Biomedical subjects

R M Schultz

Publications and source records attributed to R M Schultz.

At least 109 records · Page 6Linked to original sources

PubMed1994

Expression patterns of novel genes during mouse preimplantation embryogenesis.

Little is known about the repertoire of genes expressed following zygotic gene activation, which occurs during the two-cell stage in the mouse. As an initial attempt to isolate novel genes, we used previously prepared two-cell and two-cell subtraction cDNA libraries (Rothstein et al., Genes Dev 6:1190-1201, 1992) to isolate a panel of seven cDNA clones. Three cDNAs had no match in the current DNA sequence data banks and three others revealed sequence homology to portions of sequences in the data banks. One cDNA was 90% homologous to the ras-related gene Krev/rap 1A. The temporal patterns of expression of these genes during oocyte maturation and preimplantation development were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR) assay developed to measure relative levels of mRNAs. Three distinct temporal patterns of expression, designated Classes 1-3, were found. The two Class 1 genes displayed an actin-like pattern, with a gradual decline in expression during oocyte maturation and through the two-cell stage, followed by increases at the eight-cell and/or blastocyst stages. The four genes in Class 2 were expressed at relatively high levels during oocyte maturation and through the one-cell stage and then declined abruptly between the one- and two-cell stages; an increase then occurred at the eight-cell and/or blastocyst stages. The expression of the gene in Class 3 declined during oocyte maturation, but then showed a transient increase at the one-cell stage, with only a very slight increase in synthesis at either the eight-cell or blastocyst stage.

Actins↗

PubMed1994

Rapid, nonradioactive, and quantitative method to analyze zona pellucida modifications in single mouse eggs.

A rapid, nonradioactive method to monitor the ZP2 to ZP2f conversion in the zona pellucida of single mouse eggs has been developed. This assay is based on the chemiluminescent detection of biotinylated ZP2 and ZP2f following electrophoresis under reducing conditions and electrophoretic transfer to Immobilon P. This method is about 10 times faster and detects similar extents of ZP2 to ZP2f conversion following A23187-induced egg activation, when compared to the commonly used radioiodination procedures.

Animals↗

PubMed1994

Mouse preimplantation embryo development in vitro: effect of sodium concentration in culture media on RNA synthesis and accumulation and gene expression.

Results of previous studies indicate that culture of preimplantation mouse embryos in SOM medium containing 85 mM NaCl promotes better development in vitro, as well as supporting higher rates of protein synthesis, when compared to culture in SOM containing 125 mM NaCl (Anbari and Schultz, 1993, Mol Reprod Dev 35:24-28; Biggers et al., 1993, Mol Reprod Dev 34:380-390). In the present study we compare the effect of culturing embryos in these 2 media on several aspects of RNA synthesis and gene expression in order to determine whether the reduced development in SOM containing 125 mM NaCl and lower rates of protein synthesis are correlated with decreases in RNA synthesis and stability and changes in gene expression. Although no apparent differences in the metabolism of [3H]uridine to UMP, UDP, and UTP and its incorporation into total RNA are observed when 2-cell embryos are cultured to the morula stage in either medium, a 20% decrease in the rate of mRNA synthesis is found when embryos are cultured in SOM containing 125 mM NaCl. In addition, pulse-chase experiments reveal that total mRNA is less stable when the embryos are cultured in SOM containing 125 mM NaCl. Using a reverse transcription-polymerase chain reaction to assay for changes in the relative amounts of specific mRNAs, the relative amounts of mRNAs for IGF-I and IGF-II and their cognate receptors are dramatically reduced in embryos cultured in SOM containing 125 mM NaCl, whereas only a mild reduction is observed in the relative amount of actin mRNA. In contrast, when freshly isolated morulae are cultured to the blastocyst stage in either of these 2 media, similar amounts of these mRNAs are observed. Last, high-resolution, 2-dimensional gel electrophoresis reveals significant changes in the pattern of protein synthesis when the embryos are cultured in SOM containing 125 mM NaCl. Results of these experiments suggest that culture of embryos in medium containing lower concentrations of NaCl that are normally present in various culture media results in higher rates of mRNA synthesis and greater mRNA stability. These changes in RNA synthesis may underlie, at least in part, the improved development in vitro that is fostered by SOM containing 85 mM NaCl.

Actins↗

PubMed1994

Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes.

This paper focuses on the relationship between the developmental programs that regulate nuclear and cytoplasmic maturation of mouse oocytes. As oocytes near completion of their growth phase, they become competent to resume meiosis when isolated from the small antral follicles and cultured, but the progression of meiosis in many of these oocytes stops at metaphase I (MI). With further growth and development at the germinal vesicle (GV) stage, however, these oocytes become competent to complete nuclear maturation and progress to metaphase II (MII). In this study, about 44% of the oocytes were arrested at MI after isolation from the small antral follicles of 18-day-old mice and culture for 16 to 17 hr. Upon insemination, these MI-arrested oocytes produced the first polar body, formed pronuclei, cleaved, and developed to the blastocyst stage. Seventy five percent of these blastocysts were triploid, the remainder were diploid. Treatment of MI-arrested oocytes with calcium ionophore resulted in the completion of the first meiotic division and parthenogenetic activation. Moreover, while the pattern of proteins synthesized in MI-arrested oocytes was quite different from that of normal MI oocytes as determined by [35S]methionine labeling and two-dimensional gel electrophoresis, it was similar to that of MII-arrested oocytes. It was therefore concluded that some critical aspects of cytoplasmic maturation can occur in oocytes whose nuclear maturation is arrested at MI. In addition, triggers that promote entry of MII-arrested oocytes into anaphase II are sufficient to drive MI-arrested oocytes into anaphase I and to produce the first polar body when the triggers are generated in mature cytoplasm. The developmental capacity of MII oocytes that matured in vitro after isolation from 18- or 26-day-old mice were compared. The frequency of fertilization and cleavage to the 2-cell stage was equal in both groups. In surprising contrast, the ability to complete the 2-cell stage to blastocyst transition occurred much more frequently when the oocytes were from the 26- than the 18-day-old mice (82 and 27%, respectively). Thus, even though both groups of oocytes had completed nuclear maturation by progressing to MII, oocytes from the smaller follicles of the younger mice were deficient in maternal factors essential for development of embryos beyond the 2-cell stage. Further differentiation of these GV-stage oocytes of small antral follicles is therefore required to produce eggs competent of completing preimplantation development.

Animals↗

PubMed1994

Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs.

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

ADP Ribose Transferases↗

PubMed1994

Involvement of inositol 1,4,5-trisphosphate-mediated Ca2+ release in early and late events of mouse egg activation.

Sperm-induced activation of mammalian eggs is associated with a transient increase in the concentration of intracellular Ca2+. The role of inositol 1,4,5-trisphosphate (IP3)-mediated release of Ca2+ from intracellular stores during mouse egg activation was examined in the present study by determining the effects of microinjected monoclonal antibody (mAb) 18A10, which binds to the IP3 receptor and inhibits IP3-induced Ca2+ release, on endpoints of egg activation following insemination. The antibody inhibited in a concentration-dependent manner the ZP2 to ZP2f conversion that is involved in the zona pellucida block to polyspermy, as well as the ZP2 to ZP2f conversion promoted by microinjected IP3 in non-inseminated eggs. As anticipated, inseminated eggs that had been microinjected with the antibody were polyspermic. In addition, the antibody inhibited the fertilization-associated decrease in H1 kinase activity and pronucleus formation, and the concentration dependence for inhibition of these events was similar to that observed for inhibiting the ZP2 to ZP2f conversion. Last, the antibody inhibited the fertilization-induced recruitment of maternal mRNAs and post-translational modifications of proteins. In each case, eggs microinjected with the mAb 4C11, which also binds to the IP3 receptor but does not inhibit IP3-induced Ca2+ release, had no inhibitory effect on fertilization and egg activation. Results of these studies suggest that IP3-mediated Ca2+ release is essential for both early and late events of mouse egg activation.

Animals↗

PubMed1994

Regulation of gene expression in the mouse oocyte and early preimplantation embryo: developmental changes in Sp1 and TATA box-binding protein, TBP.

We previously demonstrated that an Sp1-dependent reporter gene is preferentially expressed in G2 of the 1-cell mouse embryo following microinjection of the male pronucleus when compared to microinjection of the female pronucleus (P.T. Ram and R.M. Schultz, 1993, Dev. Biol. 156, 552-556). We also noted that expression of the reporter gene is not observed following microinjection of the germinal vesicle of the fully grown oocyte. In the present study, we examined expression of this reporter gene during oocyte growth, as well as the nuclear concentration of two transcription factors, Sp1 and the TATA box-binding protein, TBP, during oocyte growth and the first cell cycle. The extent of reporter gene expression decreases during oocyte growth and this decrease correlates with the decrease in nuclear concentration of Sp1, as determined by confocal immunofluorescent microscopy. In addition, results of immunoblotting experiments also indicate a similar decrease in the total concentration of Sp1 during oocyte growth. The nuclear concentration of TBP also decreases during oocyte growth, as determined by confocal immunofluorescent microscopy. Following fertilization, the pronuclear concentration of these two transcription factors increases in a time-dependent fashion and the concentration of each is greater in the male pronucleus as compared to the female pronucleus. For each pronucleus and for each transcription factor, this increase in nuclear concentration is inhibited by aphidicolin, which inhibits DNA synthesis. Last, the increase in nuclear concentration of these two proteins observed between the 1-cell and 2-cell stages does not require transcription or cytokinesis.

Animals↗

PubMed1993

Regulation of zygotic gene activation in the mouse.

Zygotic gene activation (ZGA) is the critical event that governs the transition from maternal to embryonic control of development. In the mouse, ZGA occurs during the 2-cell stage and appears to be regulated by the time following fertilization, i.e. a zygotic clock, rather than by progression through the first cell cycle. The onset of ZGA must depend on maternally inherited proteins, and post-translational modification of these maternally derived proteins is likely to play a role in ZGA. Consistent with this prediction is that protein phosphorylation catalyzed by the cAMP-dependent protein kinase is involved in ZGA and that protein synthesis is not required for ZGA. Recent results suggest that ZGA may occur earlier than previously thought, i.e. not during the 2-cell stage, but rather in G2 of the 1-cell embryo. Thus ZGA may comprise a period of minor gene activation in the 1-cell embryo that is followed by a period of major gene activation in the 2-cell embryo. Following ZGA, the expression of constitutively activated genes may require an enhancer.

Animals↗

PubMed1993

Protein secretion by the mouse blastocyst: stimulatory effect on secretion into the blastocoel by transforming growth factor-alpha.

We have previously demonstrated that newly synthesized proteins are secreted into the mouse blastocoel [Dardik and Schultz (1991): Biol Reprod 45:328-333]. In the present study we examine the effect of transforming growth factor-alpha (TGF-alpha) on these proteins. We observe that TGF-alpha stimulates secretion of these newly synthesized proteins into the blastocoel and apical medium, which faces the zona pellucida, by about 65%. Although one-dimensional gel electrophoretic analysis does not reveal any marked differences in the patterns of newly synthesized proteins secreted into the blastocoel in response to TGF-alpha, zymography reveals a marked stimulation in the secretion of several gelatinases into the blastocoel and apical medium. These results suggest additional functions for TGF-alpha in mouse preimplantation development.

Animals↗

PubMed1993

Effect of sodium and betaine in culture media on development and relative rates of protein synthesis in preimplantation mouse embryos in vitro.

Results of recent experiments indicate that the improved development of mouse embryos in medium containing a low NaCl concentration (85 mM) or the inclusion of the organic osmolyte betaine in a medium containing a high NaCl concentration (125 mM) is correlated with the maintenance of intracellular sodium concentrations that more closely approximate those found in freshly isolated embryos (Biggers et al., 1993, Mol Reprod Dev 34:380-390). We examined the effect of these different culture media on the relative rates of protein synthesis since increased levels of intracellular sodium inhibit protein synthesis; a reduced rate of protein synthesis could therefore account for the differences in development in the different media, since cell division requires protein synthesis. We observe that the ability of these media to support development and to maintain more physiological concentrations of intracellular sodium is correlated with their ability to support increased relative rates of protein synthesis. Reducing the NaCl concentration from 125 mM to 85 mM leads to a greater fraction of the embryos developing from the 2-cell stage to the 8-cell stage after 1 day of culture and a substantially improves extent of development to the morula stage after 2 days of culture. This reduction in NaCl concentration also leads to a 2.4-fold increase in the relative rate of protein synthesis in 4-cell embryos. Moreover, addition of betaine to medium containing 125 mM NaCl increases the relative rate of protein synthesis. This finding provides an explanation, at least in part, for the increase in development to the blastocyst stage exhibited by mouse embryos cultured in these media.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Reporter gene expression in G2 of the 1-cell mouse embryo.

Similar amounts of expression of a luciferase reporter gene that is driven by the SV40 early promoter are detected in G2 of 1-cell mouse embryo following injection of the male pronucleus in either early or mid/late S. No luciferase activity is observed when the male pronucleus is injected in early S and the embryos analyzed for expression in late S. Last, no luciferase activity is observed when the female pronucleus is injected in mid/late S and the embryos analyzed for luciferase activity in G2 of the first cell cycle. These results suggest that zygotic gene activation initiates in the 1-cell embryo rather than in the 2-cell embryo, as previously thought, and that differences between the transcriptional activity of male and female pronuclei exist.

Animals↗

PubMed1993

Complete mouse egg activation in the absence of sperm by stimulation of an exogenous G protein-coupled receptor.

Sperm-induced egg activation has several features in common with signal transduction pathways mediated by ligand-receptor-effector systems. G proteins are postulated to act as signal transducers regulating the plethora of cellular responses constituting egg activation that occur in response to sperm. We report that acetylcholine treatment of mouse eggs expressing the human m1 muscarinic receptor, a G protein-coupled receptor, results in the full complement of events of egg activation, including ZP2 to ZP2f conversion, recruitment of maternal mRNAs, pronuclear formation with subsequent DNA synthesis, cleavage to the two-cell stage, and activation of the embryonic genome. The induction of a full complement of sperm-induced egg activation events by ACh in the absence of sperm strongly suggests that sperm-induced egg activation is mediated by egg G proteins.

Acetylcholine↗

PubMed1993

Regulation of the polyspermy block in the mouse egg: maturation-dependent differences in cortical granule exocytosis and zona pellucida modifications induced by inositol 1,4,5-trisphosphate and an activator of protein kinase C.

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 ZP2f conversion; 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 approximately 70-80%. Although a similar extent of ZP2 to ZP2f conversion was observed in oocytes and eggs after a 120-min TPA treatment (approximately 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 microM) 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 IP3 develops after maturation has resumed. We propose that the regulatory mechanism involving an IP3-mediated calcium release is deficient in GV-stage oocytes.

Animals↗

PubMed1993

Modifications of the mouse zona pellucida during oocyte maturation: inhibitory effects of follicular fluid, fetuin, and alpha 2HS-glycoprotein.

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, alpha 2-HS-glycoprotein, is present in human follicular fluid. Human follicular fluid, or purified human alpha 2-HS-glycoprotein, inhibits the maturation-associated conversion of mouse ZP2 to ZP2f in a concentration-dependent manner. Although the concentration of alpha 2HS-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 alpha 2HS-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.

Animals↗

PubMed1993

Evaluation of new anticancer agents against the MIA PaCa-2 and PANC-1 human pancreatic carcinoma xenografts.

Human pancreatic carcinoma xenograft models were developed from established MIA PaCa-2 and PANC-1 cell lines (ATCC, Rockville, MD). Tumors were maintained by serial trocar implantation in CD1 nu/nu mice, and attempts were made to test all drugs under optimal schedules at maximum tolerated doses. In both models, adriamycin, cisplatin, and 5-fluorouracil were inactive (< 60% inhibition of tumor weight), whereas gemcitabine (LY188011] produced modest activity (69% inhibition in MIA PaCa-2 and 76% inhibition in PANC-1. Major differences in tumor sensitivity were noted with diarylsulfonylureas (DSU) and taxol. The DSU (Sulofenur [LY186641] and LY295501) produced complete inhibition in the MIA PaCa-2 xenograft, but were inactive in PANC-1. Conversely, taxol produced 80% inhibition of PANC-1 tumor growth, but was inactive against MIA PaCa-2. In general, in vivo antitumor activity roughly correlated with in vitro tumor cytotoxicity with the exception of DSU. We have previously shown that DSU are extensively bound to albumin and that in vitro cytotoxic activity in serum-containing medium is not predictive of in vivo antitumor activity. The MIA PaCa-2 and PANC-1 xenograft models may be useful for selecting potential candidates for therapy of human pancreatic cancer.

Animals↗

PubMed1992

Fibroblasts transformed by different ras oncogenes show dissimilar patterns of protease gene expression and regulation.

NIH 3T3 cells transformed by different activated ras genes showed different patterns of protease gene expression, indicating the existence of least two pathways for NIH 3T3 transformation from mutated ras genes. In cells transformed by activated mammalian EJ-ras and chimeric EJ/vHa-ras, high constitutive levels of urokinase plasminogen activator (uPA) mRNA and/or phorbol-12-myristate-13-acetate (PMA) inducibility of the uPA mRNA was observed. However, PMA did not induce cathepsin L (CL) mRNA levels in these same cell lines. In contrast, NIH 3T3 cells transformed by homologous yeast RAS1Leu sequences showed low levels of uPA mRNA and a lack of PMA inducibility of uPA mRNA, but did show high constitutive levels of the mRNA for CL and/or PMA inducibility of CL mRNA expression. Based on their differences in PMA inducibility these two phenotypes are designated rasuPA+/CL- and rasCL+/uPA-, respectively. Run-on assays indicated the differences in the levels of CL and uPA mRNA with ras transformation and phorbol ester induction are due to changes in transcription rates. Based on the observation of the two ras-transformed phenotypes for protease expressions, we asked whether uPA and CL can substitute for each other in the promotion of experimental metastasis. Injection of in vitro antisense inhibited cells in nude mice showed an inhibition of lung colonization by anti-uPA only in the rasuPA+/CL- phenotype and by anti-CL only in the rasCL+/uPA- phenotype. The data thus show the existence of two distinct activated ras-transformed metastatic phenotypes induced in the same parental cell line and that uPA or CL protease expressions alternatively facilitate the metastasis of cells with one ras phenotype and not with the other.

3T3 Cells↗

PubMed1992

Zygotic gene activation in the mouse embryo: involvement of cyclic adenosine monophosphate-dependent protein kinase and appearance of an AP-1-like activity.

Protein phosphorylation catalyzed by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is implicated in regulating zygotic gene activation in the two-cell mouse embryo (Poueymirou and Schultz; Dev Biol 133:588-599, 1989). We now provide evidence that H8, which is a PKA inhibitor, inhibits expression of an hsp70-driven beta-galactosidase reporter gene and that the concentration-dependence of this inhibition is similar to that for inhibiting expression of a stage-specific gene(s) that is a product of zygotic gene activation. We also demonstrate that neither cAMP nor serum can stimulate the expression, as detected by a histochemical assay, of a cAMP response element (CRE)- or serum response element (SRE)-driven beta-galactosidase reporter gene, respectively, in either germinal vesicle-intact oocytes or aphidicolin-arrested one-cell embryos that are chronologically at the tw-cell stage. In contrast, although 12-O-tetradecanoyl phorbol-13-acetate (TPA) does not stimulate expression of a TPA response element (TRE)-driven beta-galactosidase reporter gene in germinal vesicle-intact oocytes, it stimulates such expression in aphidicolin-arrested one-cell embryos. Moreover, TPA can stimulate the expression of either a CRE- or an SRE-driven beta-galactosidase reporter gene in such embryos. Results of these studies further implicate protein phosphorylation in regulating zygotic gene activation, along with its role in modulating enhancer function in the early mouse embryo.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Changes in cAMP phosphodiesterase activity and cAMP concentration during mouse preimplantation development.

Cyclic nucleotide phosphodiesterase (PDE) activity and cAMP amounts were measured in mouse preimplantation embryos at the 1-cell, 2-cell, 8-cell/morula, and mid-blastocyst stages. PDE activity remained constant between the 1-cell and 2-cell stages. It decreased by the 8-cell stage and continued to decrease by the mid blastocyst stage to about 14% of the 1- and 2-cell values. By contrast, cAMP amounts remained essentially constant at 0.05 fmole/embryo (0.3 microM) from the 1-cell to the blastocyst stage and increased to 0.175 fmole in the fully expanded blastocyst that was close to hatching. Measurements of embryo volume indicated that intracellular volume remained essentially constant up to the blastocyst stage. The morphological changes in cell shape that accompany differentiation of the trophectoderm and that are coupled with blastocoel expansion decreased the intracellular volume. This decrease resulted in an increase in the cAMP concentration to about 0.4 microM by the mid-blastocyst stage. Previous studies indicate that either cAMP or TGF-alpha/EGF can stimulate the rate of blastocoel expansion. Although TGF-alpha/EGF can elevate cAMP levels in other cell types, TGF-alpha, at a concentration that maximally stimulates the rate of blastocoel expansion, did not elevate cAMP in blastocysts. Thus, it was unlikely that elevation of cAMP is the mechanism by which TGF-alpha stimulates the rate of blastocoel expansion.

3',5'-Cyclic-AMP Phosphodiesterases↗