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Diverse groups of chemicals in culture media are needed for successful bovine oocyte maturation and embryo development during which dramatic cytoplasmic and nuclear reprogramming events take place. In vitro embryo production (IVP) procedures frequently include supplements such as serum and/or co-culture with various types of somatic cells. However, the presence of undefined serum in culture media introduces a variation from batch to batch, increases viral or prion contamination risk, and leads to problems during fetal development. The aim of the present study was to investigate the possibility of using chemically defined-synthetic serum substitute (SSS) in place of fetal calf serum (FCS) during maturation and long-term culture to stimulate in vitro maturation (IVM), fertilization (IVF) and subsequent embryo development. In Experiment I, the effect of the protein source on in vitro maturation was tested by maturing oocytes in culture media supplemented with 10% FCS (Control Group), 10% SSS (Group I) and 10% SSS + 10 ng/ml epidermal growth factor (EGF) (Group II). In Experiment II, effects of SSS on both oocyte maturation and embryo development during in vitro culture (IVC) were tested by maturing oocytes in media supplemented with 10% FCS (FCS Group) or 10% SSS + 10 ng/ml EGF (SSS Group), followed by IVF and IVC in SOF media supplemented with 10% FCS and 10% SSS on day 4 for FCS and SSS Groups, respectively. Even though rates for cleavage and development to blastocyst stage were not different, blastocyst cell numbers were higher in Group II containing SSS and EGF. The SSS supplementation group had higher apoptotic nuclei as compared to the FCS Group in Experiment II. Transcripts for heat shock protein 70 (Hsp70), interferon tau (IF-τ), DNA methyltransferase 3a (Dnmt3a), desmosomal glycoprotein desmocollin III (DcIII) and insulin-like growth factor II receptor (Igf-2r) were altered in different culture conditions in Experiment I. However, only glucose transporter-1 (Glut-1) mRNA was different in the SSS and FCS Groups in the second experiment. In summary, SSS and EGF in maturation medium and replacement of FCS with SSS alone in culture medium on day 4 of IVC support oocyte maturation and embryo development in vitro. However, significance of culture condition induced changes on the genome-wide abundance of messenger ribonucleic acid and the significance of the apoptotic nuclei during fetal development still remain to be determined.

Global reduction of DNA methylation, a part of genome reprogramming processes, occurs in a gradual manner until before implantation and is recognized as a conserved process in mammals. Here, we reported that in bovine, satellite regions exhibited varied patterns of methylation changes when one-cell egg advanced to the blastocyst; a maintenance methylation was observed in satellite I sequences, a decrease in alpha satellites, and an increase in satellite II regions. Cloned embryos exhibited similar changes for DNA methylation in the satellite I and alpha. We also observed that the satellite I and alpha sequences were methylated more in inner cell mass region of the blastocyst whereas the satellite II showed selective demethylation in this region. Together, these findings point that individual satellite sequences carry their own methylation patterns under the pressure of global demethylation, suggesting that local methylation control system acts on the satellite regions in early bovine embryos.

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell–cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa–cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP]i was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP]i in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP]i, and cumulus and granulosa cells. Mol. Reprod. Dev. 65: 336–343, 2003. © 2003 Wiley-Liss, Inc.

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.

We report herein the establishment of three bovine pluripotent embryonic cell lines derived from 8-16-cell precompacting embryos. Two cell lines were cultured for 10 passages and underwent spontaneous differentiation. One cell line (Z2) has been cultured continuously for over 3 years and has remained undifferentiated. These cells express cell surface markers that have been used routinely to characterize embryonic stem (ES) and embryonic germ (EG) cells in other species such as stage-specific embryonic antigens SSEA-1, SSEA-3, and SSEA-4, and c-Kit receptor. In the absence of a feeder layer, these cells differentiated into a variety of cell types and formed embryoid bodies (EBs). When cultured for an extended period of time, EBs differentiated into derivatives of three EG layers - mesoderm, ectoderm, and endoderm - which were characterized by detection of specific cell surface markers. Our results indicate that the Z2 cell line is pluripotent and resembles an ES cell line. To our knowledge, this is the first bovine embryonic cell line that has remained pluripotent in culture for more than 150 passages.

Gene expression at the onset of bovine embryogenesis is developmentally regulated and histone deacetylases (HDACs) have been shown to play a key role in the control of gene expression during this period of development in other species. We determined expression pattern(s) of powerful repressors, namely histone deacetylase-1, -2 and -3, that may in part regulate gene expression during bovine oogenesis and early embryogenesis at the mRNA and protein levels. Detected fragments of the hdac genes were sequenced and comparison of the sequences showed very high homologies between DNA and amino acid sequences of bovine HDACs and those of human and mouse. RPD3, a yeast global regulator of transcription, was also detected in bovine oocytes and embryos. Results suggest that HDACs may be operative in regulation of zygotic/embryonic gene expression in cattle.

Early embryonic development is largely dependent on maternal RNAs and proteins synthesised during oogenesis. Zygotic transcription is an essential event that occurs at a species-specific time after fertilisation. In the absence of zygotic transcription the embryo dies since it can no longer support requirements for successful embryo development. Molecular genetics of gene expression during early embryogenesis, especially in the bovine species, remain one of the unsolved questions in modern biology. Earlier studies suggested that embryonic transcription in cattle begins at the late 4-cell or 8-cell stage. However, more recent studies suggest that bovine zygotes and 2-cell embryos are both transcriptionally and translationally active. Moreover, changes in chromatin structure due to acetylation of core histones and DNA replication play important roles in the regulation of zygotic/embryonic gene expression. This review will summarise results of recent studies about the timing and mechanisms of zygotic/embryonic gene expression in cattle. In addition, terminology in the literature regarding gene expression during early embryogenesis will be clarified. These terminologies include: ‘zygotic/embryonic gene expression’, ‘maternal to embryonic transition in control of development (MET)’ and ‘zygotic/embryonic genome activation (ZEGA)’.

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 birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development. Although the ethical debate has been intense, the important scientific questions raised by this work have been inadequately discussed and are still unresolved. In this essay we address three questions about nuclear transplantation in the eggs of mice and domestic animals. First, why were the recent experiments on somatic cell cloning successful, when so many others have failed? Second, were these exceptional cases, or is somatic cloning now open to all? Third, what are the future possibilities for increasing the efficiency and wider applicability of the cloning process?

Development of mammalian early embryos relies on stored maternal messenger RNAs (mRNAs) that have been synthesized during oogenesis until embryonic genome activation. Although embryonic genome acti vation in bovine embryos has been proposed to start at the late 4-cell stage, recent evidences suggest that embryonic genome activation starts earlier than the 4-cell stage, and molecular details of this event are not known. RNA polymerase II in eukaryotes is responsible for transcription of mRNA and most of the small nuclear RNAs. The unphosphorylated form of RNA polymerase II (IIA) has been shown to function in transcriptional initiation, and the hyperphosphorylated form (IIO) functions in translational elongation and mRNA splicing. In this study, we examined the changes in the amount of RNA polymerase IIA by immunoblotting in immature oocytes; mature oocytes; and 2-, 4- and 8-cell bovine embryos. We also examined the levels of IIO and the multiple intermediately phosphorylated form in the same oocytes and embryos. The IIA reached the highest level at the 2-cell stage and decreased gradually at the 4- and 8-cell stages, and IIO was at very low levels in mature oocytes and 2-cell stage embryos and was not detectable at later stages. The multiple intermediately phosphorylated form was present at the highest level in mature oocytes and was detectable at the other stages. We demonstrate that RNA polymerase IIA, which is responsible for initiation of transcription, is present in oocytes and preimplantation embryos and reaches the highest levels in the 2-cell stage embryos. Inhibition of RNA polymerase II-dependent transcription during any of the first four embryonic cell cycles has detrimental effects on progression of embryonic development beyond the 16-cell stage, indicating the importance of early transcripts for continuation of development. The results indicate that expression of all the genes whose transcription is inhibited by alpha-amanitin is essential for embryo development.

The transition from the maternal to embryonic control of early embryonic development (MET) in mammals is not fully understood. The objective of this study was to determine the amount of transcriptional activity in immature oocytes containing germinal vesicle (GV), mature metaphase II arrested oocytes (MII), 2-, 4- and 8-cell bovine embryos by labeling with 35S-UTP followed by isolation of total RNA and autoradiography. Expression of counts per minute (CPM) per cell showed that incorporation of 35S-UTP in GV oocytes was significantly higher than the background (P0.01) and decreased sharply by the time the oocytes reached MII arrest. Incorporation significantly increased during the 2-cell stage and remained at the same level during the 4- and 8-cell stages. Uptake remained constant throughout different development stages (P0.05) with the highest variability observed during the 2-cell stage. When CPM were expressed per oocyte or embryo incorporation remained high at the GV stage, decreased to the background levels at the time of MII and increased again at the 2-cell stage. It remained at the same level during the 4-cell stage but increased significantly for the second time during the 8-cell stage. Uptake remained at the same level until the 8-cell stage when a significant increase was observed. The negative controls showed a significantly lower amount of incorporation compared to the positive control (P0.05). Similar results were observed by autoradiography. Our observations suggest that MET starts as early as the 2-cell stage in bovine embryos.

This chapter focuses on the history of cloning of cattle, the comparative methodology, successes, and failures, possible causes of failures with a particular emphasis on imprint maintenance and epigenetics, and applications of cloning of cattle.

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.

This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest.

Whilst the role of Maturation or M-phase Promoting Factor (MPF) as a universal M-phase regulator is well documented, much less attention has been paid to its role in nuclear transplantation experiments and especially to its influence upon remodelling of transplanted nuclei. There is currently wide acceptance that successful nuclear transplantation using differentiated nuclei is possible only in a cytoplasmic environment that is capable of inducing rapid nuclear de-differentiation to a pronuclear-like form. In this review our purpose is firstly, to outline the conditions under which such remodelling can be induced, and secondly, to extend the debate to include a consideration of whether complete nuclear remodelling is an absolute necessity for clonal development.

Bovine sperm motility and respiration were stimulated by the triazine dye Cibacron Blue F3GA (CB), which may operate as a nucleotide mimic. CB stimulation of respiration was half-maximal at about 35 μM and respiration reached maximal levels about 1.5 minutes after CB addition. Respiratory stimulation was preceded by a transient increase in cytosolic cAMP. Sperm cAMP titers were elevated from 5 to 10 pmoles/108 cells within 30 seconds of CB addition, but rapidly dropped to a stable level of about 7.5 pmoles/108 cells. CB was a potent inhibitor of sperm membrane adenylyl cyclase and inhibited respiration in permeabilized cells. Taken together, the data indicated that CB stimulation was not manifested via the cytosol. In addition, a nonpermeant blue dextran preparation synthesized with CB also stimulated sperm respiration and motility. CB inhibited sperm membrane phosphodiesterase activity, suggesting that the transient pulse of cAMP resulted from CB interaction with this enzyme in the sperm membrane. © 1995 wiley-Liss, Inc.

Multiplying genetically superior mammalian embryos through nuclear transplantation has considerable potential for improving animal production. Today it is possible, at times, to obtain several genetically identical offspring from one original embryo. However, the production of a large number of genetically identical animals must be realized before cloning will have a major impact on animal industries. In order to realize this potential there must be continued improvements in all facets of the cloning procedure. In the past few years there has been steady progress in improving the nuclear transplantation techniques. These techniques include oocyte enucleation, blastomere transfer, cell fusion, oocyte activation, preimplantation development and embryo transfer. Owing to the multitude of steps involved in cloning, further advancements in all of these areas will be needed to obtain an efficient system of cloning embryos. Advances are also needed in methods of obtaining large quantities of high quality biological material to be used in the cloning procedure. This is a key factor in making cloning a valuable and cost-effective genetic tool for the livestock industry. The usefulness of embryo cloning will depend on scientific advancements made in the coming years.

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli β-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β-galactosidase gene under a β-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley-Liss, Inc.

Oviduct tissue-conditioned medium was evaluated for the culture of IVM-IVF bovine zygotes to the compact morula (cM) and blastocyst (BL) stages. Development was unaffected (P greater than 0.50) by freezing and thawing of conditioned medium: no. cM + BL/no. cleaved ova obtained after culture in nonfrozen, frozen-thawed, and control treatments were 31/148 (21%), 26/124 (21%) and 5/86 (6%), respectively. The greatest proportion of normal development was obtained after a conditioning period of 48 h (P less than 0.05): no. of cM + BL/no. cleaved ova in media conditioned for 5, 24, 48 and 96 h were 23/114 (20%), 38/112 (34%), 43/115 (37%) and 36/125 (29%), respectively. Development declined with increasing dilutions of conditioned media (P less than 0.005): no. cM + BL/no. cleaved ova for 100, 75, 50, 25 and 0% conditioned medium were 32/94 (34%), 29/94 (31%), 17/82 (21%), 10/94 (11%) and 11/73 (15%), respectively. The oestrous cycle stage from which oviducal tissue was obtained did not affect development (P greater than 0.75); no. cM + BL/no. cleaved ova was 21/63 (33%) at oestrus and 21/79 (27%) in the luteal phase.

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

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 requisite endpoint of mammalian oocyte maturation, whether in vivo or in vitro, is a metaphase II oocyte which is able to be fertilized and which can eventually support normal embryonic development. Oocytes which have been matured in vivo basically fulfill these criteria. On the other hand, a completely different situation exists when these cells are isolated from the ovaries and cultured in vitro. If they are too small (growing oocytes), they do not undergo maturation, or, if more advanced, will mature only to the metaphase I stage. Even in fully grown oocytes which are able to mature to metaphase II, the developmental potential after fertilization is disappointingly low, for reasons which remain unknown. The complexity of certain factors (nuclear, cytoplasmic or arising from our current culture systems) undoubtedly influences both the ability of oocytes to mature fully, as well as their developmental potential after fertilization.

Invasive adenylate cyclase (iAC) reversibly inhibits spontaneous maturation of cumulus-enclosed bovine oocytes by increasing the intracellular concentration of cAMP, [cAMP]i. In this study, physiological aspects of maintaining meiotic arrest in bovine oocytes by iAC were investigated. The maintenance of germinal vesicle arrest by iAC in both cumulus-enclosed and denuded bovine oocytes was concentration dependent (r2 = 0.857). Denuded bovine oocytes were more sensitive to maintenance of meiotic arrest by iAC then were cumulus-enclosed oocytes. At the highest concentration, 70% of the cumulus-enclosed and 90% of the denuded bovine oocytes were maintained in meiotic arrest. The iAC increased [cAMP]i in both intact cumulus-oocyte complexes and enclosed oocytes in a concentration-dependent manner (r2 = 0.795). Cumulus-enclosed oocytes maintained in meiotic arrest by iAC retained developmental competence when subsequently cultured in iAC-free medium and then fertilized. The [cAMP]i in bovine complexes decreased precipitously upon release from follicles and remained low for the next 125 min. However, the [cAMP]i of the enclosed oocytes did not change. Bovine oocytes commit to undergo meiosis in a progressive manner. Approximately 10% of the oocytes were already committed when aspirated. This proportion increased to 40% at 2 h and 70% at 5 h. Use of two inhibitors of cAMP-dependent protein kinase A provided further evidence that cAMP functions in mediating meiotic arrest in bovine oocytes. Bovine oocytes, therefore, are sensitive to different cAMP concentrations, and are developmentally competent after iAC-induced arrest, and complexes containing oocytes exhibit a decrease in [cAMP]i before spontaneous maturation. These results suggest that maintenance of meiotic arrest by iAC is accomplished through modulation of cellular machinery, and regulation of oocyte maturation by [cAMP]i may be physiologically relevant.

SummaryWe have examined the synthesis and expression of a homologue of the cell cycle protein cdc25 by early cleavage stage bovine embryos. cdc25 is the protein phosphatase responsible for activating p34cdc2 by dephosphorylating the threonine 14 (Thr 14) and tyrosine 15 (Tyr 15) residues of p34cdc2. Human cdc25 antibody was utilised in western blots immunoprecipitations to examine the presence and synthesis of cdc25 in bovine embryos. cdc25 is present as a 52 kDa non-phosphorylated and a 66 kDa presumably phosphorylated form in bovine 1−, 2−, 4− and 8−cell embryos. However, cdc25 is actively synthesised only in 8-Cell embryos, indicating that the cdc25 present prior to this stage is inherited from the oocyte. In addition, the synthesis of cdc25 was induced in 2-cell embryos in which cleavage was blocked with the DNA synthesis inhibitor aphidicolin.

Bovine sperm that were subjected to extended anoxia (2.5 h) in the absence of glycolytic substrates then diluted into oxygenated medium were immotile but metabolically active, producing ATP from lactate via oxidative phosphorylation. In response to anoxia sperm ATP titers dropped from 15-20 mumoles/10(8) cells to 1-2 mumoles/10(8) cells in the first 5 min then remained extremely low until reoxygenation. Cyclic AMP titers declined slowly over the anoxic period, but did not show the same scale of depression as ATP. After dilution and re-oxygenation ATP recovered to pre-anoxia levels within 1 min, and cAMP rose to about the pre-anoxia levels. However, motility, which varied quantitatively and qualitatively between ejaculates prior to anoxic treatment, was substantially depressed after extended anoxia in all cases; progressive motility was almost non-existent in post-anoxic sperm. Addition of isobutylmethylxanthine or Cibacron Blue F3GA, both putative phosphodiesterase inhibitors, stimulated a transient peak of cAMP, which was accompanied by motility stimulation. These techniques provide a protocol to manipulate and dissect the biochemical pathways of motility initiation in mammalian sperm.

Embryos of amphibians, fish, sheep, cattle, swine and rabbits have been multiplied by nuclear transfer. Successful nuclear transfer in these species has been accomplished by transfer of a blastomere from a late stage embryo into an enucleated oocyte or egg with large scale multiplication achieved by serial repetition of the procedure using blastomeres from nuclear transfer embryos. This allows the production of colonel lines, which when appropriately selected for performance in a given trait, can be reproduced to capture in the offspring expression of both additive and nonadditive inheritance. The efficiency of producing offspring from nuclear transfer is low in mammals in both frequency of morula or blastocyst produced and maintenance of pregnancy after embryo transfer. In domestic animals the largest number of offspring from one embryo has been eight calves. Embryos as late as the 64-cell stage in cattle and 120-cell blastocyst in sheep have been used successfully as donors of blastomeres. Recloning has also been done in cattle. Potentially, nuclear transfer provides a mechanism for multiplication and production testing of clonal lines, a method for rapid genetic improvement and a means for rapid propagation of a selected genotype.

Animal breeding is entering an exciting new era in which it will have the tools to rapidly multiply animals and to make new strains, precisely tailored to meet product and environmental demands. Many of these new tools depend on micromanipulation of embryos. The new tools include the ability to transfer new genes into the genome of domestic animals, the genetic screening of embryos by DNA marker assisted selection to directly select for genes encoding desirable traits and against genetic defects, as well as the use of embryo bisection or cloning to produce identical copies of embryos from a genetically tested high performance embryo line.

Publisher Summary This chapter discusses the nuclear transfer in mammalian embryos. Transfer of nuclei from one cell to another provides a powerful method to study the interactions of the cytoplasm of one cell with the nuclei of another. Nuclei from various differentiated states can be transferred to nondifferentiated cytoplasm and the effect on the nucleus can be monitored. Early mammalian development is characterized by an initial period of zygotic inactivity followed by a transition period during which development is directed by both maternally stored message RNA and newly synthesized RNA. One method for determining the onset of zygotically produced transcripts is to block their appearance and subsequent protein production with α-amanitin. α-amanitin blocks mRNA synthesis by binding to a subunit of polymerase II, thus, blocking chain elongation. The chapter illustrates the importance of species comparisons and the danger of extrapolating results from one species to another. Nuclear transfer for cloning has been successful in a variety of mammals, excluding the mouse. This suggests that careful between-species comparisons should be made before any overall hypothesis regarding nuclear transfer is made. The comparisons need to include an evaluation of the advanced stage of donor nuclei capable of promoting development and the length of the cell cycle of these nuclei, the components aid in promoting development, and evaluation of nonnuclear inheritance.

Summary Retroviral packaging cell lines were constructed by using the gag-pol gene of spleen necrosis virus, the gag-pol gene of Moloney murine leukaemia virus and the env gene of bovine leukaemia virus. The plasmids containing the gag-pol genes and the plasmid containing the env gene were cotransfected into NIH/3T3 and D17 cells. The cells containing the helper virus constructs were tested for their ability to package replication-defective murine leukaemia and avian reticuloendotheliosis retrovirus vectors. The titre of vector virus produced by each of the retroviral packaging cell lines was about 102 colony-forming units per ml of medium. Tests for events that might result in intact replication-competent retroviruses showed no evidence for the generation of such viruses. The vector viruses were able to infect dog and rat cells. Bovine cells were infected only after their cocultivation with the retroviral packaging cell lines producing murine leukaemia virus vectors, perhaps as a result of a low concentration of receptors.

Bovine spermatozoa accumulated a small amount of 32Pi during aerobic incubation in vitro. At least 50% of the acquired isotope rapidly entered cellular nucleotides. Both adenosine and guanosine di- and triphosphates were labeled, but contrary to expectations, the specific activity of ADP exceeded that of ATP. The uptake of phosphate and its incorporation into nucleotides were suppressed by respiratory inhibitors and were abolished by treatment with sulfhydryl-directed reagents at 10 to 20 nmol/mg of sperm protein. With fructose as an energy source for motility, glycolysis did not support phosphate uptake. Nucleotide labeling was increased 60 to 80-fold when the cells were treated with the polyene antibiotic filipin, and filipin was able to reverse the inhibition of phosphate (and succinate) entry produced by N-ethylmaleimide or mersalyl. Since filipin interacts specifically with the cholesterol-containing plasma membrane of bovine spermatozoa and increases its permeability, it is probable that the plasma membrane normally limits phosphate and succinate transport into these cells. This contention is further supported by the observation that high concentrations of extracellular Pi, the penetration of which was extremely limited under these conditions, protected against inactivation by N-ethylmaleimide. Phosphate uptake was increased 10 to 20-fold, but nucleotide labeling was inhibited, when calcium was present in the incubation medium. Ruthenium red, presumably acting extracellularly, prevented these effects of calcium. Thus, the entry of phosphate and succinate into spermatozoa is controlled by plasma membrane components that resemble the phosphate and succinate exchangers and calcium carrier found in mitochondria isolated from other sources.

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)

Radiolabeling of mammalian spermatozoa with 131 I, 67 Ga, 111 In, and 99m Tc was investigated. Spermatozoa were labeled with 99m Tc for in vivo studies because of a high labeling yield (70 to 90%) combined with the lack of impairment of sperm motility. Ovariectomized sheep were brought into estrus by sequential administration of progesterone and estradiol cyprionate. Sheep were necropsied up to 6 hours after insemination with 99m Tc-labeled ram sperm and their reproductive tracts were resected and examined with a rectilinear scanner. Radioactivity was clearly observed in the fallopian tubes, with larger amounts in the vaginas, cervices, and uteri. In contrast, when 99m Tc-spermatozoa were replaced with 99m TcO 4 − , much less radioactivity remained in the reproductive tract at resection and this was evenly distributed. Some label left the 99m Tc-spermatozoa in vivo, but radioactivity remained on the cells long enough to consider attempting to monitor sperm transport in vivo in a suitable species.

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.

An orderly series of associations was found between the cumulus and the stages of nuclear development in the hamster oocyte as the oocyte-cumulus complex underwent preovulatory maturation. Ten hours prior to ovulation meiotic resumption was first seen in the oocyte. The onset of cumulus dissociation with corresponding morphological changes in cumulus cells was observed eight hours prior to ovulation. Completion of the first meiotic division in the oocyte and full cumulus dissociation occurred 2 hours prior to ovulation. Cumulus dissociation was found to occur only in vesicular follicles destined to ovulate and not in follicles undergoing atresia. Cells with large vacuolar inclusions with distorted, hyperchromatic nuclei were found in the maturing cumulus.

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)

Hoechst dyes 33342 and 33258 were used to visualize pronuclei and nuclei of early preimplantation embryos. Murine one-cell zygotes exposed to dye stained rapidly over a range of concentrations (0, 0.02, 0.04, 0.1 or 0.2 micrograms/50 microliter of media). Development to morula and blastocyst in vitro was reduced (39/70, 56%; p less than 0.05) compared to controls (44/57, 77%) but not completely blocked. Porcine and bovine zygotes and embryos could also be stained but required incubation times up to 4 hr. Porcine embryos exposed to Hoechst 33342 had limited (p less than 0.01) in vitro development (29/74, 39%) compared to unstained controls (49/64, 76%). Hoechst dyes stain embryos from different species but suitably adjusted incubation times are required. Limited preimplantation development in vitro may be expected following staining and exposure to ultraviolet light.

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)