Endometrial Protein Synthesis Research Articles

Recombinant bovine interferon-τ enhances in vitro development of bovine embryos by upregulating expression of connexin 43 and E-cadherin

Interferon-τ (IFNT), produced in ruminants by embryonic trophoblastic cells before implantation, is involved in the maternal recognition of pregnancy. It is a pleiotropic molecule that alters the synthesis of endometrial proteins and inhibits the proliferation of some cells. The present study investigated the effects of recombinant bovine IFNT on the development of early-stage bovine embryos and the molecular mechanism underlying this effect. This study demonstrated that expression of mRNA encoding type I IFN receptor subunits was detectable from d 4 to 8 in in vitro fertilized (IVF) bovine embryos. A considerable number of IVF (n=1,941) and parthenogenetic activated (n=1,552) bovine embryos demonstrated that supplementing the culture medium with IFNT (100ng/mL) produced a greater percentage of blastocysts, and the total cell number within the resulting blastocysts was higher. In addition, IFNT upregulated the expression levels of both mRNA and protein for connexin 43 (GJA1) and E-cadherin (CDH1) and expression levels for granulocyte-macrophage colony-stimulating factor and insulin-like growth factor 2 mRNA but not for their proteins in d 8 embryos. However, IFNT inhibited mRNA expression for leukemia inhibitory factor (LIF), LIF receptor α, and the sodium/potassium-transporting ATPase subunit β-1. We concluded that IFNT promoted the development of bovine embryos by upregulating the expression of GJA1 and CDH1. Thus, supplementing embryo cultures or transfer medium with IFNT may stimulate embryo development and improve embryo transfer efficiency.

Progesterone-modulated induction of apoptosis by interferon-tau in cultured epithelial cells of bovine endometrium.

Interferon-tau (IFN-tau) is produced by the trophoblast prior to implantation in ruminants. It is involved in maternal recognition of pregnancy, and is a pleiotropic molecule that can alter the synthesis of endometrial proteins and inhibit proliferation of some cells. We have observed that IFN-tau reduces the DNA content in cultures of bovine endometrial epithelial cells; therefore, the objective of this study was to determine whether IFN-tau would induce apoptosis in bovine endometrial cells. Epithelial cells were prepared, cultured to confluence, and then incubated for 24 or 48 h in the presence or absence of 10 ng/ml progesterone, 100 ng/ml IFN-tau, or 10 microg/ml cycloheximide (CHX; an apoptosis inducer used as a positive control). Cells undergoing apoptosis exhibit such characteristics as the appearance of apoptotic bodies and DNA fragmentation. The incidence of apoptosis was assessed by using TUNEL, DNA fragmentation analysis, and Western blot analysis of Bax-alpha protein expression. The results showed that IFN-tau and CHX significantly increased the percentage of cells with apoptotic nuclei (33.6% and 44.8%, respectively) compared with controls (11.7%; P < 0.05). Progesterone treatment of the cells significantly inhibited the ability of IFN-tau to induce apoptosis (14.6%) compared with IFN-tau alone (33.6%; P < 0.05). DNA fragmentation analysis showed that INF-tau and CHX treatment resulted in an increase in the appearance of DNA laddering compared with that in untreated control cultures. Western blot analysis showed that IFN-tau and CHX treatment resulted in a greater expression of the proapoptotic protein Bax-alpha compared with that in control cultures. These data demonstrate that IFN-tau can induce apoptosis in bovine uterine epithelial cells and that this effect is modulated by progesterone. We speculate that IFN-tau might play a critical role in the remodeling of the endometrium around the time of implantation.

Conceptus-endometrial interactions and reproductive hormone profiles during embryonic diapause and reactivation of the blastocyst in the European roe deer (Capreolus capreolus)

&lt;p&gt;Abstract: Roe deer blastocysts exhibit obligate embryonic diapause between early August and late December. The blastocyst then expands and elongates rapidly before implantation. The objective of this study was to ascertain the cues for reactivation of the diapausing blastocyst. Blood samples and reproductive tracts were collected from roe does during diapause, blastocyst expansion and subsequent implantation. Peripheral concentrations of oestradiol-17/3, progesterone and prolactin were measured by radioimmunoassay. Luteal progesterone release was determined following in vitro incubation. Conceptuses and endometrial tissue were cultured with 3H-leucine for 24 hours to measure de novo synthesis of secretory proteins. Endometrial secretory proteins were separated by two-dimensional electrophoresis. Results showed that peripheral progesterone concentrations declined by 55% just prior to expansion and did not rise until a 3-fold increase after implantation. Luteal progesterone release remained constant until expansion when it declined by 50% before increasing 2-fold at elongation and implantation. Concentrations of oestradiol-17/3 remained at a consistently low level during diapause and expansion until a 30-fold increase at elongation with concentrations remaining elevated after implantation. Plasma prolactin levels remained at basal concentrations during late diapause and then increased marginally at reactivation before decreasing again at elongation and implantation. Incorporation of radiolabel into both conceptus and endometrial secretory proteins was low during diapause, but incorporation in the conceptus increased 4&amp;not;fold at expansion and by 24-fold at the expanded trophoblast stage. Incorporation into endometrial secretoty proteins remained constant until the expanded trophoblast stage and implantation when a 2-fold increase was recorded. Furthermore, the profile of endometrial secretory proteins was constant during diapause and expansion but changed qualitatively following implantation. These data indicate that both endometrial protein synthesis and sectetion did not change during late diapause and early expansion. The increase in conceptus protein synthesis not only precedes that of the endometrium but consistently low luteal progesterone release, peripheral progesterone and oestradioi-17/J concentrations at early expansion suggests that reactivation is not in response to a maternal uterine trigger.&lt;/p&gt;

Effects of guinea-pig endometrial proteins on phospholipase A 2 activity and prostaglandin synthesis

The major protein secreted by Day-15 guinea-pig endometrium cultured for 24 h had a molecular weight of 68.3 kDa on SDS-PAGE. This protein had no effects on phospholipase (PL) A 2 activity in vitro and prostaglandin (PG) production by Day-7 guinea-pig endometrium in culture. Following purification, proteins present in fractions F1, F2, F7, F7:2 and F7:3 (which comprised <15% of the total amount protein secreted) increased PLA 2 activity in vitro. The major proteins in F7:2 had molecular weights of 13.9 and 15.6 kDa on SDS-PAGE, but they had no effect on endometrial PG synthesis in the concentration used (20 mg/ml). Unpurified endometrial secreted proteins had no effect on PLA 2 activity, but stimulated endometrial PG synthesis. This stimulation was lost following purification of the proteins, and may have been due to a large amount of contaminating serum albumin. The mechanism by which oestradiol acting on a progesterone-primed guinea-pig uterus stimulates endometrial PGF 2α synthesis by a process which is dependent upon increased endometrial protein synthesis still remains obscure.

Hormonal control of proteins synthesized and secreted by guinea-pig endometrium.

The amounts of protein synthesized and secreted (as indicated by [3H]leucine incorporation) by guinea-pig endometrium cultured for 24 h increased 2.1-fold between days 7 and 15 of the oestrous cycle. This increase did not occur if the guinea-pigs were pregnant. In ovariectomized guinea-pigs, oestradiol acting on a progesterone-primed uterus was the optimum stimulus for the maximum increase in endometrial protein synthesis and secretion. The two main proteins synthesized and secreted by day-15 guinea-pig endometrium had molecular masses of 99.8 and 192.1 kDa as determined on Sephacryl S-200HR. The production of the 99.8 kDa protein increased 5.2-fold between days 7 and 15 of the cycle. The 192.1 kDa protein was not detected in secretions produced by day 7 endometrium, and the 99.8 and 192.1 kDa proteins were not detected in secretions produced by day-15 pregnant endometrium. In ovariectomized guinea-pigs, progesterone did not stimulate the synthesis of secreted proteins of molecular masses above 77 kDa. Oestradiol stimulated the synthesis of secreted proteins with molecular masses of 87.8 and 192.1 kDa. However, oestradiol acting on a progesterone-primed uterus stimulated the synthesis of the secreted proteins with molecular masses of 99.8 and 192.1 kDa, which indicates that this combination of steroid hormones may be responsible for the increase in production of these two proteins by the day-15, nonpregnant guinea-pig endometrium. This stimulation by ovarian steroids of the synthesis and secretion of the 99.8 and 192.1 kDa proteins by the guinea-pig endometrium is apparently inhibited during early pregnancy.

Endometrial proteins: a reappraisal.

Uterine factors influence reproduction at the macro-anatomy level, and the effects of hormonal steroids on endometrial morphology are well recognized in the histopathological diagnosis of dysfunctional bleeding and infertility. During the past decade, attention has been paid to endometrial protein synthesis and secretion with respect to endocrine stimuli and implantation, and to the paracrine/autocrine effects of endometrial peptide growth factors, their binding proteins and other factors. The emphasis of this presentation is on protein secretion of the secretory endometrium, in which progesterone plays a pivotal role. Insulin-like growth factors have receptors on the endometrium, and IGF-binding proteins, stimulated by progesterone, modulate the effects of IGFs locally. Also other protein products of the secretory endometrium have been reviewed in this communication, with special emphasis on studies of a progesterone-associated endometrial protein which has many names in the literature, such as PEP, PP14, alpha 2-PEG and AUP. Extensive studies are ongoing in many laboratories to elucidate the regulation, function, interplay at tissue and cellular levels, and clinical significance of these proteins.

The effects of cholera toxin, pertussis toxin, sodium fluoride and -interferon on prostaglandin production by the guinea-pig endometrium

The outputs of prostaglandin (PG) F-2 alpha, 6-keto-PGF-1 alpha and PGE-2 from Day-7 and Day-15 guinea-pig endometrium were neither stimulated nor inhibited by cholera toxin and pertussis toxin. This indicates that PG synthesis by guinea-pig endometrium is not controlled by toxin-sensitive G-proteins. Short-term treatment of guinea-pig endometrium in culture with sodium fluoride stimulated PG output, suggesting that endometrial PG synthesis may be regulated by a fluoride-sensitive G-protein. Long-term treatment of guinea-pig endometrium in culture with sodium fluoride inhibited endometrial PG synthesis, and this was due to an inhibition of endometrial protein synthesis. Human alpha-interferon had no inhibitory effect on the outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and PGE-2 from Day-15 guinea-pig endometrium in culture. It appears that the anti-luteolytic factor secreted by guinea-pig conceptus is not an alpha-interferon and is therefore probably different from ovine trophoblast protein-1.

Is protein synthesis necessary for prostaglandin production by guinea-pig endometrium?

The outputs of prostaglandin (PG) F-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from Day-7 and Day-15 guinea-pig endometrium in culture were reduced by the inclusion of actinomycin D, cycloheximide and puromycin in the culture medium, with the output of PGF-2 alpha from Day-15 endometrium being particularly affected during the first 6 h of culture. The intrauterine administration of actinomycin D on Day 10 decreased the outputs of PGF-2 alpha and PGE-2, but not of 6-keto-PGF-1 alpha, from Day-15 endometrium in culture without affecting PG output from Day-15 myometrium in culture. Actinomycin D, cycloheximide and puromycin did not reduce PG output when superfused over the Day-7 and Day-15 guinea-pig uterus in vitro for 20 min, indicating that these compounds do not have a rapid inhibitory effect on endometrial PG synthesis. In fact, they tended to stimulate PG output during this 20-min period, with cycloheximide having a pronounced effect on PGE-2 output. The synthesis of secreted proteins, but not of cellular proteins, was greater by Day-15 than by Day-7 endometrium in culture. Actinomycin D, cycloheximide and puromycin inhibited the synthesis of secreted and cellular proteins by Day-7 and Day-15 endometrium in culture. Protein synthesis and PG synthesis in the endometrium were both inhibited to a greater extent by cycloheximide and puromycin than by actinomycin D. The intrauterine administration of actinomycin D on Day 10 reduced the syntheses of secreted and cellular proteins by Day-15 endometrium in culture. These findings indicate that the endometrial synthesis of PGs, particularly of PGF-2 alpha towards the end of the oestrous cycle, is dependent upon endometrial protein synthesis.

Embryo-uterine interactions during early stages of pregnancy in domestic mammals.

The first part of this paper presents data concerning our knowledge of uterine proteins during early pregnancy in domestic mammals; the second part gives results of in vitro biochemical studies on embryo-uterine interactions in the ewe. We have developed an in vitro technique of the co-culture of ovine uterine epithelial cells with the blastocyst or its secretory proteins. The effects of a specific trophoblastic protein (oTPB), involved in the maintenance of the corpus luteum, have been particularly studied by this system. The modifications of endometrial protein synthesis have been measured by incorporation of radiolabelled amino acids and analysed by electrophoresis (SDS-PAGE or bidimensional). The results show that the presence of the blastocyst, or of its total secretory proteins or oTPB alone, decreased overall protein synthesis by the endometrial cells. The nature of the secreted proteins was apparently not affected by the blastocyst, but the addition of oTPB alone increased the production of 3 polypeptides (MW = 150.10(3); 74.10(3); 50.10(3) and pI = 7-8.2; 5.4-5.2; 6.4, respectively) and decreased the synthesis of 2 others (MW = 57.10(3); 35.10(3) and pI = 7-6.7; 5.3, respectively). We also studied the effects of co-culture with uterine cells on blastocyst DNA and protein synthesis. In no cases we obtained stimulation of blastocyst development during the co-culture period, and DNA or protein synthesis decreased in the presence of uterine cells. In conclusion, the presence of specific uterine proteins has been established in some domestic mammals (pig, rabbit) but not in all of them. Although local modifications of uterine protein synthesis are induced by the embryo or its secretory products, the nature and the role of the proteins which are affected need to be determined by further studies.

Interference of trophoblastin in ruminant embryonic mortality. A review

The endocrine factors involved in the mechanisms of pregnancy recognition, and particularly those leading to the maintenance of the progesterone environment necessary for embryonic survival, have been reviewed. An antiluteolytic protein, trophoblastin, inhibits luteolysis during early pregnancy. Some cases of embryonic mortality might result from an insufficient secretion of this embryonic signal. Trophoblastin or oTP1 (ovine trophoblastic protein 1) is secreted by the trophectoderm. It has only been demonstrated in sheep and cattle. Its presence is necessary during a short critical period (from day 12 to 22 in sheep, 16 to 24 in cattle). Daily intra-uterine injections of 14–16-day-old ruminant embryo homogenates into ewes or heifers from Day 12 of the oestrous cycle maintained progesterone secretion. Removal of 25-day-old embryos provoked the maintenance of luteal activity for several weeks or months. This persistence of corpus luteum can explain some erroneous pregnancy diagnoses based on progesterone determination. Injected into the uterine lumen, purified trophoblastin inhibited cyclic luteolysis. This 20 Kdal protein bound to its endometrial receptors which appeared during the oestrous cycle. In vitro, it stimulated the synthesis of endometrial proteins. The different hypotheses concerning the mechanisms of trophoblastin action, which might be involved in embryonic mortality, are thoroughly discussed.

Effects of superovulation, arachidonic acid or endometrium on release of prostaglandins and synthesis of proteins by bovine trophoblast

This study was conducted in vitro to examine factors that may regulate prostaglandin release by bovine trophoblast and endometrial slices. Trophoblastic tissues and endometrial slices were recovered from superovulating and normally-ovulating cattle on day 16 or 20 of pregnancy and incubated for 24 h. Release of PGF 2 α and 13,14-dihydro-15-keto-PGF 2 α (PGMF), and incorporation of [ 14C]-leucine into proteins were quantified and expressed per μg DNA, which gives a measure of cellular activity. Activity of trophoblastic tissue for synthesizing protein was decreased (P<.05) and for releasing PGMF was increased (P<.05) on day 20 compared to day 16 of pregnancy. Neither supercovulation nor day of pregnancy altered trophoblastic activity for releasing PGF 2 α . Supercovulation increased (P<.05) endometrial release of PGF 2 α . Endometrial release of PGF 2 α was less (P<.05) on day 20 than on day 16 of pregnancy. When arachidonic acid (0, 100, 200 or 400 μg) was added at the start of incubation, trophoblastic release of PGF 2 α changed (P<.05) quadratically with dose of arachidonic acid. When arachidonic acid was added 8 h after the start of incubation, triphoblastic release of PGF 2 α increased linearly (P<.01) with dose of arachidonic acid. Adding arachidonic acid to incubation medium did not affect trophoblastic or endometrial protein synthesis. Endometrial slices suppressed (P<.05) trophoblastic protein synthesis and release of PGF 2 α . Apparently, endometrium can modulate trophoblastic release of prostaglandins and synthesis of proteins in vitro, and trophoblastic tissue from supercovulated cattle 16 or 20 days pregnant can be used to study trophoblastic synthesis of prostaglandins and proteins.

Electrophoretic analyses of secreted human endometrial proteins: identification and characterization of luteal phase prolactin.

Endometrial protein synthesis and secretion throughout the menstrual cycle was studied by slab gel electrophoretic analysis of [35S]methionine incorporation into protein during short term culture of human endometrial tissue. A minimum of five protein bands that fluctuate during the menstrual cycle were identified on one-dimensional gels. Those with mol wt of 28K, 35K, 51K, and 59K decreased in the luteal phase, whereas a broad 25K band was induced in the luteal phase. This broad band was identified as two species of glycosylated PRL by antihuman PRL immunostaining and [3H]glucosamine incorporation.

Changes in the pattern of endometrial protein synthesis during decidualization in the rat.

Ovariectomized rats were given hormonal treatment mimicking progestational ovarian secretions. At maximal sensitivity a decidual reaction was induced by scratching the endometrium. After incubation of tissue with [35S]methionine, proteins were extracted from decidualizing and control endometrium and submitted to two-dimensional polyacrylamide gel electrophoresis (pH range 5--7), followed by staining or autoradiography. A total of about 800 different peptides could be distinguished on the gels. By 24 h after the decidual stimulus the most prominent changes were the appearance of 4 new peptides (mol. wt 12 000, 25 000, 42 000 and 56 000) and the disappearance of 5 others (mol. wt 31 000, 35 000, 100 000, 110 000 and 140 000). This new pattern remained grossly unchanged up to 72 h after decidual induction.

Uterine specific antigens in the ewe

Uterine flushings from ewes on days 0, 3, 6, 9, 12 and 15 of the estrous cycle were analyzed for total protein content. Flushings from days 9, 12 and 15 had greater (P<.01) amounts of protein than those from 0, 3 and 6. Antisera to uterine fluids from ewes at day 10 to 12 or day 14 to 15 of pregnancy detected two uterine-specific antigens in uterine flushings at day 7, 11 and 15 but not at days 0 and 3 of the cycle. A third uterine antigen was also detected in kidney tissue extracts. All three antigens were present in endometrial extracts at each stage examined. Progesterone, or estrogen plus progesterone, administration to ovariectomized ewes induced the appearance of the two uterine-specific antigens. The third antigen was detectable even in ovariectomized ewes. No pregnancy-specific antigens were detected in flushings from days 7, 11 or 15 of gestation. The effect of pregnancy on endometrial protein synthesis was examined in vitro . No differences were seen in the incorporation of 3H-leucine in day 11 pregnant or nonpregnant or in day 14 pregnant or nonpregnant endometrium. No differences in total uterine lumenal protein were observed. Endometrial secretions, obtained by conditioning media with endometrial explant cultures, were evaluated to assess their effect on protein synthesis in day 11 embryos cultured in vitro . No significant effects of endometrial secretions or serum were observed.

Alterations in Human Endometrial Protein Synthesis During the Menstrual Cycle and in Progestone-Stimulated Organ Culture

The cytoplasmic protein content of endometrium from women at various times in the menstrual cycle has been examined by means of polyacrylamide gel electrophoresis using a double-label isotope technique. Analysis of the gels for altered label ratios suggests an increase in the concentration of a single protein band during the immediate postovulatory period that can be duplicated by the addition of progesterone to organ cultures of preovulatory endometrium. An alteration in the protein content is also observed in the same section of these gels when they are stained and scanned. The protein band of interest migrates on sodium dodecyl sulfate gels at a rate that establishes its approximate molecular weight as 51,500.