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.     

Changes in the 17 beta-hydroxysteroid dehydrogenase activity of mouse blastocysts during delayed implantation

The rate of estrone (E1)----estradiol-17 beta (E2) or E2----E1 conversion catalyzed by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was determined for each mouse embryo in modified F-10 medium containing 0.95 microM 3H-E1 or 3H-E2. During delayed implantation, the E1----E2 conversion rate was decreased (p less than 0.005) from 5.69 +/- 0.34 fmol/h/blastocyst on Day 5 to 3.50 +/- 0.46 fmol/h/blastocyst on Day 9, whereas E2----E1 was increased (p less than 0.005) from 7.44 +/- 1.08 to 18.60 +/- 2.04 fmol/h/blastocyst. After estrogen injection, the Day 9 implanting blastocyst showed an increase (p less than 0.005) in E1----E2 conversion to 9.05 +/- 0.64 fmol/h/blastocyst but a slight, insignificant decrease in E2----E1 conversion to 14.2 +/- 1.82 fmol/h/blastocyst. A similar trend was also observed in Day 5 implanting blastocysts when compared to Day 5 delayed blastocysts. Thus, 17 beta-HSD activity in delayed blastocysts favors E2----E1 over E1----E2 conversion in a ratio of 5:1. After estrogen induction of implantation, the E1----E2 conversion rate is stimulated and the ratio of E2----E1 to E1----E2 rate is decreased to 1.5:1. The results suggest that 17 beta-HSD activity may be involved in blastocyst implantation.     

Metabolism of progesterone by hamster blastocysts and the ontogeny of progesterone metabolic capability

Progesterone (P) is required for the differentiation of reproductive tracts and maintenance of pregnancy. This study investigates whether the hamster blastocyst is capable of metabolizing P and, if so, at what stage of preimplantation development such capability becomes detectable. When the blastocysts collected from superovulated hamsters on Day 4 of pregnancy were cultured in 0.4 microM P medium, P metabolism was easily detectable at 1.25 h of culture and over half was metabolized by 7.5 h. Two major metabolites were generated: 5 alpha-pregnane-3,20-dione (or 5 alpha-dihydroprogesterone; 5 alpha-DHP) and 5 alpha-pregnane-3 beta-ol-20-one (or allopregnanolone; AP), about 90-95% and 5-10%, respectively. This indicates the activity of two enzymes: delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The rate of P metabolism increased with P concentration (0.4-6.4 microM), indicating a high capacity of the enzymes. Studies of embryos collected on Days 1-3 showed that P metabolism was not detectable up to 0100 h of Day 3 (2-4-cell), but was detectable with two metabolites, 5 alpha-DHP and AP, at 1515 h of Day 3 (morula) and thereafter. This indicates that, by the morula stage, the hamster embryo has already acquired the enzymatic capability (5 alpha-reductase and 3 beta-HSD) to metabolize P. These results, together with our earlier finding of 17 beta-hydroxysteroid dehydrogenase activity in Days 1-4 embryos, suggest that hamster preimplantation embryos can metabolize both P and estrogens, thus possibly modulating local actions of these hormones and causing local effects in the reproductive tract.     

Metabolism of estrogens by rabbit blastocysts: formation of estrogen glucosides and preferential conversion of estrone to estradiol-17 beta

When day 6 rabbit blastocysts were cultured (3 embryos/mL) in medium 199 containing 3.68 microM estradiol-17 beta (E2), 40% of E2 was metabolized in 24 h, at a rate of 18 pmol/embryo(b)/h, yielding 4 major metabolite fractions. Two of them were identified to be estrogen glucosides: 17 beta-hydroxyestra-1,3,5(10)-trien-3-yl beta-D-glucopyranoside (E(2)3G) (12 pmol/b/h) and 17-oxoestra-1,3,5(10)-trien-3-yl beta-D-glucopyranoside (E(1)3G) (0.5 pmol/b/h). If the blastocysts were cultured in 3.68 microM E1 medium, 75% of E1 was metabolized in 24 h (34.1 pmol/b/h); most of it appears as E2 (8 pmol/b/h), E(1)3G (16 pmol/b/h), and E(2)3G (6 pmol/b/h). Thus, the 17 beta-hydroxysteroid dehydrogenase activity in the rabbit blastocysts catalyzes mainly in the direction of the E1----E2 conversion, with little or no E2----E1. This may be responsible in part for the faster metabolism of E1 than E2 by the rabbit blastocyst. In comparison with the rat, mouse, and hamster blastocyst, the rabbit embryo shows an additional capability to conjugate large amounts of estrogens into glucosides by steroid glucosyltransferase.     

Estradiol as an anti-aromatase agent in human breast cancer cells

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Uptake and incorporation of myo-inositol by bovine preimplantation embryos from two-cell to early blastocyst stages

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo preimplantation cattle embryos was investigated using [(3)H] myo-inositol. Uptake of inositol was examined in two-cell and four-cell embryos (day 2 after insemination), morulae (day 6) and early blastocysts (day 7). Uptake in all stages examined was largely sodium-dependent indicating the presence of a sodium-dependent inositol transporter. Uptake of inositol did not vary significantly from two-cell to early blastocyst stages when expressed either on a per embryo or a per microg of protein basis. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP, and PtdInsP(2), was detectable at all stages examined. In contrast, incorporation of inositol into inositol phosphates was not detected until blastocyst formation at day 7. The second messenger, Ins(1,4,5)P(3), was first detected in day 7 blastocysts.     

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.     

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.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Inhibition of endoplasmic reticulum stress improves mouse embryo development

X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.     

Fucosylated glycoconjugates in mouse preimplantation embryos

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.     

Estrogen sulfotransferase and 17 beta-hydroxysteroid dehydrogenase activities in guinea-pig chorion through gestation.

Estrogen sulfotransferase (EST) activity measured under optimal in vitro conditions in the 105,000 g cytosols (HSS) of homogenized intrauterine tissues (amnion, chorion, endometrium, decidua basalis and placenta) from guinea-pigs at the 50th day of gestation indicated that the highest specific activity occurred in the chorion. EST activity in the chorion increased from day 34 (early gestation) to peak around day 45 (mid-gestation), before significantly decreasing around day 50 and further declining to barely detectable levels beyond day 60 (late gestation, the onset of parturition). 17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) activity in the chorion was almost completely membrane associated. The specific activity of the 17 beta-HSD reduction reaction in the 105,000 g pellet was 2.5-fold higher at mid-gestation than at late gestation, while the specific activity of the 17 beta-HSD oxidation reaction was 1.7-fold higher at mid-gestation as compared with late gestation. When intact pieces of chorion tissue from mid- and late gestation were incubated with 5 nM [3H]estradiol (E2), approx. 80% of the recovered free estrogen was E1 (estrone). Only chorion from animals at the onset of parturition were able to produce detectable amounts of E2 from 5 nM [3H]E1. Under the same experimental conditions the ratio of estradiol sulfate (E2S) to estrone sulfate (E1S) isolated from the media and methanol washes of late gestation chorion tissue was 3-4 times greater than for the day 45 tissue.     

Bicarbonate/chloride exchange regulates intracellular pH of embryos but not oocytes of the hamster.

The ability to regulate intracellular pH (pH(i)) is essential for normal cell development and differentiation. This study was an investigation of the regulatory system used by the hamster oocyte and preimplantation embryo to regulate pH(i) in the alkaline range. Recovery from alkalosis by late 1-cell and 2-cell embryos was rapid, and physiological pH(i) levels could be restored within 10 min. Recovery from an induced alkaline load was dependent on the chloride concentration in the external medium and sensitive to a stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-di-sulfonic acid that inhibits bicarbonate and chloride exchange. Therefore the recovery from alkalosis by hamster embryos appears to be via activity of the HCO(3)(-)/Cl(-) exchanger that was activated above a pH(i) set point of 7.24. In contrast, hamster oocytes and early 1-cell embryos (collected 3-4 h post-egg activation) could not recover from an intracellular alkalosis, and pH(i) remained elevated. Therefore, the hamster oocyte and the early 1-cell embryo still undergoing pronuclear formation lack an active HCO(3)(-)/Cl(-) exchanger for the restoration of pH(i). Inability to restore pH(i) from an alkali challenge resulted in a reduced ability of embryos to develop to the morula/blastocyst stages in culture, indicating that HCO(3)(-)/Cl(-) exchange is involved in physiological regulation of pH(i).     

Skeletal muscle 11beta-HSD1 controls glucocorticoid-induced proteolysis and expression of E3 ubiquitin ligases atrogin-1 and MuRF-1

Recent studies demonstrated expression and activity of the intracellular cortisone-cortisol shuttle 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in skeletal muscle and inhibition of 11beta-HSD1 in muscle cells improved insulin sensitivity. Glucocorticoids induce muscle atrophy via increased expression of the E3 ubiquitin ligases Atrogin-1 (Muscle Atrophy F-box (MAFbx)) and MuRF-1 (Muscle RING-Finger-1). We hypothesized that 11beta-HSD1 controls glucocorticoid-induced expression of atrophy E3 ubiquitin ligases in skeletal muscle. Primary human myoblasts were generated from healthy volunteers. 11beta-HSD1-dependent protein degradation was analyzed by [(3)H]-tyrosine release assay. RT-PCR was used to determine mRNA expression of E3 ubiquitin ligases and 11beta-HSD1 activity was measured by conversion of radioactively labeled [(3)H]-cortisone to [(3)H]-cortisol separated by thin-layer chromatography. We here demonstrate that 11beta-HSD1 is expressed and biologically active in interconverting cortisone to active cortisol in murine skeletal muscle cells (C2C12) as well as in primary human myotubes. 11Beta-HSD1 expression increased during differentiation from myoblasts to mature myotubes (p < 0.01), suggesting a role of 11beta-HSD1 in skeletal muscle growth and differentiation. Treatment with cortisone increased protein degradation by about 20% (p < 0.001), which was paralleled by an elevation of Atrogin-1 and MuRF-1 mRNA expression (p < 0.01, respectively). Notably, pre-treatment with the 11beta-HSD1 inhibitor carbenoxolone (Cbx) completely abolished the effect of cortisone on protein degradation as well as on Atrogin-1 and MuRF-1 expression. In summary, our data suggest that 11beta-HSD1 controls glucocorticoid-induced protein degradation in human and murine skeletal muscle via regulation of the E3 ubiquitin ligases Atrogin-1 and MuRF-1.     

Interleukin-1 beta induces autophagy of mouse preimplantation embryos and improves blastocyst quality

Autophagy is one of the basic cellular mechanism during preimplantation development of mammalian embryos, and it plays crucial role in several physiological processes. It is induced by interleukin (IL)-1β in mammalian cells. Our present study shows that IL-1β is important for autophagy activation in embryo development. Our in vitro culture system analysis shows effect of IL-1β in medium on the development of mouse embryos and it was found to be concentration dependent. A preimplantation embryo culture using medium containing IL-1β did not improve cleavage and blastocyst development rates of mouse embryos; however, blastocyst quality was significantly improved by increasing total cell number, especially in supplementary 20 ng/mL IL-1β. Furthermore, autophagy activation mainly occurs in 2 to 4 cell embryo and blastocyst, 20 ng/mL IL-1β into culture medium can effectively enhance levels of messenger RNA and protein of autophagy-related-factors in 2 to 4 cell embryos and blastocyst, while these factors reduce in VGX-1027 (IL-1β inhibitor) groups that also reduce the quality of blastocyst. Effects of IL-1β on the development of embryo reduced in 20 ng/mL IL-1β supplemented group when 5 mM 3-methyladenine (3-MA) was also added, which used to inhibit autophagy activation in endogenous PtdIns3Ks signal pathway. Our current results show that exogenous IL-1β can effectively induce autophagy in mouse embryos at stages of 2 to 8 cell and blastocyst, that also help to improve the quality of blastocyst.