Lactate dehydrogenase in preimplantation rabbit embryos

Unfertilized eggs and early embryos up to the 2-day (16-cell) cleavage stage of development in the rabbit contain predominantly the most cathodal lactate dehydrogenase isoenzyme made up of A-type subunits. Following early cleavage there is a progressing increase in total LDH activity in the embryo as development proceeds through 4- and 6-day blastocyst stages. This is accompanied by an increase in the amount of B-type subunits and a concomitant shift in the lactate dehydrogenase isoenzyme electrophoretic pattern toward the anodal isoenzyme types.     

Lactate dehydrogenase isoenzymatic analysis of murine lymphocyte populations: a parameter of cellular differentiation.

The lactate dehydrogenase isoenzyme pattern has been determined in different murine lymphocytic cell populations. In each cell population, the LDH activity was predominantly found in the LDH-4 and LDH-5 fractions. The percentage LDH-5 activity was significantly higher in B cells than in T cells. The same is true for lymphocytes from the spleen versus lymph node lymphocytes. The percentage LDH-5 activity is significantly higher in peripheral T lymphocytes than in thymocytes. Enrichment of the more mature thymocytes of the thymocyte cell pool by either cortisone treatment in vivo or gradient centrifugation on bovine serum albumin (BSA) results in a decrease of LDH-1 and LDH-2 fractions. In the cortisone-treated group, the shift in the LDH pattern is accompanied by a significant increase of LDH-5 and LDH-4 fractions, whereas in the BSA group only the LDH-4 fraction increases.     

Lactate dehydrogenase isozymes in the preimplantation rabbit embryo

The rabbit zygote on day 1 of development contains predominantly A-type subunits of lactate dehydrogenase. During cleavage, the isozyme pattern shows a considerable increase in the amount of B subunits present. These findings indicate that the synthesis and metabolism of lactate dehydrogenase in the rabbit embryo are markedly different from those of the mouse embryo.This research was supported by the National Institute of Child Health and Human Development Grant 03071.     

Intermediate filament protein synthesis in preimplantation murine embryos

The synthesis of two extraembryonic endodermal cytoskeletal proteins (Endo B, Mr = 50,000; Endo A, Mr = 55,000) was detected by immunoprecipitation at the 4- to 8-cell stage of preimplantation mouse development. The first detectable synthesis of both proteins occurs at about the same time as the earliest allocation of cells to the trophectodermal lineage. Both Endo A and B were identified in the two-dimensional gel pattern of blastocyst cytoskeletal proteins prepared by nonionic detergent and high-salt extraction. Endo A and B were identified as the y and x blastocyst cytoskeletal proteins, respectively, previously described by other investigators. Antibodies to Endo B are shown to react with intermediate filaments at the electron microscopic level, confirming that Endo B is an authentic intermediate filament protein. Previously, the TROMA 1 monoclonal antibody prepared by other investigators was shown to react specifically with Endo A and to decorate trophoblast cytoskeletons but did not react with the inner cell mass of blastocysts. Endo B antibodies are now also shown to decorate trophoblast cytoskeletons.     

Estradiol-17beta-hydroxysteroid dehydrogenase activity in preimplantation rat embryos

In previous studies (1–3), we have shown that Δ⁵ -3β-hydroxysteroid dehydrogenase (3β-HSD) activity in rat embryos begins on Day 4 of pregnancy (Day 1 = day of finding spermatozoa in the vagina), it peaks on Day 5, and sharply declines on Day 6. The present study investigated the presence of estradiol-17β-hydroxysteroid dehydrogenase (17β-HSD) in rat embryos recovered on Days 4, 5 and 6. The pattern of the 17β-HSD activity was similar to that of 3β-HSD. Thus, the present results strengthen our previous contention that rat morulae and blastocysts synthesize steroid hormones; moreover, the results suggest that one of the hormones synthesized is estrogen.     

Possible function of 17 beta-hydroxysteroid dehydrogenase in preimplantation hamster embryos

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) catalyzes the interconversion of estradiol-17 beta (E2) and estrone (E1). The present study is designed to investigate the following: (1) the developmental stage of hamster embryos at which 17 beta-HSD activity first becomes detectable, and (2) the E1----E2 and E2----E1 conversion rate in the preimplantation hamster embryo. Embryos obtained from superovulated hamsters on days 1-4 were cultured in medium containing 107 ng [3H]E1 or -E2/ml and the respective conversion product, [3H]E2 or -E1, was isolated and assayed. The results show that (1) E1----E2 conversion was active in all embryos at the rate of 0.57, 0.66, 0.54 and 0.48 fmol/embryo/hr for day 1 (one-cell), 2 (two-cell), 3 (eight-cell) and 4 (blastocyst), respectively, and (2) E2----E1 conversion was not detectable in hamster embryos. In long-term blastocyst culture, E2----E1 conversion becomes detectable at 25 hours and increases sharply from 25 to 47 hours. These results suggest that (1) 17 beta-HSD may function mainly to convert E1 into E2 in preimplantation hamster embryos and (2) E2----E1 conversion may become active only during and after implantation.     

Changing 17 beta-hydroxysteroid dehydrogenase activity in preimplantation rat and mouse embryos

The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity in rat and mouse preimplantation embryos was determined by measuring the interconversion of estradiol (E2) and estrone (E1). Rat and mouse embryos were cultured in medium containing 450 nM [3H]E1 or -E2 and the amount of [3H]E1 and -E2 in the medium at the end of the first hour was determined. The results showed that in both species 17 beta-HSD activity was detectable from the one-cell stage (Day 1) onward. In the rat, 17 beta-HSD effected primarily E2----E1 conversion, with the activity decreasing from Day 1 to Day 5. In the mouse, we found primarily E1----E2 conversion from Day 1 to the morning of Day 4, then E2----E1 increased sharply to near the E1----E2 rate in the evening of Day 4 and surpassed the E1----E2 rate the next morning. It seems that: 1) 17 beta-HSD is active throughout the entire preimplantation period, and 2) the enzyme activity changes during preimplantation development. Thus, the rat and mouse preimplantation embryo could regulate the E1- to -E2 ratio in the embryos and in their environment.     

Delta5-3beta-hydroxysteroid dehydrogenase and estradiol-17beta-hydroxysteroid dehydrogenase activity in preimplantation hamster embryos

Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. Δ⁵-3β-Hydroxysteroid dehydrogenase (3β-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00–09:00 hr on day 3, the activity then increased, peaked at 01:00–03:00 hr on day 4, considerably declined by 08:00–09:00 hr (day 4), and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, Dickmann and Dey, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster.In addition to 3βHSD, estradiol-17β-hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen.     

Hyperglycemia-induced apoptosis affects sex ratio of bovine and murine preimplantation embryos

The effect of glucose in the medium used during in vitro culture on both cell death by apoptosis and the sex ratio of bovine blastocysts derived from in vitro-matured and in vitro-fertilized oocytes was evaluated. Oocytes were matured, inseminated, and cultured in vitro in mSOF medium with 10% FCS with or without glucose supplementation. Exposure to high concentrations of glucose (10, 20, and 30 mM) during bovine embryo development in vitro from zygote to blastocyst resulted in a decrease in the number of cells per embryo and an increase in the frequency of apoptotic cells. A significantly higher proportion of females was found among those embryos that developed under hyperglycemic conditions in vitro. Moreover, both murine and bovine blastocysts incubated for 6 hr in 20 mM glucose had a significantly higher number of apoptotic cells in comparison to control. In this study, we also determined whether blastocyst production of the X-linked inhibitor of apoptosis protein (XIAP) differs between the sexes. Our results show that female bovine blastocysts produce significantly higher amounts of XIAP mRNA than males and this could be crucial in explaining the higher proportion of female blastocysts observed following in vitro culture under hyperglycemic conditions which induce apoptosis. Moreover, a higher proportion of female murine blastocysts cultured under hyperglycemic conditions were implanted in the uterus (65.3 of implantations from embryos cultured with 20 mM of glucose are females vs. 49% in control). This mechanism provides an explanation for the significant reduction of male children born to diabetic mothers.     

Lactate dehydrogenase binding to the mitochondrial fraction and to a mitochondrial inhibitor as a function of the isoenzymatic composition

Purified rabbit skeletal muscle LDH M4 isoenzyme, but not H4 isoenzyme, was observed to bind to either the crude mitochondrial fraction or a mitochondrial inhibitor. Several sources of LDH isoenzymes in which M-type subunits with an alkaline pI are predominant bind to this crude mitochondrial fraction and are inhibited by the mitochondrial inhibitor. Binding and inhibition have also been observed with H-type isoenzymes with a pI near 7. The binding and the inhibition processes did not occur with H-type isoenzymes with an acid pI or with M-type isoenzymes with pI near 6. The binding capacity of LDH to the mitochondrial fraction and to the mitochondrial inhibitor is very similar and depends on the net protein charge and not on whether the subunits are H- or M-type.     

Lactate dehydrogenase isoenzyme spectrum in spontaneous mouse mammary tumors and lung neoplasms]

The report deals with the LDH isoenzymatic spectrum in the mannary glands, lung, spontaneous tumours of the mammary glands and in metastases into the lung in mature female C3H mice. The difference in the metastases and the tumour LDH isoenzymatic spectrum was in the higher activity of the anode fraction of metastases. The presence of lung metastases was accompanied by a cathode drift of the LDH isoenzymatic spectrum to this organ tissue.     

Metabolism in preimplantation mouse embryos

The ability of preimplantation mouse embryos to utilize glucose oxidatively is controlled, in part at least, at the level of glycolysis. Various experimental observations are reviewed that indicate the regulatory mechanism in delayed implanting blastocysts involves the classic negative allosteric feedback of high levels of ATP on phosphofructokinase while the situation in 2-cell embryos appears to be more complicated. That is, in addition to the usual negative effect of ATP and citrate on phosphofructokinase, there appears to be a modification of hexokinase that prevents phosphorylation of adequate amounts of glucose and results in low levels of fructose-6-phosphate at the 2-cell stage and consequently there is a failure to release the inhibition of phosphofructokinase even if ATP and citrate levels decrease. Although both types of embryos have limited glycolytic activity, they do have adequate capacity for citric acid cycle activity and oxidative phosphorylation, and are able to maintain a high ATP : ADP. It is argued, therefore, that the reduced levels of macromolecular synthesis characteristic of 2-cell and delayed implanting blastocysts are not due to restricted energy substrates or regulatory controls on glycolysis and a subsequent low energy state. On the contrary, it seems that the reduction in oxidative utilization of glucose in these situations is a result of diminished energy demand because of the low level of synthetic activity. The potential significance of this relationship between energy production and utilization in terms of potential regulatory mechanisms in preimplantation embryos is discussed.