Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation.

Betaine (N,N,N-trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation. Because betaine can be synthesized in mammalian cells via choline dehydrogenase (CHDH; EC 1.1.99.1), we assessed whether this enzyme was expressed and active. Chdh transcripts and CHDH protein were expressed in oocytes. No CHDH enzyme activity was detected in GV oocyte lysate, but CHDH became highly active during oocyte meiotic maturation. It was again inactive after fertilization. We then determined whether oocytes synthesized betaine and whether CHDH was required. Isolated maturing oocytes autonomously synthesized betaine in vitro in the presence of choline, whereas this failed to occur in Chdh-/- oocytes, directly demonstrating a requirement for CHDH for betaine accumulation in oocytes. Overall, betaine accumulation is a previously unsuspected physiological process during mouse oocyte meiotic maturation whose underlying mechanism is the transient activation of CHDH.

Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.

Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.

Targeted disruption of Aurora A causes abnormal mitotic spindle assembly, chromosome misalignment and embryonic lethality.

Aurora A (also known as STK15/BTAK in humans), a putative oncoprotein naturally overexpressed in many human cancers, is a member of the conserved Aurora protein serine/threonine kinase family that is implicated in the regulation of G(2)-M phases of the cell cycle. In vitro studies utilizing antibody microinjection, siRNA silencing and small molecule inhibitors have indicated that Aurora A functions in early as well as late stages of mitosis. However, due to limitations in specificity of the techniques, exact functional roles of the kinase remain to be clearly elucidated. In order to identify the physiological functions in vivo, we have generated Aurora A null mouse embryos, which show severe defects at 3.5 d.p.c. (days post-coitus) morula/blastocyst stage and lethality before 8.5 d.p.c. Null embryos at 3.5 d.p.c. reveal growth retardation with cells in mitotic disarray manifesting disorganized spindle, misaligned and lagging chromosomes as well as micronucleated cells. These findings provide the first unequivocal genetic evidence for an essential physiological role of Aurora A in normal mitotic spindle assembly, chromosome alignment segregation and maintenance of viability in mammalian embryos.

Early embryo-endometrial signaling modulates the regulation of matrix metalloproteinase-3.

OBJECTIVE: To examine matrix metalloproteinase-3 (MMP-3) expression in human stromal cell culture after P stimulation and the effect of conditioned medium from human embryo-epithelial cells coculture on its expression and activity. DESIGN: Metabolic and endocrine studies on human tissue. SETTING: In vitro fertilization (i.v.f.) unit and endocrine research unit. PATIENT(S): Infertile patients undergoing endometrial tissue sampling for dating at the luteal phase before i.v.f. INTERVENTION(S): Endometrial sampling and collection of human embryos culture media. MAIN OUTCOME MEASURE(S): Expression and activity of secreted MMP-3 by P-induced stromal cells, and in stromal cells exposed to conditioned medium from embryo-epithelial cell coculture. RESULT(S): Expression and activity of MMP-3 in human stromal cells decrease after P induction. Following incubation of these stromal-derived decidual cells with conditioned medium from embryo-epithelial cell coculture, MMP-3 expression and activity increased in a statistically significant manner. CONCLUSION(S): Progesterone inhibition of MMP-3 expression and its support of endometrial integrity were prevented by local expression of MMP-3 in response to embryonic signaling.

Essential role for poly (ADP-ribosyl)ation in mouse preimplantation development.

BACKGROUND: Poly (ADP-ribosyl)ation is a covalent modification of many nuclear proteins. It has a strong chromatin modifying potential involved in DNA repair, transcription and replication. Its role during preimplantation development is unknown. RESULTS: We have observed strong but transient synthesis of poly ADP-ribose polymers on decondensing chromosomes of fertilized and parthenogenetically activated mouse oocytes. Inhibition of this transient upregulation with a specific enzyme inhibitor, 3-aminobenzamide, has long-term effects on the postimplantation development of the embryos. In addition, inhibition of poly (ADP-ribosyl)ation at the 4-8 cell stage selectively blocks morula compaction. CONCLUSION: These observations suggest that poly (ADP-ribosyl)ation is involved in the epigenetic chromatin remodeling in the zygote.

Genetic expression of hexokinase and glucose phosphate isomerase in late-stage mouse preimplantation embryos: transcription activities in glucose/phosphate-containing HTF and glucose/phosphate-free P1 media.

In mouse and human preimplantation development, pyruvate is consumed preferentially during early embryogenesis; however, during the morula and blastocyst stages, glucose is the preferred energy substrate. Studies have suggested that the glycolytic enzymes, hexokinase and glucose phosphate isomerase, are important enzymes in glucose metabolism during these later stages of human and mouse preimplantation development. In order to investigate the genetic activities of these enzymes in late-stage mouse embryos developing in vitro, we analysed hexokinase and glucose phosphate isomerase transcription activities by qualitative RNA assays using reverse transcriptase-nested polymerase chain reaction amplification of individual mouse morulae and early blastocysts incubated in glucose/phosphate-free preimplantation stage one (P1) medium and glucose/phosphate-containing human tubal fluid (HTF) medium. We observed an increased incidence of hexokinase transcripts in the population of blastocysts compared with morulae, and differences in transcript incidence between early blastocysts developing in HTF medium and in P1 medium. In contrast, glucose phosphate isomerase transcripts were consistantly present in all embryos analysed, and appear to be constitutively expressed during late-stage mouse embryogenesis. The different activity patterns of the two glycolytic genes may reflect different mechanisms of gene regulation or differential transcript stability during the later stages of mouse preimplantation development.

Immunohistochemical localization of prostaglandin H synthase in the embryo and uterus of the mouse from ovulation through implantation.

Prostaglandins (PGs) in the embryo and endometrium are involved in processes that are important for implantation. Although the presence of PGs (PGE2, PGF2 alpha, PGI2) in decidualized endometrium has been widely reported, less is known about the capacity of the pre-implantation embryo to synthesize PGs. Prostaglandin H (PGH) synthase is necessary for the production of PGs. Using an immunohistochemical method, PGH synthase was localized in the mouse embryo and uterus from superovulation through embryo implantation. No PGH synthase was detected in oocytes at the time of ovulation or in single-cell embryos 1 day post-fertilization (PF). Circular areas of immunostaining became evident in the cytoplasm of blastomeres at the morula stage (day 3 PF). After implantation (day 5 PF), a low level of PGH synthase reactivity was observed in embryonic cells; no PGH synthase was detected in the embryo by day 7 PF. The endometrial glands exhibited maximal immunostaining by day 3 PF, and after implantation, PGH synthase appeared in decidual cells along the border of placentation. Low levels of PGH synthase reactivity were detected in myometrial cells during the period after superovulation through day 7 PF. This is the first demonstration of PGH synthase in the mouse embryo prior to apposition with glandular endometrial epithelium, supporting the hypothesis that the embryo has the potential to produce PGs that may mediate autocrine and/or paracrine responses at the time of nidation.

Immunofluorescence assessment of the timing of appearance and cellular distribution of Na/K-ATPase during mouse embryogenesis.

We have employed immunofluorescence with a rat kidney Na+/K+-ATPase polyclonal antibody to investigate the cellular distribution and timing of appearance of this enzyme during preimplantation development. The enzyme is first detected in the late morula within the cytoplasm of each blastomere. When cavitation begins this distribution changes dramatically to a ring encircling the blastocoel, restricted to the basolateral cell margins. Using this enzyme as a marker for cavitation, we examined its expression in embryos that had been treated with wheat germ agglutinin (WGA), which causes cleavage arrest and was reported to trigger premature compaction- and cavitation-like events in early cleavage stages (L. V. Johnson, 1986, Dev. Biol. 113, 1-9). Although WGA-treated 2-,4-, and 8-cell embryos quickly underwent compaction- and cavitation-like events, no Na+/K+-ATPase expression was observed. Thus the WGA effect does not likely involve acceleration of the developmental program for cavitation. Embryos arrested at the 8-cell stage but cultured overnight to Day 4, however, expressed the enzyme in the typical blastocyst pattern (around each fluid-filled cavity). We conclude that Na+/K+-ATPase expression is initiated or increases dramatically in the late morula and is independent of cytokinesis. The enzyme assumes a distribution during cavitation consistent with its presumed role in transtrophectodermal fluid transport.

Effects of cadmium on lactate dehydrogenase activities in mouse pre-embryos at various stages.

A decline in LDH activity from four-cell to late blastocyst was demonstrated in pre-embryos from F1 (C57 female X A2G male) female mice. An exposure to 0.5 or 1 microgram/ml cadmium did not affect the in vitro development of the four-cell pre-embryos and morulae or their LDH activities. At 5 or 10 micrograms/ml cadmium, the in vitro development of the treated pre-embryos was affected. Although most of the treated four-cell pre-embryos had proceeded to compaction, they became degenerated 24 h after treatment. The LDH activity of these degenerated pre-embryos was higher than that in the control blastocysts. We propose that cadmium may interfere with the general energy metabolism of the cells. This causes a reduced rate of LDH degradation, leading to a slower decline in LDH activity in cadmium-treated pre-embryos. Failure of some critical biochemical processes after cadmium treatment may ultimately lead to the subsequent degeneration of the treated pre-embryos.

Development of activatable adenylate cyclase in the preimplantation mouse embryo and a role for cyclic AMP in blastocoel formation.

Forskolin- and cholera toxin-activated adenylate cyclase activity increases between the morula and blastocyst stages of mouse preimplantation development, as assessed by the ability of these agents to increase embryonic cAMP levels. Development of activatable adenylate cyclase requires transcription but is independent of the fifth nuclear replication, cell division, and compaction. Early cavitating embryos treated with cholera toxin and forskolin or with N6-monobutyryl-cAMP display an increase in the rate of fluid accumulation in comparison with untreated controls. The stimulatory effect is specific for cAMP, since neither the inactive cAMP analogue N6-monobutyryl-2'-deoxy-cAMP nor N2-monobutyryl-cGMP stimulates the rate of fluid accumulation. These results constitute the first report of a possible physiological function for cAMP in preimplantation development, namely, in blastocoel formation.

A fine analysis of glucose-phosphate-isomerase patterns in single preimplantation mouse embryos.

Mouse embryos were derived from eggs heterozygous for alleles of the dimeric enzyme glucose phosphate isomerase (Gpi-1a/Gpi-1b) that had been fertilized with sperm carrying a third allele (Gpi-1c). This particular combination makes it possible to study the activity of the paternally derived as well as the maternally derived genes, the persistence of oocyte-coded enzyme throughout early development and the possible simultaneous expression of both the paternally derived allele and the maternal message. The different isozymes present in single embryos were separated by electrophoresis. The results show that the oocyte-coded glucose phosphate isomerase is gradually replaced by embryo-coded enzyme. Expression of the paternally derived allele was first detected at the morula stage, during which the translation of the maternally derived message seemed to be either exhausted or below the detection limit of our system. Some oocyte-coded enzyme persisted until after implantation.

Cavitation in the mouse preimplantation embryo: Na/K-ATPase and the origin of nascent blastocoele fluid.

This study tested the proposition that Na/K-ATPase activity could be involved in the morphogenetic aspects of mouse blastocyst formation by facilitating the localization of certain organelles to apposed borders, the production of nascent blastocoele fluid, and cavitation. It was assumed that such Na/K-ATPase activity would be sensitive to varying concentrations of external K (Ko)--which would alter plasma membrane potentials--and to ouabain--which would directly alter Na/K-ATPase function. Morulae were cultured for 40 hr in varying concentrations of Ko and/or ouabain and were observed for their ability to form blastocoeles (cavitate) and to localize mitochondria to apposed cell borders. Cavitation was accelerated when Ko was decreased from the control value of 6.0 to 0.6 mM and was delayed when Ko was increased to 25 mM. With Ko at 6.0 mM, 10(-5) M ouabain accelerated cavitation while 10(-4) M ouabain delayed cavitation and reduced the total number of embryos that cavitated by the end of the 40-hr culture period. With Ko at 0.6 mM, 10(-5) M ouabain now delayed cavitation while 10(-4) M ouabain almost completely inhibited it. When Ko was increased to 25 mM, 10(-5) M ouabain again accelerated cavitation while 10(-4) M ouabain delayed-rather than inhibited--cavitation. Morphometric analyses at the electron microscopic level showed changes in the distances of mitochondria from apposed cell borders with conditions that accelerated or delayed cavitation and these changes differed for inside and outside cells of the morula. These observations are consistent with the proposition that Na/K-ATPase activity could be involved in the localization of organelles to apposed cell borders, the production of nascent blastocoele fluid, and in cavitation during mouse blastocyst development.

Immunological detection of cell surface galactosyltransferase in preimplantation mouse embryos.

Presence of cell surface galactosyltransferase was surveyed in preimplantation mouse embryos by indirect immunofluorescence staining using an affinity-purified antibody against galactosyltransferase from human milk. Distinct fluorescence staining was observed in embryos ranging from late 8-cell stage to early blastocysts, while the embryos at other stages were stained only weakly. The cell surface enzyme was also present in F9 embryonal carcinoma cells, in a fraction of bone marrow cells of the mouse, and in a few percent of testicular sperm.

Adenosinetriphosphatase (Mg-ATPase) activity in the plasma membrane of preimplantation mouse embryo as revealed by electron microscopy.

The ultrastructural localization of Mg2+-ATPase activity was investigated in unfertilized eggs and in preimplantation mouse embryos. Enzyme activity was detected on the entire surface of unfertilized eggs and in one-cell embryos. From the two-cell stage up to the 12-cell embryo the reaction product was localized both at the exposed surface of the embryo and between blastomeres. At the late morula stage, activity was only present on the free surface of the peripheral cells outside the apical tight junctions. In the early blastocyst the membranar reaction was observed both on the outside membrane of the trophoblastic cells and on the inner surface facing the blastocoele, whereas in the late blastocyst the reaction remained unchanged on the outer surface of the embryo but was absent from the membranes bordering the blastocoele. These results are discussed with reference to the modification of the cell surface membrane during preimplantation development.