Postmortem recovery and cryopreservation of spermatozoa from the vas deferens of rhesus macaques (Macaca mulatta).

To determine whether sperm derived from the vas deferens could be retrieved and successfully cryopreserved, testes were collected from 20 rhesus macaques (Macaca mulatta). The males ranged in age from 3 to 19 yr with an average age of 8.5 yr. No sperm was obtained from three animals that were younger than 4 yr. The remaining 17 samples contained sperm with an average sperm cell number of 421.8+/-88.7x10(6) and an average motility of 72.8+/-4.4%. After 24h of culture in TALP medium at 37 degrees C in 5% CO(2) and 95% air, the overall motility decreased significantly in all samples regardless of treatment. Freezing in TEST (TES-Tris buffer)-yolk buffer containing 6% (vol/vol) glycerol had a significant effect on sperm, reducing the immediate postthaw motility to 42.4% in nontreated samples. Treatment with dibutyryl-cAMP and caffeine further reduced sperm motility after 4h in fresh sperm (72.8% vs. 50.4%) but increased motility in sperm that had been frozen (14.0% vs. 23.2%). The age of the male did not influence sperm concentration or grade but proved to be a significant factor in determining motility of frozen-thawed treated sperm, with lower motility found in samples from older males. Overall, the study demonstrates that motile sperm can be obtained from postmortem males, although subsequent studies will be needed to determine whether the quality is sufficient to facilitate its use in assisted reproduction.

Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryos.

A series of experiments was performed to determine the dynamics of pronuclear development as well as the efficiency of either adenovirus-associated (AAV) or lentivirus-derived vectors to introduce a green fluorescent protein (GFP) reporter gene into rhesus macaque (Macaca mulatta) embryos. Assessment of pronuclear development at various times after fertilization revealed that the appearance of pronuclei was determined by the presence of the first and the timing of the second polar body. The dynamics of pronuclear formation was a significant determinant of whether an oocyte reached the blastocyst stage, however, when the percentage of blastocysts were based on the number of zygotes, the timing of the appearance of polar bodies did not appear to have any effect on subsequent development. Injection of different AAV-derived vectors showed that the serotype of the vector did not affect development or the proportion of transgenic embryos. Moreover, all putative transgenic embryos proved to be expression mosaics. Injection of embryos with lentiviral vectors showed that timing of injection (before or after fertilization) had no effect on subsequent transgene expression, but that the type of reporter gene determined post-injection development and rate of transgenesis. The transfer of embryos following injection of a lentiviral vector into three recipients resulted in one pregnancy which was lost during the second trimester. Analysis of fetal tissues showed ubiquitous presence of the transgene and GFP expression in all tissues examined. These results show that lentivirus-derived vectors can efficiently transform rhesus embryos and are suitable for the generation of transgenic rhesus monkeys.

The effects of blastomere biopsy and oxygen tension on bovine embryo development, rate of apoptosis and interferon-tau secretion.

A series of experiments was performed to examine the effects of blastomere biopsies on subsequent development of IVF-derived bovine embryos. The first experiment was designed to assess the optimal time for blastomere removal. One blastomere was removed either 48 or 72 h after IVF. Biopsy at 48 h resulted in 17.2% of embryos proceeding to the blastocyst stage, which was lower than when biopsies were performed at 72 h (37.5%, p < 0.05). In the second experiment, embryos were cultured either under atmospheric or 5% O(2) following blastomere removal. Biopsies had no effect on rate of blastocyst formation with 36% of controls and 33.7% of biopsied embryos proceeding to that stage. However, culture under 5% O(2) significantly increased the number of blastocysts from 29.9% to 40.3% (p < 0.05). This effect was significant in both biopsied and control embryos. In the final experiment, biopsied embryos were again cultured under different oxygen tension. Blastocysts were collected and cultured individually for 48 h in medium droplets in their respective O(2) concentration after which time the medium was assayed for concentration of interferon-tau (IFN-tau). Reduced O(2) concentration again significantly increased blastocyst formation from 24.9% to 41.9% (p < 0.05). IFN-tau secretion was not affected by biopsies, but culture under atmospheric O(2) resulted in significantly increased IFN-tau concentration in medium droplets (12274.0 +/- 2825.9 pM vs 5046.5 +/- 2562.2 pM; p < 0.05).

In vitro fertilization in the pigtailed macaque (Macaca nemestrina).

The objective of this study was to investigate the possibility of collecting oocytes and semen from pigtailed macaques (Macaca nemestrina) and to establish a protocol for the production of viable embryos that would be suitable for transfer into surrogate females. A total of 82 oocytes were collected from a total of four females (on 2 d with two females each). Semen was collected from the same male on both occasions with respective ejaculate volumes of 0.55 and 0.1 mL containing 2 x 10(9) and 6.6 x 10(8)sperm/mL. Following insemination and after 48 h in culture, 42 (51.2%) of the oocytes had cleaved. Of these, 21 were selected based on developmental stage and their morphology and cryopreserved. The remainder was kept in culture for an additional 5 d, at which time three had reached the expanded blastocyst stage. A total of five transfers were performed with frozen-thawed embryos; two of these resulted in pregnancies and the birth of infants. The results of this study demonstrated that oocytes can be retrieved from pigtailed macaques and that such oocytes can be inseminated and cultured in vitro to the blastocyst stage and give rise to viable offspring after transfer into surrogate females.

Mitochondrial DNA deletions in rhesus macaque oocytes and embryos.

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Mitochondrial DNA (mtDNA) mutations, including the common deletion, have been found in skeletal muscle fibres from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in rhesus oocytes after hormonal stimulation and in embryos generated by in vitro production, or whether this deletion is already present in the immature oocyte. Using a nested primer PCR strategy, we found a significant increase in the proportion of mtDNA deletions in stimulated oocytes and embryos from rhesus macaques, compared with mtDNA deletions in immature, unstimulated oocytes derived from necropsied ovaries of age-matched monkeys. The common deletion is larger in the rhesus (5704 bp) than in humans (4977 bp). Accumulation of mtDNA deletions in oocytes may contribute to mitochondrial dysfunction and impaired ATP production. We propose the rhesus to be an excellent model to assess the quality of gametes and embryos and their developmental competence in primates, including humans.

Ovarian senescence in the rhesus monkey (Macaca mulatta).

BACKGROUND: A decline in fertility is evident in human females past their middle thirties. This 'reproductive senescence', marked by a sharp decline in pregnancy rates, may be attributed to reductions in numbers of available oocytes and their quality. Because Old World primates exhibit ovarian morphology and physiological control and timing of menstrual cycles closely resembling those of humans, the current study investigated the rhesus macaque as a potential model for human reproductive senescence. METHODS: Ovaries collected from females aged 1-25 years and divided into five age groups were analysed histologically. RESULTS: General ovarian morphology demonstrated significant changes as the females approached menopause. The proportions of primordial and primary follicles all demonstrated significant differences across age groups (primordial: 77.1, 79.9, 69.7, 62.9, 55.1%; primary: 21.5, 18.8, 28.5, 35.2, 43.1% for age groups 1 to 5 respectively; P<0.0001 for both). Samples from females approaching or undergoing the menopausal transition (aged 20-25 years) demonstrated evidence of ovarian senescence, having scattered and atretic follicles, low numbers of primordial follicles and reduced stromal tissue. CONCLUSION: This study supports the value of the rhesus monkey as a model for reproductive ageing because its ovary undergoes follicular reservoir depletion similar to that seen in humans.

Effects of developmental stage, embryonic interferon-tau secretion and recipient synchrony on pregnancy rate after transfer of in vitro produced bovine blastocysts.

Three separate trials of bovine embryo transfers were performed consisting of 32, 41 and 33 transfers, respectively, to examine the effects of (a) the developmental stage of in vitro-derived blastocysts, (b) the amount of interferon-tau (IFN-tau) they secreted during culture and (c) the cyclic stage of the recipient at the time of transfer on the probability of establishment of pregnancy. One blastocyst was transferred into the ipsilateral uterine horn to the CL. At the time of transfer, blastocysts were classified into one of three developmental stages (early blastocyst, blastocyst and expanded blastocyst) and the cyclic stage of each cow was assessed (-12 h, on time, +12 h, +24 h, >24 h). Prior to the second and third trials, blastocysts were individually cultured for 24 h in 50 microl medium droplets and the IFN-tau concentration in the droplet was determined. Logistic regression analyses revealed that expanded blastocysts had a significantly higher likelihood of establishing pregnancy (p = 0.009), and that there was a significant interaction with the cyclic stage of the recipient in this group with lower rates of pregnancy resulting from decreasing synchrony with the recipient (p = 0.033). IFN-tau secretion during culture was significantly higher in expanded blastocysts than in the other two groups (p < 0.05). A significant effect of the pre-transfer level of IFN-tau secretion was found only in the 'Blastocyst' group where transfer of embryos with lower IFN-tau production prior to transfer resulted in higher pregnancy rates (p = 0.047). These results demonstrate that IFN-tau secretion may be a useful tool to predict pregnancy outcome, but only within certain developmental stages.

Evolution of the interferon tau genes and their promoters, and maternal-trophoblast interactions in control of their expression.

It is well established that the interferon tau (IFN-tau) family of proteins play a major role in preventing the regression of the corpus luteum during early pregnancy in ruminants, such as cattle, sheep and goats, but not in other mammals. These interferons, which are structurally and functionally related to type I interferon, such as IFN-alpha and -omega, arose from a duplication of an IFN-omega gene approximately 36 million years ago. The IFN-tau genes have continued to duplicate since that time and have acquired the ability to be transcribed uniquely in the trophectoderm. Low expression is first detectable at the blastocyst stage, but massive transcriptional upregulation occurs a few days later during the initial stages of conceptus elongation. Expression is finally terminated upon trophectoderm attachment to uterine endometrium. The major promoter element that controls expression is an Ets-2/AP-1 enhancer element. Growth factors and cytokines released by the maternal endometrium that, possibly in response to progesterone, act through Ras and the mitogen-activated protein kinase (MAP-kinase) signal transduction pathway have been implicated in controlling IFN-tau gene transcription by activating Ets-2. This timely expression of IFN-tau is not only required to rescue the corpus luteum of pregnancy but may also be an indicator of conceptus fitness, thereby serving as a critical factor that dictates the continuation of pregnancy in ruminants.

Sexual dimorphism among bovine embryos in their ability to make the transition to expanded blastocyst and in the expression of the signaling molecule IFN-tau.

IFN-tau is a secretory product of trophectoderm of cattle, sheep, and their relatives and is expressed for a few days in early pregnancy after the blastocyst first forms. It serves to alert the mother that she is pregnant. A delayed or less than robust IFN-tau signal is a likely cause of embryonic loss. Here we have determined whether blastocyst production of IFN-tau, which is encoded by a cluster of genes on chromosome 9, differs between the sexes in cattle, as assessed by culture of in vitro-derived embryos on two different media, one complex (tissue culture medium 199 supplemented with serum) with coculture support, the other relatively simple (synthetic oviductal fluid plus albumin). With both media, female blastocysts produced approximately double the amount of IFN-tau as males, regardless of such variables as oocyte batch, blastocyst quality, hatching, and length of time in culture. However, in either tissue culture medium 199, which contains 5.5 mM d-glucose, or in synthetic oviductal fluid, in the presence but not in the absence of added glucose, significantly fewer female than male embryos were able to progress from the morula/early blastocyst stage to more advanced stages of development. It is possible that the differences between male and female embryos both in their production of IFN-tau and in their ability to progress in development in glucose-rich media are manifestations of phenomena that occur in vivo and provide plasticity in embryo selection during early pregnancy.

Polymorphic forms of expressed bovine interferon-tau genes: relative transcript abundance during early placental development, promoter sequences of genes and biological activity of protein products.

Multiple interferon (IFN)-tau genes exist in cattle, but it has remained unclear how many are expressed, the extent of their variation, and whether different genes exhibit similar patterns of expression and code for proteins with similar biological activities. A total of 118 complementary DNA (cDNA) were bi-directionally sequenced from reverse-transcribed bovine (bo) conceptus RNA over the period from blastocyst formation until day 25 of pregnancy. Fourteen different cDNAs, encoding eight different IFN-tau, were confirmed unique. All showed high sequence conservation (>98% nucleotide identity; >96% amino acid identity). The cDNA fell into three, recently evolved, phylogenetic groups (tau1, 2, and 3). Mean concentrations of IFN-tau messenger RNA were greater at day 17 and day 19 than at day 14 and day 25, with different genes showing comparable expression patterns, although there appeared to be a major bias in expression of two genes (for boIFN-tau1c and tau3a) in blastocysts. Genes representing members of the three boIFN-tau groups were cloned. Their promoter regions were conserved over regions considered important for transcriptional activation. Recombinant protein generated in Escherichia coli from representative genes in the three groups had similar but not identical antiviral activities. In summary, many IFN-tau genes, which are probably under similar transcriptional control, are expressed in bovine trophoblast during the peri-implantation period of development.

Genetic and environmental determinants of interferon-tau secretion by in vivo- and in vitro-derived bovine blastocysts.

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-tau secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo's ability to develop to the blastocyst stage and of subsequent interferon-tau secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-tau. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-tau was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-tau by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.

Control of interferon-tau secretion by in vitro-derived bovine blastocysts during extended culture and outgrowth formation.

A series of experiments was conducted to examine the pattern of interferon-tau (IFN-tau) secretion by bovine blastocysts during extended culture in vitro. In the first experiment, blastocysts were cultured individually for three 48-hour periods. The day of blastocyst formation affected how much IFN-tau was produced during the first two culture periods, but not during the third period. The overall secretion of IFN-tau during the 6-day period increased significantly and well beyond what could be accounted for by the concomitant increase in cell numbers. In the second experiment, blastocysts were initially cultured in individual droplets for 48 hr, then plated into 48-well plates. Medium concentrations of IFN-tau were determined after 48 hr and again after 6 and 12 days of culture. Initial IFN-tau secretion did not affect the ability to form outgrowths or their final size, and initial differences in secretion between groups of blastocysts had disappeared by the second and third analyses. In the third experiment, blastocysts were cultured individually for 48 hr in droplets containing the medium that had been flushed through the uteri of non-pregnant sheep on days 10, 12, and 15 of the estrous cycle. Culture in the medium obtained from the Day 15 flush significantly increased the number of cells that blastocysts contained, as well as IFN-tau secretion.

The effects of group size on development and interferon-tau secretion by in-vitro fertilized and cultured bovine blastocysts.

The effects of culturing bovine embryos in groups were investigated. In the first experiment, 1000 oocytes were matured, fertilized and then cultured in groups of 40 in 25 microl of medium. From half of these groups, blastocysts were removed and cultured separately, while in the other half blastocysts were allowed to remain in the group culture microdrop. Blastocysts developed equally well in both groups, although hatching was reduced in those blastocysts removed from the culture droplet. In the second experiment, 1000 zygotes were cultured from the 8-cell stage to the blastocyst stage either individually or in groups of 40. Culture in groups increased the formation of blastocysts, the percentage of hatching blastocysts, the number of cells within blastocysts and the production of interferon-tau. In the final experiment, 1000 zygotes were cultured in groups up to the blastocyst stage. Two-thirds of these blastocysts were then cultured in groups of three, while the remaining blastocysts were cultured individually. Co-culture did not affect hatching or cell number but significantly elevated interferon-tau secretion. These results demonstrate that group culture either before or after blastocyst formation can alter the expression of a specific gene important for the establishment of pregnancy.

Embryo transfer in the rat as a tool to determine genetic components of the gestational environment.

Embryo transfer, with the recipient dam nursing the transferred progeny, was used to study the impact of the gestational environment on adult blood pressure (BP) in three inbred rat strains, the hypertensive Dahl salt-sensitive SS/JrCtr, the normotensive Dahl salt-hypertension resistant SR/Jr, and the normotensive Dark Agouti rat. Rats that had been cross-fostered within 6 h of birth were included as a control for lactational and nurturing factors. Systolic BP was measured by tail-cuff plethysmography twice a week in rats after the age of 7 weeks. Embryo transfer success, measured as the percentage of embryos transferred resulting in pups weaned at 4 weeks, was 27% between the SS/JrCtr and SR/Jr and 53% for the SS/JrCtr and Dark Agouti. This assessment included all failures, some of which probably were not associated with the transfer. If only the number of embryos transferred to dams with successful pregnancies was included, the success rate was 48% between the SS/JrCtr and SR/Jr and 82% between the SS/JrCtr and Dark Agouti strains. Anomalies in pups were not evident. In contrast to the lactational environment, the gestational milieu had a profound effect on basal blood pressure of the hypertensive SS/JrCtr progeny, less of an effect on that of the Dark Agouti, and no effect on that of the SR/Jr. Although the SS/JrCtr strain is significantly larger than the SR/Jr and Dark Agouti strains, neither embryo transfer nor cross-fostering altered body weight of rats at the age of 6 weeks. These data indicate that embryo transfer can be an easy and efficient method of isolating genetically determined factors of the gestational environment.

Relationship between age of blastocyst formation and interferon-tau secretion by in vitro-derived bovine embryos.

This study was designed to examine the relationship between the speed at which bovine embryos reach the blastocyst stage, their cell number, and interferon-tau production. A total of 800 oocytes were fertilized by frozen-thawed semen. On day 2, 44 hr after exposure to sperm, 78, 320, and 296 embryos were at the two-, four-, and eight-cell stages, respectively, with an overall cleavage rate of 86.8%. Within these three groups 15 (19.2%), 106 (33.1%), and 158 (53.4%) embryos proceeded to the blastocyst stage. Of these 46.7%, 65.1%, and 63.3% hatched in the three groups, respectively. Blastocysts began to appear at day 7, but a few did not form until as late as day 13. Expanded blastocysts (n = 279) were cultured individually for 48 hr in 50-microliter droplets of medium, fixed for cell counts, and the concentration of interferon-tau in the medium was determined. Blastocysts originating from two-cell embryos had significantly fewer cells (46.5 +/- 23.3) than either four-cell- (97.2 +/- 13.5) or eight-cell-derived embryos (113.8 +/- 13.6; P < 0.05). Hatching was accompanied by an increase in cell number (129.8 +/- 15.5 versus 41.9 +/- 14.4; P < 0.01). Blastocysts derived from embryos that had reached the eight- or four-cell stage 44 hr after insemination produced significantly more interferon than embryos derived from two-cell embryos (941.7 +/- 92.1, 930.1 +/- 163.1, versus 232.8 +/- 70.1 pM). In contrast, hatching, ovary batch, the speed of early cleavage, cell number, and quality grade had no effect on interferon-tau secretion. The embryo's age at blastocyst formation was not related to the number of its cells but did have a significant effect (P < 0.001) on interferon-tau production, with mean concentrations in the medium of 294.8 +/- 57.9, 563.3 +/- 82.0, 1126.3 +/- 133.6, 1778.5 +/- 297.2, 512.9 +/- 82.0, 315.0 +/- 157.5, and 157.5 pM among blastocysts appearing from days 7 to 13, respectively. These data suggest that blastocysts that form at days 7 and 8 produce less interferon-tau than those that form on days 9 or 10. Since early-forming blastocysts are generally considered more developmentally competent than those which form late, there may be a negative relationship between early interferon-tau production and competence.

Modulation of blood pressure in the Dahl SS/jr rat by embryo transfer.

Gestational hypertension and malnutrition are associated with hypertension and ischemic heart disease in the adult human. The impact of the gestational environment on the adult blood pressure in two well-characterized genetically homogeneous rat strains, the hypertensive SS/jr and normotensive SR/jr, was studied by cross-fostering within 6 hours of birth and by embryo transplantation with the recipient dam nursing the transplanted pups. Systolic blood pressure (BP) was measured by tail-cuff plethysmography twice a week after the age of 7 weeks. The lactational environment (cross-fostering) had no effect on blood pressure. Embryo transfer between like strains had no effect on the development of hypertension, nor did the BP of R transferred to S (RetS) differ from that of normal R or RetR. At 7 weeks of age, the BP of SetR was significantly lower than that of S or SetS (P<.01) and was similar to that of RetR and R. With age, the blood pressures of the S, SetS and SetR increased at approximately the same rate but from a significantly different baseline. Salt-sensitivity in the S and resistance in the R were not altered. The protective effect of the R gestational environment on SetR female BP was abrogated during whelping and lactation. Embryo transfer and cross-fostering did not alter the weight of rats older than 7 weeks. Because the BP of the R dams were significantly lower than that of the S dams, these studies do not distinguish between the effects of the R dams' lower blood pressure per se and hormonal influences of the R uterus on the S blood pressure phenotype.

Targeted overexpression of Cu/Zn superoxide dismutase protects pancreatic beta-cells against oxidative stress.

Current evidence suggests that reactive oxygen species (ROS) may participate in the destruction of pancreatic beta-cells leading to type 1 diabetes. Genetic factors pre-disposing individual susceptibility to type 1 diabetes might therefore include those affecting the efficacy of ROS metabolism. In a direct in vivo test of this hypothesis, we have generated strains of mice carrying transgenes that supplement basal levels of the radical-scavenging enzyme Cu/Zn superoxide dismutase in the pancreas via directed expression in beta-cells. Expression of these transgenes significantly enhances resistance to alloxan-induced diabetogenesis above that of control animals, thereby providing direct in vivo evidence that genetic variation in ROS metabolism can affect susceptibility to oxidative stress-mediated diabetogenesis.

Adenovirus-mediated gene transfer by perivitelline microinjection of mouse, rat, and cow embryos.

To determine the fate of an episomally expressed transgene, mouse, rat, and cow zygotes were injected into the perivitelline space with approximately 100 pl of buffer containing the replication-defective human adenovirus, AdCMVLacZ/sub360. Viral concentrations ranged from 2.5 to 2.5 x 10(5) plaque-forming units (pfu)/100 pl. As viral titer increased, fewer embryos were able to develop to blastocysts. In the mouse, the percentage of blastocysts formed ranged from 82% in controls to 16% after injection at the highest titer. In the rat and cow, a similar decrease in blastocyst formation was noted (62% to 6% and 26% to 4%, respectively). Reporter gene (galactosidase, LacZ) activity could be detected in mouse embryos after injection at a concentration of only 25 pfu/100 pl, whereas a tenfold higher titer was required in the other two species to observe the blue LacZ reaction product. When examined after 5 (mouse), 6 (rat), or 9 (cow) days of in vitro culture, the proportion of LacZ-positive embryos ranged from 15% to 96%, 6% to 76%, and 18% to 58% in mouse, rat, and cow embryos, respectively, depending upon viral concentration. However, a large percentage of positive embryos proved to be expression mosaics, the degree of which was likewise dependent on titer. While none of the embryos showed LacZ activity at 30 h after injection, 70% of mouse, 8% of rat, and 20% of cow embryos expressed the reporter gene at 42 h. Delaying the timing of injection revealed that the efficiency with which mouse and rat embryos could be infected decreased with increasing degree of differentiation. Only 35% and 18% of mouse embryos expressed the reporter gene after injection at the morula or blastocyst stage, respectively. A similar drop in efficiency was noted in rat embryos when injections took place at the 8-cell, morula, or blastocyst stage, with 70%, 33%, and 9% of embryos, respectively, subsequently showing LacZ activity. Likewise, advanced development resulted in a decrease in the efficiency of viral-mediated gene transfer in cow embryos, with 100%, 78%, and 68% of embryos being positive after injection at the 8-cell, morula, or blastocyst stage, respectively. These results demonstrate that a human adenovirus can be used to express a reporter gene transiently in nonhuman embryos.

Increasing the rate of blastocyst formation and hatching from in vitro-produced bovine zygotes.

This study was designed to identify parameters that would facilitate early selection of superior embryos, as well as to define culture conditions that could increase the proportion of embryos proceeding to the blastocyst stage. In the first experiment, the developmental potential of bovine embryos that had reached different stages of development after 60 h of culture following insemination was assessed. No 2-cell embryos underwent further cleavage. Of the 4-cell embryos (n = 188) only 12.2% progressed to the blastocyst stage, while 62.5% of 8-cell embryos (n = 480) did so (P < 0.01). In a further experiment, the effects of conditioning the culture medium (TCM 199) either with Buffalo rat liver cells (BRLC) or bovine oviductal epithelial cells (BOEC) and the effects of co-culture with either of these 2 cell types were examined. The percentage of 8-cell embryos proceeding to the morula and blastocyst stages was independent of cell type and culture system. However, BOEC-conditioned medium supported significantly lower production of blastocysts than any of the other culture methods. Only 24.1% of the former proceeded to the blastocyst stage after the full 10 d of culture, and only 3% hatched, values that were significantly lower than in the other 3 groups (P < 0.01). Among the latter, 44% progressed to the blastocyst stage in BRLC-conditioned medium while 44 and 46% reached that endpoint after co-culture with BOEC or BRL cells, respectively. The percentages that hatched among these were 28.2, 31 and 28.5%, respectively.

Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos.

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.