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The preimplantation embryo is highly resilient to experimental manipulations. A specific manipulation that has revealed many clues to the developmental process is chimera production. Chimeras have been used to describe the importance of developmental characteristics of embryonic cells and how these characteristics are involved with developmental fate. These characteristics have been monopolized in the production of interspecific chimeras and the production of transgenic animals. This review attempts to discuss the major factors affecting preimplantation mammalian embryo chimera production.

The regulation of acrosomal exocytosis in capacitated bovine spermatozoa by soluble extracts of zonae pellucidae was examined. Kinetic studies demonstrated that zonae pellucidae stimulated synchronous acrosome reactions. The t12 of this process was 5–10 min and response was maximal at 20 min. The apparent initial rate of exocytosis in sperm populations was dependent upon the concentration of zona pellucida protein, with an ED50 and a maximally effective dosage of 20 and 50 ng protein/μl, respectively. Zonae pellucidae caused up to a 48-fold increase in the apparent initial rate and a 3- to 4-fold stimulation in the net occurrence of exocytosis. In contrast, solubilized zonae pellucidae did not induce acrosome reactions in uncapacitated sperm. The development of a capacitated state, as assayed by the ability of sperm to fertilize eggs in vitro, was compared to the expression of zona pellucida-regulated acrosome reactions in a series of kinetic experiments. Both activities were manifest with similar kinetics and displayed identical dependencies toward stimulatory and inhibitory agents in vitro. It is concluded that capacitation is an essential prerequisite for the induction of acrosomal exocytosis in bovine sperm by the zona pellucida.

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

Mammalian sperm cells contain most of the components of a cyclic AMP-mediation system. To determine if the cyclic AMP-dependent protein kinase has a role in the control of bovine sperm motility, a sperm model was developed that was permeable to exogenously added ATP. Treatment of bovine epididymal spermatozoa with dithiothreitol and Brij-35 (polyoxyethylene alcohol), a nonionic surfactant, resulted in a sperm model with caffeine-stimulated, ATP-reactivatable motility. The results of the data obtained using this sperm model can be summarized as follows. (1) Brij partially solubilized the cyclic AMP-dependent protein kinase activity and released nearly half of the total acid-extractable nucleotides of the cells. (2) Brij treatment severely damaged the sperm mitochondria as judged by their lack of respiration. (3) Brij-treated spermatozoa lose their motility but were reactivated with ATP; the reactivated motility was stimulated by caffeine. (4) Despite the caffeine stimulation of motility in Brij-treated spermatozoa, increased protein phosphorylation did not accompany reactivation of motility, nor could a cyclic AMP effect be demonstrated on reactivated motility or on kinase activity in the sperm model.

Bovine epididymal and ejaculated spermatozoa were incubated for 22 and 9.5 h respectively, in a chemically defined medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. Addition of bovine follicular proteoglycan or chondroitin sulfates ABC significantly increased the incidence of acrosome reaction. The stimulatory effects of the proteoglycan or chondroitin sulfates were negated by exposure to the enzyme chondroitinase ABC. Viability of spermatozoa was not affected by the various experimental treatments. Transmission electron microscopy of spermatozoa showed that vesiculation had occurred between the plasma and outer acrosomal membrane. These results suggest that proteoglycan present in follicular fluid at the time of ovulation may promote the acrosome reaction which precedes the ability of sperm to fertilize an ovum.

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.

Three protein kinases were separated from bovine epididymal spermatozoa by using DEAE-Sephadex ion exchange chromatography and Sephadex G-100 gel filtration. The molecular weights of these three kinases were estimated to be 120,000, 78,000 and 56,000 by the use of Sephadex G-100 gel filtration. The activity of all three kinases was greatly enhanced by cyclic adenosine 3',5'-monophosphate (cyclic AMP). In the presence of 10-6 m cyclic AMP, a catalytic subunit of molecular weight 30,000 to 35,000 was separated from each of the three protein kinases. Under these same conditions, cyclic AMP-binding proteins were located in fractions corresponding to molecular weights of 78,000, 35,000 to 40,000, and 17,000 to 18,000; the latter may result from proteolysis for it was not observed in fresh preparations. The cyclic AMP-binding protein of molecular weight 78,000 combined with the isolated catalytic subunits of all three protein kinases. The apparent Km for casein (approximately 3 mg per ml) appeared to be neither different for the three kinases nor affected by the addition of cyclic AMP. The apparent Km for ATP varied from 3.5 to 8.7 µm, but it appeared to be the same for all three protein kinases and also appeared to be unaffected by cyclic AMP. The isolated catalytic subunit of the kinase of molecular weight 120,000 exhibited a Km for ATP of 5.5 µm, which agreed with the Km for the intact kinase. Maximum kinase activity was at about pH 6.5 with casein as substrate and at about pH 7.5 with histone as substrate for all three kinases. The isolated catalytic subunit of the 120,000 molecular weight kinase showed identical responses to pH. Cyclic AMP activated all three kinases half-maximally in the 10-7 m range, whereas cyclic GMP activated the kinases half-maximally in the 10-6 m range. 8-Methylthio-cyclic AMP appeared to be more potent than cyclic AMP in activating the kinases. The data suggest that the catalytic subunits of the three kinases are either similar or identical.

Proteoglycan production by granulosa cells in vitro is regulated by gonadotropins. The objective of this study was to determine if FSH stimulation of proteoglycan synthesis was modulated by calmodulin or calcium. Assay for calmodulin using an ATPase assay dependent on calmodulin yielded concentrations of 7.7 microM. Bovine granulosa cells from follicles 1-9 mm diameter were incubated for 45 minutes in a chemically defined medium containing 5 microCi/ml 3H-glucosamine and various phenothiazine drugs which are inhibitors of calmodulin. In response to oFSH or rFSH at equivalent biological potencies, proteoglycan production decreased with increasing concentrations of phenothiazines from 1 to 50 microM. Addition of EGTA at 0.0, 0.5, 1.0 or 2.0 mM showed decreased proteoglycan production with increased amounts of the chelator. These data suggest that calmodulin and calcium are necessary for proteoglycan production by granulosa cells in response to FSH in vitro.

The present study shows that there is communication between reaggregated asynchronous cleavage stage blastomeres that regulates blastocoele formation. Individual blastomeres from eight-cell murine embryos were transferred to empty zonae pellucidae, intact two-cell embryos, or enucleated two-cell embryos, and were examined over a period of 75 hours for development of cavitation. It was found that the isolated blastomeres cavitated concurrently with intact control eight-cell embryos, while intact control two-cell embryos cavitated 24 hours later. However, the embryos resulting from combining a two-cell embryo and a blastomere from an eight-cell embryo cavitated at a time in between the eight- and two-cell controls.

Embryonic stem cells (derived from 129/Ola mice) containing a mutant hypoxanthine phosphoribosyltransferase gene that had been corrected in vitro in a planned manner by homologous recombination were injected into blastocysts obtained from C57BL/6J mice. The injected blastocysts were introduced into pseudopregnant female mice to complete their development. Eleven surviving pups were obtained. Nine were chimeras: six males and three females. Two of the males transmitted the embryonic stem cell genome containing the alteration in the hypoxanthine phosphoribosyltransferase gene to their offspring at high frequencies. These experiments demonstrate that a preplanned alteration in a chosen gene can be made in the germ line of an experimental animal by homologous recombination in an embryonic stem cell.

Bovine cumulus-oocyte complexes from small (1-5 mm) follicles were cultured for 24 h in 0.25 ml minimum essential medium supplemented with 10% fetal bovine serum and 20 microCi [3H]glucosamine. Treatment groups consisted of supplementing the culture medium with no hormone (control), 0.5 IU/ml follicle-stimulating hormone (FSH) or 10 mM 8-Br-adenosine cyclic monophosphate (cAMP). After culture, the complexes were fixed for light and scanning electron microscopy. Electron photomicrographs revealed that complexes induced to expand with FSH or cAMP contained a copious glycosaminoglycan (GAG) matrix extending between and around the cumulus cells. Control complexes did not exhibit expansion or an extracellular matrix. The radiolabeled GAG material was isolated for chemical identification. Chemical analyses included: (1) electrophoresis of GAG material, (2) electrophoresis of GAG material after enzyme or nitrous acid treatment, (3) thin-layer chromatography of GAG hydrolysates. The results from electrophoresis showed that the radiolabeled GAG co- migrated with hyaluronic acid. The GAG material was resistant to chondroitinase ABC and nitrous acid degradation but was digested by hyaluronidase. Complexes treated with FSH and cAMP incorporated higher (P less than 0.1 and P less than 0.025 respectively) amounts of [3H]glucosamine into hyaluronic acid than control cultures. Thin-layer chromatography identified the primary amino sugar of the GAG to be glucosamine. These data collectively showed that the radioactive GAG produced by bovine cumulus-oocyte complexes was hyaluronic acid.

The developmental fate of single blastomeres from eight-cell murine embryos reaggregated with intact two-cell embryos was evaluated after culture. Fluorescein isothiocyanate was used to follow developmental fate in preblastocyst chimeric embryos. Expression of stage-specific embryonic antigen 3 was used to assay developmental fate at the blastocyst stage, and glucosephosphate isomerase variants were used to assay at the blastocyst stage and after implantation. The results suggest that the descendents of the 1/8 component stay in a patch area and do not selectively migrate to the inner cell mass (ICM). This is in contrast to many studies that indicate that smaller blastomeres, which are more advanced in development, migrate to the ICM. The differences in experimental designs are discussed. Possible mechanisms for this phenomena are that the eight-cell blastomere is physically excluded from the ICM by position or polarization, or that it is differentiating ahead of the two-cell component and becomes trophectoderm.

The effectiveness of inhibiting bovine sperm migration through cervical mucus in vitro by prior treatment of semen with 45 to 150 micrograms of soybean trypsin inhibitor, univalent (papain-digested, nonagglutinating) and bivalent (undigested) rabbit anti-bovine sperm immunoglobulin, and heat-treated heifer serum was studied. Sperm head-to-head agglutination resulted from treatment of semen with bivalent immune antibody and heat-treated heifer serum. Migration through cervical mucus was inhibited only by treatment resulting in spermagglutination. It is postulated that in vivo inhibition of sperm migration may be influenced by secretory immunoglobulins from the cervix.

This report is to review and summarize various aspects of maturation and fertilization of bovine oocytes. Reference also is made to other species where pertinent data for the bovine are unavailable. Finally, factors important to success of in vitro maturation and fertilization as well as applications of these procedures to the animal industry are addressed.

Lowering the external concentrations of both Mg+2 and Ca+2 caused failure of meiotic resumption in vitro of bovine, oocyte-cumulus complexes. Lowering of external Ca+2 levels singly had no effect on either meiotic resumption or completion of the first meiotic division. Lowering of external Mg+2 concentrations alone, although having no effect on meiotic resumption in vitro when Ca+2 was present, did interfere with the completion of the first meiotic division. The result was arrest of oocyte maturation between germinal vesicle breakdown and formation of the first metaphase plate.

The effects of high intracellular cAMP concentrations ([cAMP]i) on germinal vesicle maintenance of bovine cumulus-oocyte complexes were investigated, using 8-bromo-3',5'-cAMP (8-Br-cAMP) or an invasive adenylate cyclase from Bordetella pertussis to increase the [cAMP]i. The effects of interactions of these agents with macromolecular supplements in culture medium (fetal calf serum, FCS; polyvinylpyrrolidone, PVP; BSA), and different methods of processing complexes before culture, on subsequent germinal vesicle maintenance by invasive adenylate cyclase were studied. While 8-Br-cAMP was unable to maintain germinal vesicle arrest in the majority of oocytes for 20 h (36% with FCS, 24% with BSA, 18% with PVP), it maintained germinal vesicle arrest in a high proportion of cumulus-enclosed oocytes when BSA or PVP was used (37% with FCS, 52% with BSA, 53% with PVP). The difference in frequency of germinal vesicle maintenance between macromolecular supplements was not related to [cAMP]i when assayed after culture for 2 h with invasive adenylate cyclase. Complexes processed in whole follicular fluid were not maintained in meiotic arrest (26%) when cultured with invasive adenylate cyclase and PVP. Complexes processed in follicular fluid with 3-isobutyl 1-methylxanthine (IBMX) plus invasive adenylate cyclase were arrested at the germinal vesicle stage at high frequencies (65%), while those processed in IBMX or IBMX plus 8-Br-cAMP-supplemented follicular fluid had intermediate (43% and 49%, respectively) frequencies of intact germinal vesicles. Oocyte complexes processed in follicular fluid supplemented with IBMX and invasive adenylate cyclase formed morulae and blastocysts (27.2%), as did oocytes processed in follicular fluid alone (26%). Phosphoprotein profiles showed that control oocytes and 8-Br-cAMP-treated oocytes share a profile that is different from that of oocytes treated with invasive adenylate cyclase. These results show that increased [cAMP]i reversibly maintains bovine oocytes in meiotic arrest for an extended period without the occurrence of the post-translational protein modifications observed during meiotic resumption or transient arrest.