Expression of 17ß-hydroxysteroid dehydrogenase and the effects of LH, FSH and prolactin on oestrone and 17ß-oestradiol secretion in the endometrium of pigs during early pregnancy and the oestrous cycle.

The endometrium of pregnant and cyclic pigs is a source of oestrone (E1) and 17ß-oestradiol (E2). However, the roles of LH, FSH and prolactin (PRL) as regulators of endometrial steroidogenesis, and the presence of 17ß-hydroxysteroid dehydrogenase (17ß-HSD) in the porcine endometrium, remain unknown. Therefore, in the present study we examined 17ß-HSD expression and the effects of LH, FSH and PRL on E1 and E2 release in vitro in endometrial explants harvested from gravid pigs on Days 10-11 (embryo migration within the uterus), 12-13 (maternal recognition of pregnancy) and 15-16 (beginning of implantation) and compared them with results obtained in non-gravid pigs. The results show that: (1) endometrial 17ß-HSD activity was decreased on Days 15-16 in pregnant and cyclic pigs compared with the preceding days; (2) LH, FSH and PRL increased endometrial E1 secretion on Days 10-11 and 15-16 of pregnancy and on Days 12-13 and 15-16 of the oestrous cycle; and (3) LH, FSH and PRL increased endometrial E2 secretion on Days 15-16 of pregnancy and during the days studied in the oestrous cycle. In conclusion, data suggest that LH, FSH and PRL affect endometrial secretion of estrogens in pigs.

Periconceptional undernutrition affects in utero methyltransferase expression and steroid hormone concentrations in uterine flushings and blood plasma during the peri-implantation period in domestic pigs.

Female undernutrition during early pregnancy may affect the physiological pattern of genomic DNA methylation. We hypothesised that in utero DNA methylation may be impaired in females fed a restrictive diet in early pregnancy. In this study we evaluated whether poor maternal nutritional status, induced by applying a restricted diet during the peri-conceptional period, may influence: (1) the potential for in utero DNA methylation, expressed as changes in the mRNA expression and protein abundance of methyltransferases: DNA methyltransferase 1 (DNMT1) and DNMT3a in the endometrium and the myometrium, (2) the intrauterine microenvironment, measured as oestradiol 17ß (E2) and progesterone (P4) concentrations in uterine flushings and (3) plasma concentration of E2 and P4 during the peri-implantation period. Our results indicate that maternal peri-conceptional undernutrition affects maintenance and de novo DNA methylation in the endometrium, de novo methylation in the myometrium and a results in a decrease in intrauterine E2 concentration during the peri-implantation period. The intrauterine concentration of P4 and plasma concentrations of E2 and P4 did not change. These findings suggest that undernutrition during the earliest period of pregnancy, and perhaps the pre-pregnancy period, may create changes in epigenetic mechanisms in the uterus and intrauterine milieu of E2 during the peri-implantation period.

Genistein and daidzein affect in vitro steroidogenesis but not gene expression of steroidogenic enzymes in adrenals of pigs.

Phytoestrogens (PEs), including genistein and daidzein, are plant-derived substances that mimic or antagonize estrogen action in animals. The majority of studies investigated the effects of PEs on reproduction in humans and laboratory animals. The mechanisms of phytoestrogen action on reproductive processes in domesticated animals, including pigs, are garnering increasing attention. However, very few in vivo and in vitro studies investigating the effects of PEs on adrenal glands have been carried out on models other than humans and rats. The aim of the present study was to determine whether the effects of genistein and daidzein on adrenal in vitro steroidogenesis are accompanied by changes in expression of genes encoding key steroidogenic enzymes in porcine adrenocortical cells. The following genes were analyzed: cholesterol side-chain cleavage enzyme (P450scc, CYP11A1 gene), 3ß-hydroxysteroid dehydrogenase (3ß-HSD, HSD3B1 gene), 17α-hydroxylase/C17-20 lyase (P450c17, CYP17A1 gene) and 21-hydroxylase (P450c21, CYP21A2 gene). Porcine adrenocortical cells collected from both luteal- and follicular-phase gilts were exposed for eight hours to genistein (10 µM), or daidzein (10 µM), in the absence or presence of ACTH (5 nM). Genistein and daidzein inhibited basal and ACTH-stimulated secretion of cortisol and corticosterone and stimulated secretion of androstenedione. PEs did not affect the expression of CYP11A1, HSD3B1, CYP17A1 and CYP21A2 in the adrenocortical cells of luteal- and follicular-phase gilts. It can be concluded that the influence of PEs on steroid secretion in porcine adrenal glands is not mediated by changes in the expression of genes encoding major steroidogenic enzymes. More studies are needed to elucidate the intracellular mechanisms leading to the PE-induced changes in adrenal steroidogenesis in pigs.

The effect of interleukin-1ß, interleukin-6, and tumor necrosis factor-α on estradiol-17ß release in the myometrium: the in vitro study on the pig model.

Estradiol-17ß (E2) is a potent regulator of early pregnancy and the estrous cycle in pigs. Production of E2 occurs in the porcine myometrium, but the factors involved in its regulation are unknown. In this in vitro study, it was investigated whether interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α affect the release of E2 from the porcine myometrium on Days 10 to 11, 12 to 13, and 15 to 16 of pregnancy and the estrous cycle. The expression of the cytochrome P450 family 19 (CYP19) gene and the presence of the aromatase cytochrome P450 protein in the myometrium confirmed the ability of the tissue to produce E2. In gravid pigs, the expression of IL1RI mRNA and IL6R mRNA was markedly increased on Days 15 to 16 of gestation, whereas TNFRI mRNA was increased on Days 10 to 11 of gestation. In cyclic pigs, the expression of myometrial IL1RI mRNA did not differ among the studied days, although the expression of IL6R and TNFRI mRNAs was increased on Days 15 to 16. In gravid pigs, IL-1ß, IL-6, and TNF-α increased myometrial E2 secretion on Days 15 to 16 but did not affect E2 release on Days 10 to 11 and 12 to 13 of pregnancy. In cyclic pigs, IL-1ß, IL-6, and TNF-α did not increase myometrial E2 release. In conclusion, IL-1ß, IL-6, and TNF-α affected myometrial E2 release in a manner that is dependent on the physiologic status of the female. The porcine myometrium expresses IL1RI, IL6R, and TNFRI genes and is the target tissue for IL-1ß, IL-6, and TNF-α. In gravid pigs, IL-1ß, IL-6, and TNF-α may increase myometrial release of E2 in vitro specifically on Days 15 to 16 of pregnancy. These findings may be of interest to researchers using pigs as an animal model for fetal programming.

The effect of interleukin 1ß and interleukin 6 on estradiol-17ß secretion in the endometrium of pig during early pregnancy and the estrous cycle.

Estrogens are produced by porcine embryos during early pregnancy. It was found that the uterus of pigs might be a source of steroid hormones, including estrogens. However, the factors involved in the regulation of endometrial steroidogenesis remain unknown. We hypothesize that interleukin (IL) 1ß and IL6, which are cytokines produced by porcine embryos and uterine cells, might be involved in the regulation of endometrial estrogen synthesis. Porcine endometrial explants were harvested from gravid (N = 15) and cyclic (N = 15) pigs on days 10 to 11, 12 to 13, and 15 to 16. Samples were analyzed to determine: (1) the expression of CYP19 mRNA and the presence of aromatase cytochrome P450 protein in the tissue; and (2) the release of endometrial estradiol-17ß (E2) in response to IL1ß and IL6 after 6 and 12 hours of in vitro incubation. The effects observed in pregnant gilts were compared with the effects in nonpregnant gilts on corresponding days of the estrous cycle. On days 15 to 16 of pregnancy the expression of CYP19 in the endometrium was markedly decreased when compared with other periods, and the quantity of endometrial P450 aromatase protein was higher on these days than on days 12 to 13. In nongravid pigs, the expression of CYP19 was lower on days 15 to 16 when compared with days 12 to 13 and the quantity of P450 aromatase protein did not differ during the studied days of the estrous cycle. Basal endometrial E2 release was higher in pregnant gilts when compared with cyclic gilts only on days 15 to 16. In gravid pigs IL1ß and IL6 did not affect endometrial E2 release on days 10 to 11 and 12 to 13 of pregnancy (P > 0.05); however, increased E2 release was observed on days 15 to 16 (P < 0.05). In cyclic pigs neither IL1ß, nor IL6 affected endometrial E2 release (P > 0.05). These results provide evidence that: (1) the endometrium possesses a potential for steroidogenesis and produces E2in vitro in gravid and nongravid pigs between days 10 to 16; and (2) IL1ß and IL6 increase in vitro endometrial synthesis of E2 in pregnant pigs on days 15 to 16. Therefore, IL1ß and IL6 might act as stimulators of endometrial E2 secretion in vitro during the time of decreased production of embryonic estrogens. This correlates with a rapid remodeling of the endometrial tissue and the beginning of hemotrophic nutrition of the embryos occurring on days 15 to 16 of pregnancy.

The effect of tumor necrosis factor α (TNFα), interleukin 1ß (IL1ß) and interleukin 6 (IL6) on endometrial PGF2α synthesis, metabolism and release in early-pregnant pigs.

Cytokines produced by the porcine uterus and embryos may be involved in the regulation of endometrial prostaglandin synthesis, metabolism, and release. We studied the effect of tumor necrosis factor α (TNFα), interleukin 1ß (IL1ß) and interleukin 6 (IL6) on: 1) endometrial release of prostaglandin F2α (PGF2α), 2) expression of the terminal enzyme of PGF2α synthesis--PGF synthase mRNA (PGFS mRNA), 3) secretion of PGF(2)α metabolite--13,14-dihydro-15-keto PGF2α (PGFM) by the endometrium and 4) presence and activity of endometrial NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The effects of cytokines were determined on days 10-11 and days 12-13, e.g., before and during maternal recognition of pregnancy, and on days 15-16, e.g., during the peri-implantation period and compared with its effect in cyclic gilts on corresponding days of the estrous cycle. TNFα did not affect endometrial release of PGF2α in pregnant and cyclic pigs. IL1ß enhanced endometrial PGF2α release on days 12-13 and 15-16 in pregnant and cyclic pigs, respectively. IL6 increased PGF2α release mainly on days 15-16 of pregnancy. Expression of PGFS mRNA was decreased by IL1ß on days 12-13 of pregnancy (P<0.05) and increased in response to IL1ß, TNFα and IL6 on 12-13 (P<0.05) and 15-16 (P<0.01) of the estrous cycle. IL1ß increased release of PGFM in gravid pigs on days 12-13, 15-16 and in non-gravid pigs 10-11 and 15-16 of the cycle. On days 15-16 of pregnancy TNFα and IL6 increased endometrial secretion of PGFM. We determined that in porcine endometrium NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is present. In gravid pigs, the highest expression of endometrial 15-PGDH occurred during days 12-13 of pregnancy, while in non-gravid pigs during days 10-11 of the estrous cycle. These data provide new evidence that TNFα, IL1ß, IL6 are involved in the regulation of endometrial synthesis, release and metabolism of PGF2α to protect CL during early pregnancy or to facilitate its regression in cyclic females.

Interleukin 1ß-induced synthesis and secretion of prostaglandin E2 in the porcine uterus during various periods of pregnancy and the estrous cycle.

Peri-implantation porcine embryos express interleukin-1ß (IL-1ß), which could affect uterine activity during early pregnancy. In vitro studies were conducted to determine if IL-1ß stimulates secretion of PGE2 and expression of cyclooxygenase-2 (COX-2) mRNA and microsomal PGE synthase-1 (mPGES-1) mRNA in uterine tissues harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. IL-1ß (10 ng/ml) increased PGE2 secretion and mPGES-1 mRNA expression in uterine tissues isolated from pigs between days 10 to 13 of pregnancy and the estrous cycle. IL-1ß stimulated PGE2 and mPGES-1 mRNA expression only in cyclic uterine tissues on days 15 to 16. Interleukin-1ß increased COX-2 mRNA expression in the endometrial tissues of pregnant and cyclic pigs harvested on days 10 to 13. It stimulated COX-2 expression in pregnant pigs' myometrial tissues on days 10 to 11, and on days 15 to 16 in tissues from both pregnant and cyclic pigs. The uterine secretion of PGE2 in response to IL-1ß was determined by local intrauterine concentrations of P4 and E2. This study demonstrates that IL-1ß activates expression of mPGES-1 mRNA in uterine tissues to stimulate synthesis and secretion of PGE2 on days 10 to 13 of both pregnancy and the estrous cycle. The profile of COX-2 mRNA expression in the myometrium differs from its profile in the endometrium. This work provides new insight on the role of IL-1ß in PGE2 production to overcome luteolysis in pregnant pigs.