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To gain a better understanding of global methylation differences associated with development of nuclear transfer (NT)-generated cattle, we analyzed the genome-wide methylation status of spontaneously aborted cloned fetuses, cloned fetuses, and adult clones that were derived from transgenic and nontransgenic cumulus, genital ridge, and body cell lines. Cloned fetuses were recovered from ongoing normal pregnancies and were morphologically normal. Fetuses generated by artificial insemination (AI) were used as controls. In vitro fertilization (IVF) fetuses were compared with AI controls to assess effects of in vitro culture on the 5-methylcytosine content of fetal genomes. All of the fetuses were female. Skin biopsies were obtained from cloned and AI-generated adult cows. All of the adult clones were phenotypically normal and lactating and had no history of health or reproductive disorders. Genome-wide cytosine methylation levels were monitored by reverse-phase HPLC, and results indicated reduced levels of methylated cytosine in NT-generated fetuses. In contrast, no differences were observed between adult, lactating clones and similarly aged lactating cows produced by AI. These data imply that survivability of cloned cattle may be closely related to the global DNA methylation status. This is the first report to indicate that global methylation losses may contribute to the developmental failure of cloned bovine fetuses.

The objective of this study was to examine the timing and mechanisms involved in transcription initiation in bovine embryos. Transcriptional activity and its regulation were explored by labeling 1-cell zygotes and 2-cell embryos with [ 3 H]uridine in the presence or absence of alpha-amanitin, aphidicolin, and tricostatin A (TSA) (inhibitors of mRNA synthesis, DNA replication, and histone deacetylases, respectively) followed by a total RNA isolation and determination of [ 3 H]uridine incorporation. We also analyzed translation of zygotic/embryonic mRNAs by labeling zygotes and 2-cell embryos with [ 35 S]methionine in the presence or absence of alpha-amanitin, aphidicolin, and TSA followed by two-dimensional PAGE and autoradiography. We show that bovine 1-cell zygotes and 2-cell embryos are transcriptionally and translationally active. The first and second rounds of DNA replication are important regulators of early gene expression as the inhibition of DNA replication resulted in a dramatic decrease in both transcriptional and translational activity. Moreover, acetylation of histones plays an important role in this early gene activation at the onset of embryonic development in the cow.

The transfer of nuclei from one cell to another provides a powerful tool for studying the interactions between the cytoplasm of one cell and the nucleus of another. This study was designed to examine the ability of the bovine metaphase oocyte cytoplasm to support mitotic cell cycles under the direction of differentiated somatic cell nuclei of various mammalian species. Skin fibroblast cells from cows, sheep, pigs, monkeys, and rats were used as sources of donor nuclei. Nuclear transfer units produced by fusion of enucleated bovine oocytes and individual fibroblasts from all species examined underwent transition to interphase accompanied by nuclear swelling, further progression through the cell cycle, and completion of the first mitosis. Regardless of the species of donor fibroblasts used, some cleaving units progressed further and developed to advanced stages, as evidenced by continuation of cell proliferation and formation of a blastocoele cavity at the time appropriate for the donor fibroblast species. Although no pregnancies have been carried to term after transfer of embryos into surrogate animals, these observations suggest that mechanisms regulating early embryonic development may be conserved among mammalian species and that bovine oocyte cytoplasm can support the introduced differentiated nucleus regardless of chromosome number, species, or age of the donor fibroblast.

The presence of checkpoint mechanisms which are able to recognize damaged chromatin and thereafter to prevent exit from metaphase I has been investigated in giant mouse oocytes produced by fusion of a normal metaphase I oocyte with an equivalent oocyte with damaged chromatin. The presence of damaged chromatin did not prevent the onset of anaphase I in both sets of chromatin in the fused cells. Interestingly, fused or unfused cells containing only damaged chromatin failed to enter anaphase and persisted instead in a metaphase-like state. These results demonstrate the fragility of checkpoint controls in mammalian female germ cells.

Neither bovine follicular fluid nor granulosa cells affected the completion of the first meiotic division in vitro of bovine oocytes. Porcine follicular fluid also had no effect on maturation of bovine oocytes in vitro. Porcine follicular fluid and granulosa cells did not inhibit maturation of porcine oocytes. The results are discussed in relation to current literature on intrafollicular control BIOLOGY OF REPRODUCTION 23, 699-704 (1980)

Organ culture methods using segments of follicle wall and oocytes were employed to study the follicular control of meiosis in porcine oocytes. Segments of follicle wall maintained dictyate arrest in oocytes, an action which was specific to the follicle hemisections and not shown by control tissue. LH was shown to induce meiotic resumption in this in vitro system. The cumulus oophorus was found necessary to prevent oocyte maturation in vitro consistently when follicle wall was present.

Brief exposure to a medium of elevated ionic strength combined with subsequent incubation in dialyzed fluid collected from bovine ovarian follicles results in an enhanced uptake of Ca2+ by bovine ejaculated sperm. An uptake of Ca2 + by either epididymal or ejaculated sperm is also induced by treatment with the divalent cation ionophore A23187 and is followed by release of the aerosomal enzyme hyaluronidase. A similar release of enzyme from ejaculated sperm is produced by hypertonic treatment and follicular fluid, but not by either alone. Uptake of Ca2 + and release of hyaluronidase likewise follow ionophore treatment of epididymal sperm from the guinea pig. rabbit, and hamster. In control experiments, hyaluronidase release is not induced by treatment with the protonophore, carbonylcyanide-in-chlorophenylhydrazone, by the respiratory inhibitor, rotenone, or by treatment with A23 187 in a Ca2 +.free medium. It is concluded that two presumptive components of the capacitation sequence of bovine spermatozoa, enhanced influx of Ca2 + and subsequent release of acrosomal contents, are produced either by ionophore treatment or by hypertonic extraction and exposure to follicular fluid, procedures that produce capacitation in vitro for the sperm of several other mammalian species.

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine follicular oocytes (N = 5991) were exposed to an analog of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cyclic AMP (db-cAMP) (2.5, 5, and 10 mM), the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX, 0.2 mM), or the purine, hypoxanthine (0.5, 1.0, 2.0 mM), and the nucleoside, adenosine (0.05, 0.1, 0.2 mM), for 6 or 21 h to assess their effects on oocyte nuclear maturation. Potential effects of bovine follicular fluid (BFF) were also evaluated after different preculture washing procedures. In a separate experiment, denuded oocytes were used to study the effect of cumulus removal on meiotic inhibition. Db-cAMP decreased the frequency of germinal vesicle breakdown (GVBD) at 6 h (88% for control and 51%, 45%, and 32% for 2.5, 5, and 10 mM concentrations, respectively). IBMX had a comparable effect with only 41% of the oocytes resuming meiosis. Hypoxanthine and adenosine alone or in combinations also decreased the number of oocytes undergoing GVBD at 6 h. Only 22% GVBD occurred when the combined highest concentration of both substances was used compared to 88% in controls. If oocytes were incubated in 50% BFF after a wash in control medium during processing, 56% would resume meiosis at 6 h vs. 35% if the washing procedure included inhibitors (db-cAMP + IBMX). Total BFF (100%) during washing and maturation prevented 72% of the oocytes from resuming meiosis. Db-cAMP and IMBX combined or BFF also inhibited meiotic resumption of denuded oocytes. At 21 h, the inhibitory effects were less pronounced, with most oocytes only delayed in completing the first reduction division.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY One hundred and .tWO puberal gilts from nine genetic groups were used to determine the association of two morphological types of Ovarian follicular loss (hemorrhagic and milky) with genetic background of ~gilts, level of feed intake and stage of estrous cycle. At estrus (day 0 of estrous cycle) a lesser prqportion of gilts (P

Bovine epididymal spermatozoan motility and respiration are enhanced by cyclic nucleotide phosphodiesterase inhibitors such as caffeine (Garbers et al., 1971). This report deals with additional aspects of the relationship of these cyclic nucleotide phosphodiesterase inhibitors and bovine epididymal sperm metabolism. Caffeine induced elevated cyclic3’,5’-adenosine monophosphate (cyclic AMP) concentrations within 1 mm after addition, with either endogenous substrate or 10 mM fructose as substrate. Although the stimulation of motility was rapid, the maximal respiratory rate with either pyruvate or glucose as substrate did not occur until 1 to 4 mm. After incubation of sperm with caffeine for a period of time, no respiratoly lag occurred on addition of pyruvate. With fructose as substrate, ATP concentrations declined significantly between 3 to 5 mm after addition of caffeine, whereas ATP concentrations were significantly reduced by 2 mm in the absence of exogenous substrate. ADP and AMP concentrations increased as ATP decreased, resulting in a marked decline in the phosphate potential. Bongkrekic acid (BKA), an inhibitor of mitochondrial adenine nucleotide translocase, blocked sperm motility and respiration with acetate or pyruvate as substrate, whereas it had no effect on motility in the presence of glucose. Caffeine was capable of stimulating motility in the presence of glucose and BKA. Caffeine also increased glucose utilization rates, and lactate accumulation from glucose, in the absence or presence of BKA. Pyruvate utilization was either not affected or was reduced by caffeine, whereas lactate accumulation from pyruvate was consistently reduced. These results suggest a general stimulation of sperm metabolism by cyclic nucleotide phosphodiesterase inhibitors. The increase in sperm motility and cyclic AMP is rapid and precedes any significant decline in ATP concentrations or increases in respiratory rate. The data suggest that the fall in ATP concentrations, and therefore the decline in “phosphate potential,” is a causal factor responsible for the glycolytic and respiratory rate enhancement observed in sperm treated with cyclic nucleotide phosphodiesterase inhibitors.