stage were transferred (1 to each oviduct) to 17 synchronized recipients; 8 pregnancies were established and each of 5 recipients delivered a single lamb. The remaining 3 recipients aborted at the third month of gestation. These results show that sheep embryos can be produced in vitro from randomly chosen oocytes and by using relatively simple procedures. However, the viability of the embryos was low, with approximately only 15% developing to term after transfer at the 2-cell stage.     

Localization of nucleic acids in the nucleoli of oocytes and early embryos of mouse and hamster: An autoradiographic study

Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either “maternal” or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5-³H]uridine radioactivity accumulated (after a 2-hr pulse) in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6-³H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4- to 10-hr culture with [8-³H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte—embryo system may explain the specific morphology of both oocyte and early embryo “nucleoli.” © 1995 Wiley-Liss, Inc.     

Regulation of intracellular pH in bovine oocytes and cleavage stage embryos.

This study investigated the mechanisms for the regulation of intracellular pH in bovine oocytes and embryos. Na(+)/H(+) antiporter activity for the regulation of intracellular pH in the acid to neutral range was detected in both in vitro matured bovine oocytes and in vitro produced embryos. However, the activity of the antiporter was significantly reduced in oocytes compared to 2-cell, 4-cell, and 8-cell embryos. HCO(3)(-)/Cl(-) exchanger activity could be detected in oocytes and embryos using the chloride removal method, however the ability of this transporter to regulate intracellular pH against an alkaline load was poor and intracellular pH could not be re-established. The inability of the HCO(3)(-)/Cl(-) exchanger to adequately regulate intracellular pH was further highlighted by the arrest of embryos at the 8-16 cell stage when challenged with a small alkaline load. Therefore, bovine embryos are extremely sensitive to alterations in intracellular pH above the resting value of around 7.2. This sensitivity could account in part for impaired development and viability of bovine embryos produced in vitro.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

Ovarian stimulation of marmoset monkeys (Callithrix jacchus) using recombinant human follicle stimulating hormone

To reduce the number of animals required for controlled studies of marmoset oocytes and early embryos, a superovulation protocol was developed for the common marmoset. Females were given up to 50 i.u./day recombinant human follicle stimulating hormone (FSH)--(r-hFSH) for 6 days. Ovaries were visualized by a modified laparoscopic technique and follicular aspiration was performed using a needle and suction apparatus inserted directly through an otoscope speculum. The number of follicles + ovulation points (+/- S.E.) was 2.9 (+/- 0.2) in controls and 14.1 (+/- 1.6; P < or = 0.001) in the 50 i.u. r-hFSH per day animals. Oocytes, typically at the germinal vesicle stage at collection, extruded a first polar body within 26 hours. In vitro fertilization was performed and embryos developed to the hatched blastocyst stage (34%). With many high quality oocytes and the ability to synchronize cycles, the marmoset is a valuable primate model for examining nuclear reprograming and early embryonic events.     

Effects of in vitro growth culture duration and prematuration culture on maturational and developmental competences of bovine oocytes derived from early antral follicles

Bovine ovaries offer a large pool of oocytes that could be used for in vitro production of embryos of genetically valuable animals. The effects of in vitro growth (IVG) culture duration (10, 12, and 14 days) on the viability and growth of bovine oocytes derived from early antral follicles (0.5–1 mm diameter) in this study. In addition, the effect of pre-IVM culture with phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) on nuclear maturation of IVG oocytes was also evaluated. In experiment 1, oocyte viability observed after 10 or 12 days of IVG culture was greater (P < 0.05) than that observed after 14 days of culture. Oocyte diameters and proportions of oocytes at metaphase II stage were greater (P < 0.05) when 12 or 14 days of IVG culture where used when compared with 10 days culture. In addition, the proportion of oocytes at metaphase II stage was greater (P < 0.05) when pre-IVM culture was performed for oocytes derived from 12 and 14 days of IVG culture. When 12 and 14 days of IVG culture followed by pre-IVM culture were compared in experiment 2, cumulus cell membrane integrity was greater (P < 0.05) after 12 days. Blastocyst production rate for oocytes obtained after 12 days of IVG culture (24.5%) was greater (P < 0.05) than for oocytes obtained after 14 days (9.9%). In conclusion, 12 days IVG followed by pre-IVM culture was considered the optimal processing system for bovine oocytes derived from early antral follicles when oocyte viability, diameter, maturation, and development competences were considered.     

Method for producing transgenic bovine embryos from in vitro matured and fertilized oocytes

Microinjection and in vitro culture procedures were developed to produce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM-199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embryos in vitro for 10 d. In Experiment II, microinjected embryos were cultured 1) in rabbit oviducts, 2) in vitro in a 5% CO(2) in air, or 3) in a 5% CO(2) / 5% O(2) / 90% N(2) incubator. There were no significant differences between the 2 in vitro culture environments. The in vitro culture systems supported development of embryos significantly better than the rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitro vs 10% in vivo; 98% of transferred ova were recovered from the rabbit oviducts. From both experiments, 6 of 92 blastocysts were positive for the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis.