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MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB-SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB-SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein-protein interactions or promote degradation of meiosis-specific proteins.

The oocyte-to-embryo transition (OET) arguably initiates with formation of a primordial follicle and culminates with reprogramming of gene expression during the course of zygotic genome activation. This transition results in converting a highly differentiated cell, i.e. oocyte, to undifferentiated cells, i.e. initial blastomeres of a preimplantation embryo. A plethora of changes occur during the OET and include, but are not limited to, changes in transcription, chromatin structure, and protein synthesis; accumulation of macromolecules and organelles that will comprise the oocyte's maternal contribution to the early embryo; sequential acquisition of meiotic and developmental competence to name but a few. This review will focus on transcriptional and post-transcriptional changes that occur during OET in mouse because such changes are likely the major driving force for OET. We often take a historical and personal perspective, and highlight how advances in experimental methods often catalyzed conceptual advances in understanding the molecular bases for OET. We also point out questions that remain open and therefore represent topics of interest for future investigation.We review, often with a historical perspective, transcriptional and post-transcriptional changes that underlie the oocyte-to-embryo transition in mouse.

Ahypothesis to explain the maternal age-dependent increase in formation of aneuploid eggs is deterioration of chromosome cohesion. Although several lines of evidence are consistent with this hypothesis, whether cohesion is actually reduced in naturally aged oocytes has not been directly tested by any experimental perturbation. To directly target cohesion, we increased the activity of separase, the protease that cleaves the meiotic cohesin REC8, in oocytes.We show that cohesion is more susceptible to premature separase activation in old oocytes than in young oocytes, demonstrating that cohesion is significantly reduced. Furthermore, cohesion is protected by two independent mechanisms that inhibit separase, securin and an inhibitory phosphorylation of separase by CDK1; both mechanisms must be disrupted to prematurely activate separase.With the continual loss of cohesins from chromosomes that occurs throughout the natural reproductive lifespan, tight regulation of separase in oocytes may be particularly important to maintain cohesion and prevent aneuploidy.

MessengerRNA is remarkably stable during oocyte growth, thus enabling mRNAs to accumulate during the growth phase and thereby provide mRNAs that support early embryonic development. MSY2, a germ cell-specific RNA-binding protein, is implicated in regulating mRNA stability. MSY2 is essential for development because female Msy2−/−mice are infertile. We describe here the characterization of Msy2−/−oocytes. Mutant oocytes grow more slowly during the first wave of folliculogenesis, and maturation to and arrest at metaphase II is severely compromised because of aberrant spindle formation and chromosome congression. Consistent with MSY2 conferring mRNA stability is that the amount of poly(A)-containing RNA is reduced by ∼25% in mutant oocytes. Stability of an exogenous mRNA injected into mutant oocytes is lower than when compared to their wild-type counterparts, and moreover, expression of wild-type MSY2 in mutant oocytes increases mRNA stability, whereas injection of a mutant form of MSY2 not capable of binding RNA does not. Transcription quiescence that normally occurs during the course of oocyte growth is not observed in mutant oocytes, and the transcriptome of mutant oocytes is markedly perturbed. These results, and those of previous studies, strongly implicate a central role of MSY2 in regulating mRNA stability.

Nearly all male cystic fibrosis (CF) patients exhibit tissue abnormalities in the reproductive tract, a condition that renders them azoospermic and infertile. Two swine CF models have been reported recently that include respiratory and digestive manifestations that are comparable to human CF. The goal of this study was to determine the phenotypic changes that may be present in the vas deferens of these swine CF models. Tracts from CFTR−/−and CFTRΔF508/ΔF508neonates revealed partial or total vas deferens and/or epididymis atresia at birth, while wild-type littermates were normal. Histopathological analysis revealed a range of tissue abnormalities and disruptions in tubular organization. Vas deferens epithelial cells were isolated and electrophysiological results support that CFTR−/−monolayers can exhibit Na+reabsorption but reveal no anion secretion following exposure to cAMP-generating compounds, suggesting that CFTR-dependent Cl−and/or HCO3−transport is completely impaired. SLC26A3 and SLC26A6 immunoreactivities were detected in all experimental groups, indicating that these two chloride-bicarbonate exchangers were present, but were either unable to function or their activity is electroneutral. In addition, no signs of increased mucus synthesis and/or secretion were present in the male excurrent ducts of these CF models. Results demonstrate a causal link between CFTRmutations and duct abnormalities that are manifested at birth.

Sox2is a key gene that controls transcriptional networks required for pluripotency. The role of Sox2in the developmental transition of a highly differentiated oocyte to totipotent blastomeres of the early preimplantation embryo, however, is not known. We report that Sox2, which is localized in the nucleus, is first zygotically expressed during the 2-cell stage and that its expression dramatically increases between the morula and blastocyst stages. Injecting a cRNA encoding Sox2into 1-cell embryos resulted in overexpression of SOX2 by approximately 70% and developmental arrest at the 2-cell stage, whereas injecting cRNAs encoding Pou5f1, Myc(also known as c-Myc), or Klf4has little effect on the ability of 2-cell embryos to cleave to the 4-cell stage. Global transcription assessed by bromo uridine triphosphate incorporation is reduced by approximately 15%, and transcript profiling revealed that approximately 15% of zygotically expressed genes are dramatically repressed in 2-cell embryos overexpressing SOX2. Furthermore, overexpressing a dominant-negative SOX2perturbs reprogramming of gene expression in 2-cell embryos, though to a much lesser extent than that observed following overexpression of SOX2, and leads to developmental failure after the 2-cell stage but before the 8-cell stage. Results of these experiments implicate Sox2as a critical transcriptional regulator in the oocyte-to-embryo transition that entails formation of totipotent blastomeres and indicate that the amount of Sox2is critical for successful execution of this transition.

Culture systems that support development and maturation of oocytes in vitro with a high efficiency would have great impact not only on research addressed at underlying mechanisms of oocyte development but also on preservation of fertility. Recently, attention has turned to using culture systems that preserve follicle integrity, in contrast to existing systems that do not maintain follicle integrity, with the hope of improving oocyte development. We report that an alginate-based follicle culture system supports both follicular and oocyte growth in vitro, with little effect on the oocyte transcriptome. Nevertheless, oocytes obtained from these follicles exhibit an increased incidence of defects in spindle formation and chromosome alignment as well as pronounced abnormalities in cortical granule biogenesis. Developmental competence is also highly compromised, because few matured oocytes develop into 1-cell embryos with pronuclei. This situation contrasts with a high incidence of pronuclear formation following development using an existing in vitro culture system that does not preserve follicle integrity.

Targets of steroidogenic factor 1 (SF1; also known as NR5A1 and AD4BP) have been identified within cells at every level of the hypothalamic-pituitary-gonadal and -adrenal axes, revealing SF1 to be a master regulator of major endocrine systems. Mouse embryos express SF1 in the genital ridge until Embryonic Day 13.5 (E13.5). Thereafter, expression persists in the male and is substantially lower in the female gonad until birth. We hypothesize that the sexually dimorphic expression of Sf1during gonadogenesis is mediated by sex-specific regulation of its promoter. To investigate dimorphic regulation within the fetal gonad, we developed an experimental strategy using transient transfection of E13.5 gonad explant cultures and evaluated various Sf1promoter constructs for sexually dimorphic DNA elements. The proximal Sf1promoter correctly targeted reporter activity to SF1-expressing cells in both XY and XX gonads. Stepwise deletion of sequences from the Sf1promoter revealed two regions that affected regulation within female gonads. Mutation of both sequences together did not cause further disruption of reporter activity, suggesting the two sites might work in concert to promote activity in female somatic cells. Results from gel mobility shift assays and fetal gonad-chromatin immunoprecipitation showed that TCFAP2 binds to one of the two female-specific sites within the proximal promoter of Sf1. Together, we show that transient transfection experiments performed within developing testes and ovaries are a powerful tool to uncover elements within the Sf1promoter that contribute to sex-specific expression.

The epigenetic mechanisms involved in establishing and maintaining genomic imprinting are steadily being unmasked. The nucleosome remodeling and histone deacetylation (NuRD) complex is implicated in regulating DNA methylation and expression of the maternally expressed H19gene in preimplantation mouse embryos. To dissect further the function of the NuRD complex in genomic imprinting, we employed an RNA interference (RNAi) strategy to deplete the NuRD complex component Metastasis Tumor Antigen 2 (MTA2). We found that Mta2is the only zygotically expressed Mtagene prior to the blastocyst stage, and that RNAi-mediated knockdown of Mta2transcript leads to biallelic H19expression and loss of DNA methylation in the differentially methylated region in blastocysts. In addition, biallelic expression of the paternally expressed Peg3gene, but not Snrpn, is also observed in blastocysts following Mta2knockdown. Loss of MTA2 protein does not result in a decrease in abundance of other NuRD components, including methyl-binding-CpG-binding domain protein 3 (MBD3), histone deacetylases 1 and 2 (HDACs 1 and 2), and chromodomain helicase DNA-binding protein 4 (CHD4). Taken together, our results support a role for MTA2 within the NuRD complex in genomic imprinting.

In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.

Data are accumulating to demonstrate that pH regulation in the male reproductive tract has a vital role in modulating sperm cell fertilizing capacity, and therefore male fertility. Bicarbonate uptake by sperm cells is required for the achievement of motility levels required for fertilization. Vas deferens epithelial cells can carry out measurable bicarbonate secretion, but the available literature to date reports that the vas deferens luminal content is typically acidic. This study aimed to determine pH in the boar vas deferens lumen and whether modulatory mechanisms exist for regulation of pH in this compartment of the male reproductive tract. A fiberoptic pH probe was used to assess pH in the vas deferens of anesthetized adult boars. The mean pH, derived from multiple measurements at variable positions along the vas deferens lumen, was 7.39 ± 0.09. Furthermore, administration of xylazine, an alpha-2 adrenergic receptor agonist rapidly (<10 min) alkalinized the vas deferens lumen in most cases. Because the duct was transected proximal to the site of measurements, the observations rule out the possibility that alkalinization resulted from secretion in more proximal portions of the duct. These results indicate that the boar vas deferens lumen can be alkaline, and they suggest that porcine vas deferens epithelia increase net bicarbonate secretion in vivo after systemic alpha-2 adrenergic stimulation. This secretory response greatly changes the luminal environment to which sperm cells are exposed, which will initiate or enhance motility, and is expected to modulate male fertility.

Advanced maternal age is unequivocally associated with increased aneuploidy in human eggs and infertility, but the molecular basis for this phenomenon is unknown. An age-dependent deterioration of the spindle assembly checkpoint (SAC) has been proposed as a probable cause of aneuploidy. Accurate chromosome segregation depends on correct chromosome attachment to spindle microtubules, and the SAC provides time for this process by delaying anaphase onset until all chromosomes are stably attached. If SAC function decreases with age, oocytes from reproductively old mice would enter anaphase of meiosis I (AI) prematurely, leading to chromosome segregation errors and aneuploid eggs. Although intuitively appealing, this hypothesis is largely untested. We used a natural reproductive aging mouse model to determine if a defective SAC is the primary cause of aneuploidy in eggs. We tracked the progress of individual oocytes from young and old mice through meiosis I by time-lapse microscopy and counted chromosomes in the resulting eggs. This data set allowed us to correlate the timing of AI onset with aneuploidy in individual oocytes. We found that oocytes from old mice do not enter AI prematurely compared to young counterparts despite a 4-fold increase in the incidence of aneuploidy. Moreover, we did not observe a correlation between the timing of AI onset and aneuploidy in individual oocytes. When SAC function was challenged with a low concentration of the spindle toxin nocodazole, oocytes from both young and old mice arrested at meiosis I, which is indicative of a functional checkpoint. These findings indicate that a defective SAC is unlikely the primary cause of aneuploidy associated with maternal age.

Testosterone induces and maintains prostaglandin endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase 2) expression in vas deferens epithelial cells, but it remains unknown whether this has a physiological role in the context of male reproductive biology. Prostaglandins induce concentration-dependent increases in anion secretion in porcine vas deferens epithelial cell (1°PVD) monolayers, where bicarbonate contributes to cAMP-stimulated anion secretion. Moreover, bradykinin induces anion secretion across 1°PVD monolayers that is indomethacin sensitive, and both PTGS2and PTGS1are expressed in this model system. Therefore, it was hypothesized that testosterone modulates anion secretion across vas deferens epithelia via PTGS-dependent pathways and prostaglandin synthesis. Porcine vas deferens epithelial cells were isolated and cultured as monolayers on permeable supports until assayed in modified Ussing chambers. RNA and protein were isolated concurrently for semiquantitative expression analysis. Testosterone upregulated basal and bradykinin-induced short-circuit current across 1°PVD monolayers, indicative of anion secretion. Testosterone also induced greater transepithelial electrical resistance. Increases in anion secretion were associated with preferential upregulation of PTGS2 at the mRNA and protein levels. In addition, testosterone induced greater basal and bradykinin-induced anion secretion across vas deferens epithelial cells isolated from the distal segment of the duct. Taken together, these results suggest that testosterone upregulates epithelial responsiveness to acute modulations of anion secretion (likely bicarbonate secretion), which ultimately modifies the environment to which sperm are exposed.

Meiotic maturation in oocytes is a prolonged process that is unique because of cell cycle arrests at prophase of meiosis I (MI) and at metaphase of meiosis II (MII). Fluctuations in cyclin-dependent kinase 1 (CDK1/CDC2A) activity govern meiotic progression, yet little is known about how these fluctuations are achieved. CDC14 is a highly conserved dual-specificity phosphatase that counteracts the function of proteins phosphorylated by CDK. Mammals contain two CDC14 homologs, CDC14A and CDC14B. We report that CDC14B localizes with the meiotic spindle in mouse oocytes, and (unlike somatic cells) it does not localize in the nucleolus. Oocytes that overexpress CDC14B are significantly delayed in resuming meiosis and fail to progress to MII, whereas oocytes depleted of CDC14B spontaneously resume meiosis under conditions that normally inhibit meiotic resumption. Depletion of FZR1 (CDH1), a regulatory subunit of the anaphase-promoting complex/cyclosome that targets cyclin B1 (CCNB1) for ubiquitin-mediated proteolysis, partially restores normal timing of meiotic resumption in oocytes with excess CDC14B. These studies also reveal that experimentally altering CDC14B levels generates eggs with abnormal spindles and with chromosome alignment perturbations. Our data indicate that CDC14B is a negative regulator of meiotic resumption and may regulate MI in mouse oocytes.

Sumoylation is a posttranslational modification in which SUMO (small ubiquitin-related modifier) proteins are covalently attached to their substrates. In vertebrates, developmental roles for sumoylation have been studied, but the function of sumoylation during mammalian oocyte growth and maturation is not known. As a prelude to conducting studies on the role of sumoylation during oocyte development, we analyzed the temporal and spatial pattern of expression of UBE2I, a SUMO-conjugating E2 enzyme. Immunocytochemical analysis of UBE2I revealed a punctate nuclear staining pattern, with transcriptionally quiescent, fully grown, GV-intact oocytes having larger UBE2I-containing bodies than transcriptionally active, meiotically incompetent growing oocytes. Inhibiting transcription in incompetent oocytes resulted in an increase in the size of the UBE2I-containing bodies. Overexpression of either wild-type UBE2I or catalytically inactive UBE2I resulted in an increase in the size of the UBE2I-containing bodies but also an increase in BrUTP incorporation, suggesting that transcriptional activation by UBE2I is independent of its catalytic activity. Although UBE2I-containing bodies did not completely colocalize with SUMO1 or SUMO2 and SUMO3, which were localized mainly on the nuclear membrane and in the nucleoplasm, UBE2I strikingly colocalized with SFRS2, which is a component of nuclear speckles and critical for mRNA processing. These results suggest a novel function for UBE2I and therefore sumoylation in gene expression..

Epithelia lining the male reproductive duct modulate fertility by altering the luminal environment to which sperm are exposed. Although vas deferens epithelial cells reportedly express high levels of cyclooxygenases (Ptgs), and activation of bradykinin (BK) receptors can lead to upregulation of PTGS activity in epididymal epithelia, it remains unknown whether BKs and/or PTGSs have any role in modulating epithelial ion transport across vas deferens epithelia. Porcine and human vas deferens epithelial cell primary cultures and the PVD9902 cell line responded to lysylbradykinin with an increase in short circuit current (ISC; indicating net anion secretion), an effect that was 60%–93% reduced by indomethacin. The BK effect was inhibited by the B2 receptor subtype (BDKRB2) antagonist HOE140, whereas the B1 receptor subtype agonist des-Arg9-BK had no effect. BDKRB2 immunoreactivity was documented in most epithelial cells composing the native epithelium and on Western blots derived from cultured cells. Gene expression analysis revealed that the PTGS2transcript is 20 times more abundant than its PTGS1counterpart in cultured porcine vas deferens epithelia and that BDKRB2mRNA is likewise highly expressed. Subsequent experiments revealed that prostaglandin E2, 1-OH prostaglandin E1 (prostaglandin E receptor 4 [PTGER4] agonist) and butaprost (PTGER2 agonist) increase ISCin a concentration-dependent manner, whereas sulprostone (mixed PTGER1 and PTGER3 agonist) produced no change in ISC. These results demonstrate that autacoids can affect epithelial cells to acutely modulate the luminal environment to which sperm are exposed in the vas deferens by enhancing PTGS activity, leading to the production of prostaglandins that act at PTGER4 and/or PTGER2 to induce or enhance anion secretion.

Experiments were conducted to characterize the effects of oxytocin (OT) and vasopressin (VP) on epithelial cells isolated from human (1°HVD) and porcine (1°PVD) vas deferens and an immortalized epithelial cell line derived from porcine vas deferens (PVD9902 cells). Cultured monolayers were assessed in modified Ussing flux chambers and the OT- or VP-induced change in short circuit current (ISC) was recorded. All cell types responded to basolateral OT or VP with a transient increase in ISCthat reached a peak of 3–5 μA cm−2. Concentration-response curves constructed with 1°PVD and PVD9902 cells revealed that the apparent KD(kapp) for OT was ∼100-fold less than the kappfor VP. Amplicons for the OT receptor (OXTR) and vasopressin type 2 and type 1a receptors (AVPR2and AVPR1A) were generated with RT-PCR and the identification of each amplicon confirmed by sequence analysis. A selective antagonist for OXTR and AVPR1A fully blocked the effects of OT and partially blocked the effects of VP when assessed in both 1°PVD and PVD9902 monolayers. APVR2 antagonists blocked the effects of low (≤30 nM) but not high concentrations of VP, indicating that VP was affecting both AVPR2 and a second receptor subtype, likely OXTR or AVPR1A. Experiments employing chelerythrine demonstrated that OT stimulation of vas deferens monolayers requires PKC activity. Alternatively, VP (but not OT) increased the accumulation of cytosolic cAMP in vas deferens epithelial cells. Results from this study demonstrate that OT and VP can modulate ion transport across vas deferens epithelia by independent mechanisms. OT and VP have the potential to acutely change the environment to which sperm are exposed and thus, have the potential to affect male fertility.

MSY2 is a member of the Y-box family of proteins solely expressed in male and female germ cells. In the male, MSY2 serves as a coactivator of transcription by binding to a consensus promoter element present in many germ cell-specific genes. In the nucleus, MSY2 marks specific mRNAs for cytoplasmic storage, stabilization, and suppression of translation. The inactivation of MSY2 by gene targeting leads to spermatogenic arrest and infertility. In testes of mice lacking MSY2, incomplete nuclear condensation is prominent in later-stage spermatids at the time of massive spermatid loss. Because MSY2 interacts with DNA and mRNAs, there are several distinct sites of action, which could be disrupted in mice that lack MSY2, resulting in the arrest of spermatogenesis. To define the molecular cause(s) of the spermatogenic arrest in mice lacking MSY2, transcriptional and posttranscriptional processes were assayed. Transcription, mRNA processing, and mRNA intracellular transport appear normal in the absence of MSY2. However, a redistribution of mRNAs from ribonucleoprotein particles to polysomes and marked decreases were detected for many meiotic and postmeiotic germ cell mRNAs, including the mRNAs encoding the transition proteins and protamines. This suggests that increased mRNA instability is a likely cause of the male infertility in Msy2-null mice.

The step-wise assembly of a functional nucleolus, which occurs over the first few cell cycles during preimplantation development, is poorly understood. In this study, we examined the function of the evolutionary conserved nucleolar protein SURF6 in preimplantation mouse embryo development. Immunocytochemical analyses revealed that the localization of SURF6 was similar but not identical to those of fibrillarin and B23/nucleophosmin 1, which are involved in rRNA processing and ribosome biogenesis in mammalian somatic cells. Surf6mRNA, which is expressed in oocytes and maternally inherited in the zygote, reached a peak level of expression during the 8-cell stage of embryo development, at which time rDNA is highly transcribed. Knock-down of Surf6mRNA by RNAi led to a decrease in both the mRNA and protein levels, and resulted in developmental arrest at the 8-cell/morula stage, as well as a decrease in the level of 18S rRNA. These results suggest that Surf6is essential for mouse preimplantation development, presumably by regulating ribosome biogenesis.

A sperm-specific phospholipase (PL) C, termed PLCζ, is proposed to be the soluble sperm factor that induces Ca2+oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCζ mRNA have reduced amounts of PLCζ protein. Sperm derived from these transgenic mice trigger patterns of Ca2+oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca2+oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCζ is the physiological trigger of Ca2+oscillations required for activation of development.

Using an interwoven-loop experimental design in conjunction with highly conservative linear mixed model methodology using estimated variance components, 18 genes differentially expressed between nuclear transfer (NT)- and in vitro fertilization (IVF)-produced embryos were identified. The set is comprised of three intermediate-filament protein genes (cytokeratin 8, cytokeratin 19, and vimentin), three metabolic genes (phosphoribosyl pyrophosphate synthetase 1, mitochondrial acetoacetyl-coenzyme A thiolase, and α-glucosidase), two lysosomal-related genes (prosaposin and lysosomal-associated membrane protein 2), and a gene associated with stress responses (heat shock protein 27) along with major histocompatibility complex class I, nidogen 2, a putative transport protein, heterogeneous nuclear ribonuclear protein K, mitochondrial 16S rRNA, and ES1 (a zebrafish orthologue of unknown function). The three remaining genes are novel. To our knowledge, this is the first report comparing individual embryos produced by NT and IVF using cDNA microarray technology for any species, and it uses a rigorous experimental design that emphasizes statistical significance to identify differentially expressed genes between NT and IVF embryos in cattle.

A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication. Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals. Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G2/M-phase transition. We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G2/M-phase border. Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis. The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.

We previously reported the discovery of a novel mammalian H1 linker histone termed H1FOO (formerly H1OO), a replacement H1, the expression of which is restricted to the growing/maturing oocyte and to the zygote. The significance of this pre-embryonic H1 draws on its substantial orthologous conservation, singular structural attributes, selectivity for the germ cell lineage, prolonged nucleosomal residence, and apparent predominance among germ cell H1s. Herein, we report that the intronic, single-copy, five-exon (≥5301 base pair) H1foogene maps to chromosome 6 and that the corresponding primary H1footranscript gives rise to two distinct, alternatively spliced mRNA species (H1fooαand H1fooβ). The expression of the oocytic H1FOOtranscript and protein proved temporally coupled to the recruitment of resting primordial follicles into a developing primary follicular cohort and thus to the critical transition marking the onset of oocytic growth. The corresponding potential protein isoforms (H1FOOαand H1FOOβ), both nuclear localization sequence-endowed but export consensus sequence-free and possessing a significant net positive charge, localized primarily to perinucleolar heterochromatin in the oocytic germinal vesicle. Further investigation will be required to define the functional role of the H1FOO protein in the ordering of the chromatin of early mammalian development as well as its potential role in defining the primordial-to-primary follicle transition.

Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.

Experiments were conducted to determine the responsiveness of human vas deferens epithelial cell monolayers to adenosine and related agonists. Human abdominal vas deferens epithelial cells have been isolated from adult tissues and grown to confluence on permeable supports. All cells exhibit intense ZO-1 and cytokeratin immunoreactivity. Cultured cell monolayers exhibit high electrical resistance with a lumen-negative potential difference and short circuit current (Isc) indicative of anion secretion and/or cation absorption. A portion of the basal Iscis inhibited by amiloride. Amiloride-sensitive Iscis enhanced by exposure to glucocorticoids and is Na+dependent, indicating the presence of epithelial sodium channel-mediated Na+absorption. Epithelial anion secretion and intracellular generation of cAMP are acutely stimulated by adenosine and the adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine (NECA), with these effects being fully blocked by 8-phenyltheophylline. Adenosine receptors are localized to the apical membrane of the epithelial cells, as basolateral adenosine is without effect. Freshly excised human vas deferens recapitulate observations made on cultured epithelia when evaluated with the self-referencing vibrating probe: amiloride inhibition of basal ion transport, stimulation by adenosine, and inhibition by 8-phenyltheophyline. These results demonstrate that adult human vas deferens epithelium actively transports ions to generate the luminal environment of the deferent duct. Thus, vas deferens epithelium likely plays an active role in male fertility, and interventions that modulate epithelial function might be exploited to treat male-factor infertility or in contraception.

The paucity of biological material has inhibited identifying genes that are differentially expressed during mammalian oogenesis and preimplantation development. We report here the linear amplification of mRNA from small numbers of mouse oocytes and preimplantation embryos to generate amounts of sense RNA that are sufficient for suppression subtractive hybridization. The resulting oocyte-specific and 8-cell-specific cDNA libraries were partially characterized, and the known oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1oogenes were found in the oocyte-specific cDNA library but not in the 8-cell-specific library. Further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries should furnish a trove of information regarding temporal changes in gene expression during oogenesis and preimplantation development in the mouse.

The large amount of MSY2 protein, a mouse germ cell-specific Y-box protein, in oocytes and its degradation by the late two-cell stage suggest that MSY2 may stabilize and/or regulate the translation of maternal mRNAs. We report here the ability of bacterially expressed recombinant MSY2 protein to bind to mRNA and repress translation in vitro. Although MSY2 displays some sequence specificity in binding to short RNA sequences derived from the 3′ untranslated region (UTR) of the protamine 1 (Prm1) mRNA, as determined by both gel shift and filter binding assays, essentially no sequence specificity is observed when full-length Prm1 mRNA is used. The binding of MSY2 is ∼10-fold greater to the full-length Prm1 mRNA than to a 37-nucleotide sequence derived from the 3′ UTR, and gel shift assays indicate that multiple MSY2 molecules bind to a single Prm1 mRNA. MSY2 binding to luciferase mRNA at ratios of protein to mRNA that are likely to exist in the oocyte also leads to a moderate inhibition of protein synthesis in vitro. Given the abundance of MSY2 in mouse oocytes (2% of total oocyte protein), these data suggest that MSY2 packages mRNAs in vivo with relatively little sequence specificity, which may lead to both stabilization and translation repression of maternal mRNAs.

We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluoresecent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluoresence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available.

Epithelial cells were isolated from adult porcine vas deferens and grown in the absence or presence of steroid hormones. Transepithelial resistance (Rte), basal short circuit current (Isc), and the effects of selected ion transport modulators on these parameters were evaluated in modified Ussing chambers at three time points (5–8, 11–14, and 18–22 days postseeding). At the earliest time point, no significant differences were observed. At the middle time point, when compared with Rtein untreated control monolayers, Rtein monolayers exposed to 17β-estradiol, aldosterone, cortisol, cortisone, prednisolone, prednisone, and dexamethasone was significantly lower; in contrast, Rtein monolayers exposed to testosterone, dihydrotestosterone, or progesterone did not differ from that in control monolayers. Treatments with cortisol, prednisolone, and dexamethasone were associated with an elevated basal Iscthat was amiloride sensitive, indicating ongoing Na+absorption by these monolayers. Rtewas increased by amiloride treatment in glucocorticoid-treated monolayers but remained significantly less than that of control monolayers. At the third time point, the postamiloride Rteof glucocorticoid-treated monolayers was not different from that of control monolayers. Responses to ATP, forskolin, bumetanide, and DASU-02 were not affected by steroid treatment at any time point. Taken together, these results suggest that estrogens and corticosteroids can modulate epithelial function in the distal excurrent duct of the adult male reproductive system. At physiological or pharmacological concentrations, these hormones would be expected to modify the luminal environment (both the ionic composition and pH) to which sperm are exposed and thus affect male fertility.

Translational control plays a central role during oocyte maturation and early embryogenesis, as these processes occur in the absence of transcription. MSY2, a member of a multifunctional Y-box protein family, is implicated in repressing the translation of paternal mRNAs. Here, we characterize MSY2 expression in mouse oocytes and preimplantation embryos. Northern blot analysis indicates that MSY2 expression is highly restricted and essentially confined to the oocyte in the female mouse. MSY2 transcript and protein, as assessed by reverse transcription-polymerase chain reaction and immunoblotting, respectively, are expressed in growing oocytes, metaphase II-arrested eggs, and 1-cell embryos, but then are degraded by the late 2-cell stage; no expression is detectable in the blastocysts. During oocyte maturation, MSY2 is phosphorylated and following fertilization it is dephosphorylated. Quantification of the mass amount of MSY2 reveals that it represents 2% of the total protein in the fully grown oocyte, i.e., it is a very abundant protein. Both endogenous MSY2 and MSY2-enhanced green fluorescent protein (EGFP), which is synthesized following microinjection of an mRNA encoding MSY2-EGFP, are primarily localized in the cytoplasm, and about 75% of the MSY2 remains associated with oocyte cytoskeletal preparations. Results of these studies are consistent with the proposal that MSY2 functions by stabilizing and/or repressing the translation of maternal mRNAs.

A shift from a meiotic cell cycle to a mitotic cell cycle occurs following fertilization. The molecular basis for this transition, however, is poorly understood. Although cyclin A1 is proposed to regulate M phase in the meiotic cell cycle, and cyclin A2 is proposed to regulate S and M phases in the mitotic cell cycle, little is known about changes in the expression levels of cyclin A1 and A2 during meiotic and mitotic cell cycles in mammalian oocytes. We report that the mRNA levels of both cyclins A1 and A2 decrease during oocyte maturation. The amount of cyclin A1 mRNA then increases between the one-cell and blastocyst stages, whereas that of cyclin A2 remains relatively constant. The amount of cyclin A1 protein declines during maturation and is not readily detected from the two-cell to the blastocyst stage. In contrast, cyclin A2 is not readily detected in the oocyte and metaphase II-arrested egg but is detected following fertilization and throughout the subsequent stages of preimplantation development. The appearance of cyclin A2 protein following fertilization positively correlates with an increase in the size of the mRNA. This increase, as well as the increase in the amount of cyclin A2 protein, is prevented by 3′-deoxyadenosine (3′-dA), an inhibitor of polyadenylation. Consistent with a role for cyclin A2 in regulating the G1/S transition, 3′-dA also inhibits DNA replication in treated one-cell embryos. These results suggest that regulation of expression of cyclins A1 and A2 is under posttranscriptional regulation and that the observed changes in their expression may be involved in the transformation of a meiotic cell cycle to a mitotic cell cycle following fertilization.

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A (an inhibitor of histone deacetylases) or aphidicolin (an inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn’t correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.

The objective of the present study was to identify and characterize transcripts whose levels are increased in the mouse uterus during decidualization. Using the method of suppression subtractive hybridization, we identified a novel transcript. This transcript contained a potential open reading frame that coded for a 196-amino-acid protein that shows homologies to the heat shock protein 20 family of genes. This transcript was expressed in several adult tissues and in the embryo. Its steady-state level was significantly greater in implantation segments of the uterus compared to nonimplantation segments. Furthermore, the steady-state levels of this novel transcript were significantly greater in uterine horns undergoing artificially induced decidualization compared to control contralateral horns. Using in situ hybridization methods, signals for the transcript were localized to the endometrial stromal cells that were undergoing decidualization. Finally, we found that progesterone caused a significant increase in the steady-state level of this novel transcript in the uterus when administered to ovariectomized mice. In the presence of estradiol-17β, this effect was significantly reduced. In conclusion, we have identified a novel transcript of a potential heat shock protein whose level is significantly increased in the uterus during decidualization and in response to progesterone.

M-Phase promoting factor (MPF) is a complex of p34cdc2and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34cdc2, rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34cdc2is in excess to that of cyclin B, i.e., the accumulation of p34cdc2is not a limiting factor. We report measurements of the absolute mass of p34cdc2and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34cdc2, cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 × 106, 11.3 × 106, 24.6 × 106, 15.6 × 106, respectively, and in meiotically competent oocytes the numbers are 14.3 × 106, 95.5 × 106, 80.0 × 106, 40.1 × 106, respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34cdc2, and this is consistent with the hypothesis that the accumulation of p34cdc2plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34cdc2in both meiotically incompetent and competent oocytes.

The H19gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpnwas maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24–48 h of culture when 100 μM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34cdc2kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 μM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34cdc2kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34cdc2kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34cdc2and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

The mouse germinal vesicle (GV)-intact oocyte is a symmetric cell, with the GV centrally localized and with components of the plasma membrane and cortex symmetrically distributed around the periphery of the oocyte. During oocyte maturation, two distinct regions of the egg plasma membrane and cortex develop: the amicrovillar region overlying the meiotic spindle and the microvillar region. The development of this polarity is significant, since sperm bind to and fuse with the microvillar region. We are interested in the development of egg polarity and have characterized the localizations of several markers for egg polarity in normal metaphase II eggs and GV-intact oocytes. The asymmetric distributions of these markers (including actin, cortical granules, binding sites for the sperm proteins fertilin α and fertilin β, and two different β1integrin epitopes) develop during oocyte maturation in vitro, and this polarity can be perturbed by treatments that disrupt the actin microfilaments or microtubules. In addition, immunoelectron microscopy reveals that binding sites for recombinant fertilin β are specifically localized to the microvillar region, suggesting that the binding sites for this sperm ligand are either specifically localized or activated in this region. These results indicate that structural remodeling of the mouse egg plasma membrane is accompanied by molecular remodeling, resulting in the localization or activation of specific molecules in subdomains of the plasma membrane.

The pattern of expression of major histocompatibility complex class I H-2K in early mouse embryos was determined through reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization methods. H-2K transcripts were detected in all stages of preimplantation embryos from oocyte to blastocyst. In the blastocyst, transcripts were found in both trophectoderm and inner cell mass. Quantitative RT-PCR revealed that in CD-1 mice H-2Kqtranscript levels per oocyte/embryo were 71 ± 13 in oocyte, 150 ± 9 in 2-cell embryos, 220 ± 22 in 8-cell embryos, and 560 ± 86 in blastocysts. By RT-PCR, H-2KqmRNA was also detected in postimplantation staged (7.5-, 8.5-, and 9.5-day) embryos. Examination of components of the 7.5-day conceptus revealed the highest levels in the ectoplacental cone (EPC), followed by the decidua, and finally the embryo. The spatial distribution of H-2K transcripts in decidua was very heterogeneous, whereas the low levels in the embryo were found to be uniform throughout. Both cell types of the EPC contained H-2K transcripts. In the 9.5-day embryo, high transcript levels were observed over the developing liver above a background of constitutive expression everywhere else.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor α (TGFα) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGFα, bFGF, TGFβ1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.

To extend our previous finding that ethanol stimulates murine preimplantation development, we focused in the current study on the cavitation and expansion of the blastocyst. Ethanol stimulation of blastocyst cavitation and expansion was optimal at a concentration of 0.1% and required only a 5-min exposure. Because intracellular levels of calcium were transiently increased in the ethanol-exposed embryos, we determined the effect of directly increasing calcium on the rates of blastocyst cavitation and expansion. Treatment with the calcium ionophore, A23187, altered development much as did ethanol, indicating that ethanol may stimulate preimplantation development by increasing the intracellular calcium concentration. The relationship between intracellular calcium levels and blastocoel volume suggests that morphogenetic events during preimplantation development, particularly cavitation and blastocyst formation, may be regulated through modulation of intracellular calcium levels.

Mouse oocyte maturation in vivo or in vitro is accompanied by a precocious release of cortical granules. When oocytes are matured in vitro in the absence of serum, cortical granule exocytosis results in modifications of the zona pellucida (ZP) that are known to constitute the zona pellucida block to polyspermy. Fetuin, a glycoprotein found in newborn calf serum, can inhibit these maturation-associated changes in the zona pellucida. In this report we demonstrate that the human fetuin homologue, α2-HS-glycoprotein, is present in human follicular fluid. Human follicular fluid, or purified human α2-HS-glycoprotein, inhibits the maturation-associated conversion of mouse ZP2 to ZP2fin a concentration-dependent manner. Although the concentration of α2HS-glycoprotein in human follicular fluid is high enough to account for the inhibitory effect of human follicular fluid on this maturation-associated conversion, human follicular fluid immunodepleted of α2HS-glycoprotein retains its full inhibitory activity. Thus, other inhibitory molecules are likely to be present in follicular fluid and contribute to the inhibition of the maturation-associated conversion of ZP2.

Germinal vesicle (GV)-intact fully grown mouse oocytes do not undergo cortical granule (CG) exocytosis in response to A23187 treatment, whereas metaphase II (MII)-arrested eggs do. This differential response may reflect the development of the ability of the egg to undergo CG exocytosis, which is responsible for the biochemical modification of the glycoprotein ZP2 in the zona pellucida. Accordingly, we compared in these two stages the ability of 12-O-tetradecanoyl phorbol 13-acetate (TPA) or inositol 1,4,5-trisphosphate (IP3) to promote CG exocytosis and/or the ZP2 to ZP2fconversion; these agents are known to stimulate early events of mouse egg activation. TPA (10 ng/ml) treatment for 60 and 120 min resulted in a 25% and 52% CG loss in GV-intact oocytes and a 38% and 76% loss in MII eggs, respectively; fertilization resulted in a CG loss of ~ 70–80%. Although a similar extent of ZP2 to ZP2fconversion was observed in oocytes and eggs after a 120-min TPA treatment (~ 70–80%), a greater extent of conversion was observed in oocytes after a 60-min treatment (80% for oocytes, 50% for eggs). Microinjection of IP3(final concentration 1 μM) into MII eggs resulted in an extent of ZP2 conversion similar to that observed following fertilization, whereas little conversion occurred in GV-intact oocytes similarly injected. These results indicate that a protein kinase C sensitivity develops prior to meiotic maturation, whereas responsiveness to IP3develops after maturation has resumed. We propose that the regulatory mechanism involving an IP3-mediated calcium release is deficient in GV-stage oocytes.

Expression of the trophoblast interferon, bovine trophoblast protein-1 (bTP-1), has been studied in embryos produced by in vitro maturation-in vitro fertilization (IVM-IVF). No bTP-1 production was noted until after embryos had reached the expanded blastocyst stage and had begun to hatch (Days 8–9 post-fertilization). Single blastocysts comprising 115 ± 22 cells released 1.0 ± 0.1 units of interferon activity/24 h. Amplification of conceptus mRNA by reverse transcription-polymerase chain reaction procedure with bTP-1-specific oligonucleotides confirmed that bTP-1 transcripts were present in blastocysts but were not detectable at earlier stages. Although cultured blastocysts produced by IVM-IVF procedures continued to secrete bTP-1 for a few days, they failed to attach to the substratum and form outgrowths, and soon lost structural integrity. However, when Day 8 blastocysts/morulae were transferred to the uteri of synchronized cows, recovered 4 days later, and placed into individual cultures, they attached and formed outgrowths that produced large amounts of bTP-1 (> 2000 units/culture/24 h after 14 days). Embryos thus first expressed bTP-1 when a functional trophectoderm was first formed, and induction did not require a period of in vivo development. However, continued viability of the blastocyst and bTP-1 production were not sustained in vitro and may require some exposure to the uterine environment.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3and detecting all known pertussis toxin-sensitive α-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against α-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the β2-subunit of G proteins and an antiserum reacting with both β1- and β2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa β1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α-subunits nor G protein β-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein α-subunits, whereas the putative β-subunit could be identified as a β2-subunit.

A precocious but limited loss of cortical granules (CG) occurs during mouse oocyte maturation both in vivo and in vitro. Although CG loss during maturation in vivo is not associated with changes in the zona pellucida (ZP), a maturation-associated conversion of ZP2 to ZP2foccurs during oocyte maturation in vitro in serum-free medium. We now demonstrate that a maturation-associated change of ZP3 to ZP3f, as assessed by a reduction in sperm binding, also occurs during maturation in vitro in serum-free medium, and that both newborn calf serum (NCS) and fetuin, each of which inhibits the ZP2 conversion, also inhibit the ZP3 conversion. The concentration-dependence of the NCS- and fetuin-mediated inhibition of the ZP2 conversion, coupled with the concentration of fetuin present in NCS, is consistent with fetuin being the component present in NCS that is primarily responsible for this inhibition. Although NCS can inhibit the ZP modifications that occur during oocyte maturation in vitro, ionophore treatment of eggs, which results in an extensive release of CGs over a short period of time, overcomes the inhibitory effect of NCS on the ZP2 conversion. Results of these studies suggest a potential regulatory function of serum-derived components in the formation of a fertilizable egg.

To initiate studies on the protein microenvironment of the mouse blastocoel, we examined the electrophoretic profile of newly synthesized proteins secreted into the blastocoel, as well as those secreted into the medium. Although most polypeptides reveal no relative enrichment, some proteins (e.g., proteins of Mr= 155 000 and 33 000) are enriched in the blastocoel relative to those secreted apically into the medium. In addition, some proteins (e.g., proteins of Mr= 102 000 and 40 000) are enriched in the medium relative to the blastocoel. The relative amount of newly synthesized protein secreted into the blastocoel is about 2.5% of total protein synthesis. In addition, the trophectoderm and inner cell mass contribute to these proteins. Results of these studies suggest a potential function for these blastocoel-enriched proteins in inner cell mass development.

As a companion to amino acid transport and protein synthetic studies, it was of interest to quantify the amino acid pools in embryos and reproductive tract fluids during preimplantation development. Primary amines in the acid-soluble extracts of embryo and fluid samples were separated by high-performance liquid chromatography, reacted with o-phthalaldehyde, and quantified by fluorescence emission. The amino acid compositions of embryos were like those of corresponding reproductive tract fluids. Taurine was high in eggs and fluids but declined with development, while glycine levels rose. Glycine was highest in concentration in all samples (except the egg), followed by glutamate and alanine, while most other amino acids were consistently of low abundance.

Possible roles of cell-to-cell communication mediated by intercellular bridges and gap junctions in development of the female gamete and embryo are discussed. Synchronization of cell cycle events is presumably a role for intercellular bridges between germ cells. The follicle of the Cecropiamoth reveals that an electrical polarity exists between nurse cells and oocytes which are connected by intercellular bridges and this polarity may generate differences that result in differentiation of the oogonia to become either the oocyte or nurse cells. Gap junction-mediated transfer of cyclic AMP, made in response to gonadotropin stimulation, between granulosa cells is discussed as a mechanism that allows cells within a tissue to respond to an external stimulus even though all cells in that tissue may not be exposed to the stimulus. A nutritional role for heterologous cell communication between follicle cells and the oocyte in oocyte growth is presented as an example of how gap junction-mediated communication can allow one cell type to influence the behavior of another cell type. During development, a restriction in communication between differentiating cells is frequently observed. Examples of this phenomenon in a mammal and an insect are presented.

In the rhesus monkey, progesterone (P) given after a small dose of estradiol benzoate (EB) induces a luteinizing hormone (LH) surge with a short latency and short duration (Terasawa et al., 1982). In the present study, effects of P injection in relation to the interval after EB and to the time of day were investigated. Nine long-term ovariectomized female rhesus monkeys (which were implanted with an estradiol-17β(E2) capsule 2 wk prior to the experiments) were injected with EB (10 µg) and P (2.5 mg). In order to determine the period of estrogen priming necessary to induce the facilitatory effects of P, P was injected at 30, 24, 12 or 0 h after EB in the first four experiments. The time of EB injections was fixed at 0830 h. To determine whether there is any diurnal influence on the action of P, the EB injection was moved to 2030 h, followed by P 30 h later.Administration of P 30 h after EB induced a typical LH surge with peak latency (6.7 ± 0.5 h) and duration (16.0 ± 1.4 h) in all animals. Similarly, P injection 24 h after EB induced a LH surge with peak latency (7.4 ± 0.4 h) and duration (18.0 ± 1.2 h) in all animals. The amplitudes of the P-induced LH surges at both 30 h and 24 h after EB were also similar (P 30 h; 65.0 ± 18.7 ng/ml, P 24 h; 59.8 ± 24.4 ng/ml). In contrast, administration of P 12 h or 0 h after EB resulted in a LH surge in only 4 and 3 of 9 animals, respectively, and these numbers of responders were significantly less (P 12 h: P=0.0147, P 0 h: P=0.0045) than those of P 30 h and P 24 h. The amplitudes of the LH surge in animals responding to P 12 h and P 0 h after EB were much smaller than those of P 30 and P 24 h after EB (P<0.01), although the peak latency and duration of the response were similar. Overall responses to P 12 h and P 0 h after EB were significantly (P<0.005) different from those of P 30 h and P 24 h after EB. A 12-h shift of the injection time of both EB and P did not alter the LH response. Reversed timing of EB and P injections induced a LH surge with peak latency, 7.7 ± 0.5 h; duration, 16.3 ± 1.7 h; and amplitude, 59.2 ± 20.7 ng/ml. These results indicate that 1) P injection 24 h or 30 h after EB reliably induces a LH surge with a short latency and duration, 2) P injection 12 h or 0 h after EB is not effective in inducing a LH surge, and 3) there is no diurnal influence on the P-induced LH surge.Therefore, the time interval of estrogen priming required for progesterone action in the rhesus monkey is similar to that required in rodents for LH release as well as for lordosis behavior. However, in the rhesus monkey, unlike the rat, the circadian signal seems not to interact with the action of P exogenously administered.