Bartosz Wojciechowicz

Transcriptomic analysis of the porcine endometrium during early pregnancy and the estrous cycle

The goal of this study was to describe the alterations in the transcriptome of the endometrium in pigs during the beginning of implantation (days 15-16 of pregnancy) compared to cyclic pigs during the onset of luteolysis (days 15-16 of the estrous cycle). The global expression of genes in porcine gravid and non-gravid endometria was investigated using the Porcine (V2) Two-color gene expression microarray, 4 × 44 (Agilent, USA). Analysis of the microarray data showed that, of 589 accurately annotated genes, the expression of 266 genes was up-regulated and expression of 323 was down-regulated in the endometrium harvested during early pregnancy compared with the endometrium during the estrous cycle. In pregnant pigs, genes with the most significantly altered expression were involved in the following biological processes: the metabolic process, cellular process, cell communication, immune system process, developmental process, cell adhesion, antigen processing and presentation, antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class II, immune response, and the polysaccharide metabolic process. In the pregnant endometrium, cell adhesion molecules and steroid hormone biosynthesis pathways were the most significantly enriched biological pathways. Analysis of the interaction network among selected genes showed that androgen receptor (AR) encoding genes interact with genes involved in important processes occurring during early pregnancy. The bioinformatic analysis revealed information about the meaning of differentially expressed genes. The data provided new insight into the dynamic changes of the endometrial gene expression profile during days 15-16 of pregnancy.

The effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) 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 1β (IL1β) 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. IL1β 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 IL1β on days 12-13 of pregnancy (P<0.05) and increased in response to IL1β, TNFα and IL6 on 12-13 (P<0.05) and 15-16 (P<0.01) of the estrous cycle. IL1β 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, IL1β, 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.

The activity and localization of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase and release of androstenedione and progesterone by uterine tissues during early pregnancy and the estrous cycle in pigs.

Abstract Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A4 and P4 by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A4 by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P4 did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P4 in pregnant pigs on days 12 to 13. The release of P4 by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.

The effect of interleukin 1β and interleukin 6 on estradiol-17β 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) 1β 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-17β (E2) in response to IL1β 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 IL1β 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 IL1β, 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) IL1β and IL6 increase in vitro endometrial synthesis of E2 in pregnant pigs on days 15 to 16. Therefore, IL1β 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.

Steroids in the equine oviduct: synthesis, local concentrations and receptor expression

Steroids play an important role in mammalian reproduction and early pregnancy. Although systemic changes in steroid concentrations have been well documented, it is not clear how these correlate with local steroid concentrations in the genital tract. We hypothesised that, in the horse, the preimplantation embryo may be subjected to high local steroid concentrations for several days. Therefore, we measured progesterone, 17-hydroxyprogesterone, 17?-oestradiol, testosterone and 17?-testosterone concentrations in equine oviductal tissue by ultra-HPLC coupled with tandem mass spectrometry, and progesterone, 17?-oestradiol, oestrone and testosterone concentrations in oviduct fluid by radioimmunoassay, with reference to cycle stage and side of ovulation. Progesterone concentrations were high in oviductal tissue and fluid ipsilateral to the ovulation side during dioestrus, whereas other steroid hormone concentrations were not influenced by the side of ovulation. These results suggest that the high ipsilateral progesterone concentration is caused by: (1) contributions from the follicular fluid in the oviduct and diffusion of follicular fluid steroids after ovulation; (2) local transfer of steroids via blood or lymph; (3) local synthesis of progesterone in the oviduct, as evidenced by the expression of steroidogenic enzymes; and (4) a paracrine contribution from follicular cells. These data provide a basis for the study of the importance of endocrine and paracrine signalling during early embryonic development in the horse.

Transcriptomic analysis of the myometrium during peri-implantation period and luteolysis–the study on the pig model

In pigs, implantation begins with the attachment of embryos to the endometrium. As the process is regulated by the expression of numerous genes, endometrial transcriptomic profiles have been extensively studied in early gravid pigs. However, the myometrium, a secretory tissue, should not be neglected, as it can also participate in the regulation of implantation in early pregnant pigs. To clarify this issue, the transcriptomic profile of the porcine myometrium during the peri-implantation period (i.e. on days 15 to 16 of pregnancy) was compared with the profile observed during luteolysis (i.e. on days 15 to 16 of the oestrous cycle) with an Agilent’s Porcine (V2) Two-Colour Gene Expression Microarray 4 × 44 (Agilent, USA). Analysis of the microarray data revealed that of 526 unique, accurately annotated genes, the expression of 271 unique genes was upregulated, while the expression of 255 genes was downregulated in pregnant versus cyclic myometrium. The in-depth data analysis revealed differential expression of genes encoding for factors involved in immunomodulation, tissue growth and differentiation, and prostaglandin and steroid biosynthesis and action. Moreover, the comparison of the obtained data on the myometrial transcriptome with our previously published results on the endometrial transcriptome allowed us to determine substantial differences in the regulatory function of both tissues. The new insights into the function of the myometrium of early pregnant pigs obtained here are in agreement with our previous results that suggest that this tissue plays an important role in providing optimal conditions for developing embryos. Therefore, the importance of the myometrium as an active embryo signal-responsive tissue during early pregnancy cannot be underestimated.

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

Estradiol-17β (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)-1β, 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-1β, 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-1β, IL-6, and TNF-α did not increase myometrial E2 release. In conclusion, IL-1β, 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-1β, IL-6, and TNF-α. In gravid pigs, IL-1β, 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.

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 17β (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.

Expression of 17β-hydroxysteroid dehydrogenase and the effects of LH, FSH and prolactin on oestrone and 17β-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 17β-oestradiol (E2). However, the roles of LH, FSH and prolactin (PRL) as regulators of endometrial steroidogenesis, and the presence of 17β-hydroxysteroid dehydrogenase (17β-HSD) in the porcine endometrium, remain unknown. Therefore, in the present study we examined 17β-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 17β-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.

Steroids affect gene expression, ciliary activity, glucose uptake, progesterone receptor expression and immunoreactive steroidogenic protein expression in equine oviduct explants in vitro.

The oviduct undergoes dramatic functional and morphological changes throughout the oestrous cycle of the mare. To unravel the effects of steroids on the morphology, functionality and gene expression of the equine oviduct, an in vitro oviduct explant culture system was stimulated with physiological concentrations of progesterone and 17β-oestradiol. Four conditions were compared: unsupplemented preovulatory explants, preovulatory explants that were stimulated with postovulatory hormone concentrations, unsupplemented postovulatory explants and postovulatory explants that were stimulated with preovulatory hormone concentrations. The modulating effects of both steroids on oviduct explants were investigated and the following parameters examined: (1) ciliary activity, (2) glucose consumption and lactate production pattern, (3) ultrastructure, (4) mRNA expression of embryotrophic genes, (5) steroidogenic capacities of oviductal explants and (6) progesterone receptor expression. The present paper shows that the equine oviduct is an organ with potential steroidogenic capacities, which is highly responsive to local changes in progesterone and 17β-oestradiol concentrations at the level of morphology, functionality and gene expression of the oviduct. These data provide a basis to study the importance of endocrine and paracrine signalling during early embryonic development in the horse.