Neal L. First

An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis☆

Solubilized oocyte zonae pellucidae promoted acrosomal exocytosis in fura-2- or carboxyfluorescein-loaded, mature bovine sperm. Associated elevations of internal [Ca2+] and pH in sperm suspensions were first detectable at 2-5 min, without apparent temporal resolution, and increased monotonically thereafter. Video imaging of fura-2-loaded, single cells identified a responsive subpopulation, destined to undergo exocytosis, that displayed no early transient but manifested lags of 1-7 min then sustained elevations of internal [Ca2+]. Both the zona-induced exocytosis and dye responses were diminished for functionally immature sperm and for mature sperm treated preliminarily with pertussis toxin. Together, these results indicate that a developmentally regulated mechanism of signal transduction employs G protein(s) to couple the physiological (zona) agonist to alterations of the internal ionic mediators of acrosomal exocytosis.

Onset of transcription in bovine oocytes and preimplantation embryos

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 (P < 0.01) 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 (P > 0.05) 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 (P < 0.05). Similar results were observed by autoradiography. Our observations suggest that MET starts as early as the 2‐cell stage in bovine embryos. Mol. Reprod. Dev. 51:36–41, 1998.

The regulation of acrosomal exocytosis: I. Sperm capacitation is required for the induction of acrosome reactions by the bovine Zona pellucida in vitro☆

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 t1/2 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/microliter, 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.

Genome-Wide Epigenetic Alterations in Cloned Bovine Fetuses

Abstract 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.

Pluripotency of bovine embryonic cell line derived from precompacting embryos.

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.

Developmental changes in RNA polymerase II in bovine oocytes, early embryos, and effect of α-amanitin on embryo development

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 activation 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 α‐amanitin is essential for embryo development. Mol. Reprod. Dev. 51:381–389, 1998.

Regulation of acrosomal exocytosis: II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is controlled by extrinsic positive regulatory elements☆

The effects of accessory sex gland secretions on the zona pellucida-induced acrosome reaction of bovine spermatozoa were investigated. Soluble extracts of zonae pellucidae initiated exocytosis in ejaculated spermatozoa. This process had an ED50 of 20 ng/microliter zona pellucida protein and saturated at 50 ng/microliter (Florman and First, 1988. Dev. Biol. 128, 453-463). In epididymal sperm this dose-response relationship was shifted toward greater agonist concentrations by at least a factor of 10(3). Reconstitution of high potency agonist response was achieved in vitro by incubation of epididymal sperm with bovine seminal plasma. Reconstitution was dependent on the seminal plasma protein concentration. The ED50 of this process was 62 micrograms protein/10(8) sperm and saturation was observed with 124 micrograms protein/10(8) sperm. Agonist responses in reconstituted epididymal sperm and in ejaculated sperm were indistinguishable with regard to dependence on the zona pellucida protein concentration and the kinetics of induced acrosome reactions. Kinetic studies suggest that reconstitution is due to adsorption of regulatory factors from seminal plasma. In addition to the positive regulatory elements responsible for reconstituting activity, seminal plasma also contains negative regulatory elements which inhibit agonist response. These negative factors are inactivated during sperm capacitation, permitting the expression of positive regulators. Acting together, these regulatory elements could coordinate high affinity agonist response with the availability of eggs in vivo.

Cloning by somatic cell nuclear transfer

The birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development.(1) 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? BioEssays 20:847–851, 1998.

Use of A Fluorescent Stain for Visualization of Nuclear Material in Living Oocytes and Early Embryos

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.

Expression patterns of histone deacetylases in bovine oocytes and early embryos, and the effect of their inhibition on embryo development.

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.