Marta Wasielak

The influence of embryo presence on prostaglandins synthesis and prostaglandin E2 and F2α content in corpora lutea during periimplantation period in the pig

We determined the expression of PGE2 synthase (mPGES‐1), PGF synthase (PGFS), carbonyl reductase/prostaglandin 9‐ketoreductase (CBR1) genes and the content of PGE2, PGF2α in porcine corpora lutea on Days 12–14 of pregnancy and Days 12–14 of the estrous cycle. For this study we used a surgically‐generated model in which one of the uterine horns was cut transversely and a part of this horn was detached from the uterine corpus. The expression of mPGES‐1, PGFS, and CBR1 genes and mPGES‐1/PGFS ratio were significantly higher in corpora lutea of the pregnant gilts compared to the corpora lutea from the parallel ovaries of the cyclic gilts. There was no difference in mPGES‐1, PGFS, CBR1 genes expression and mPGES‐1/PGFS ratio between corpora lutea ipsi‐(CL1) and contralateral (CL2) to the uterine horn with the developing embryos. The highest content of PGE2 was found in CL1 of the pregnant gilts. The PGE2/PGF2α ratio was significantly higher in CL1 of the pregnant gilts compared to corpora lutea from parallel ovary of the cyclic gilts. We suggest that the activity of the investigated genes is induced by compounds of embryonic origin which are not distributed only to the ipsilateral ovary but are transported within the mesometrium to both ovaries in a more systemic manner. Mol. Reprod. Dev. 75: 1208–1216, 2008.

Proteomic analysis of porcine endometrial tissue during peri-implantation period reveals altered protein abundance.

UNLABELLED In mammals, successful pregnancy depends upon the readiness of uterus for implantation, followed by correct communication between the endometrium and the developing conceptus. The objective of this study was to elucidate changes in protein abundance associated with progression of estrous cycle and pregnancy from Day 9 to Day 12. We analyzed porcine endometrial tissue lysates by 2D-DIGE. Abundance of several proteins was altered depending upon the pregnancy status of animals. MALDI-TOF/TOF was used to identify a number of these proteins. Endometrial proteins that increased from Day 9 to Day 12 of cycle included annexin A4, beta-actin, apolipoprotein, ceruloplasmin and afamin. Changes in protein abundances associated with conceptus secreted factors, including haptoglobin, prolyl-4-hydroxylase, aldose-reductase and transthyretin, were also observed. Functional analysis revealed that endometrial proteins with altered abundance on Day 12 irrespective of the reproductive status were related to growth and remodeling, acute phase response and free radical scavenging, whereas transport and small molecule biochemistry were the functions activated in the pregnant endometrium as compared to the cyclic endometrium. These data provide information on dynamic physiological processes associated with uterine endometrial function of the cyclic and pregnant endometrium during period of maternal recognition of pregnancy in pigs and may potentially demonstrate a protein profile associated with successful pregnancy. BIOLOGICAL SIGNIFICANCE In pigs, the fertility rates are generally very high but the early embryonic loss that occurs during the second and third weeks of gestation critically affects the potential litter size. Temporal changes that take place in the uterine environment during the period of early pregnancy in pigs and a cross-talk between the uterus and the embryo play an important role in embryonic survival and successful pregnancy. A better understanding of the molecular changes associated with these processes will pave way for understanding of endometrial functions and help towards increasing embryo survival. In this study, we present a 2D-DIGE based analysis of changes in porcine endometrial proteome that are associated with progression of cycle and progression of pregnancy. The network analysis of the results clearly revealed the pathways that are involved in rendering the endometrium receptive to the presence of embryo and also the changes that are result of molecular communication between the endometrium and the conceptuses. This comprehensive identification of proteomic changes in the porcine endometrium could be a foundation for targeted studies of proteins and pathways potentially involved in abnormal endometrial receptivity, placentation and embryo loss.

Expression of peroxisome proliferator activated receptor (PPAR) genes in porcine endometrium exposed in vitro to IL-6 and INFγ

The aim of the present study was to examine the effects of interferon gamma (INFγ) or interleukin 6 (IL-6) on gene expression of PPARs in the porcine endometrium on day 14 of the estrous cycle and pregnancy. Endometrial tissue (200-210 mg), after 18 h of pre-incubation, was incubated for 6 or 12 h in the presence of INFγ (5 or 50 ng/ml) or IL-6 (1 or 10 ng/ml). Gene expression was analyzed by quantitative real time RT-PCR. During the estrous cycle, neither INFγ nor IL-6 affected PPARα and PPARβ/δ transcript levels in the endometrium of the cyclic pigs incubated for 6 or 12 hours. The presence of INFγ (5 ng/ml) significantly (p<0.05) increased PPARγ1 gene expression in the tissue incubated for 12 h. During pregnancy, INFγ (50 ng/ml) significantly (p<0.05) enhanced PPARα and PPARβ/δ mRNA levels in the endometrium incubated for 6 h, whereas IL-6 (1 or 10 ng/ml) did not change their expression at any incubation time. The effect of both cytokines on PPARγ1 transcript level differed and was dependent on the incubation time. We observed an inhibitory (after 6 h of incubation, p<0.0001) and a stimulatory (after 12 h of incubation, p<0.05) effect of INFγ (5 ng/ml) or IL-6 (10 ng/ml) on PPARγ1 gene expression. The present study indicates that INFγ and IL-6 modulate PPARs gene expression in the porcine endometrium during the estrous cycle and pregnancy. The effect depends on the reproductive status of animals and the length of in vitro incubation of endometrial tissue with the treatments.

The effect of hormonal estrus induction on maternal effect and apoptosis-related genes expression in porcine cumulus-oocyte complexes

BackgroundThe effect of hormonal estrus induction on maternal effect (MATER - maternal antigen that embryo requires, ZAR-1 - zygote arrest 1, and BMP15 - bone morphogenetic protein 15) and apoptosis-related genes expression (BCL-2 and BAX) in porcine cumulus-oocyte complexes (COCs) and selected follicular parameters was investigated in this study.MethodsGilts were divided into three groups: (I) with natural estrus; (II) stimulated with PMSG/hCG; and (III) with PMSG/hCG + PGF2alpha. Analysis of maternal effect and apoptosis-related transcripts expression in COCs, and progesterone synthesis pathway genes expression (P450scc and 3betaHSD) in granulosa cells was performed by qPCR. BMP15 protein expression in follicular fluid (FF) was analyzed by western blot. Oocyte nuclear maturation was assessed by aceto-orcein staining. Progesterone (P4) and estradiol (E2) concentrations in FF and serum were measured by ELISA. Data were analyzed with the one-way ANOVA and Bonferroni post-test or Kruskal-Wallis test and Dunns post-test.ResultsThe highest expression of MATER, ZAR-1, and BMP15 genes was found in COCs recovered from gilts treated with PMSG/hCG when compared to PMSG/hCG + PGF2alpha-stimulated or non-stimulated gilts. Hormonal treatment did not affect the BMP15 protein expression in FF, but increased the expression of genes participating in P4 synthesis in granulosa cells. The higher percentage of immature oocytes was found in PMSG/hCG-treated when compared to the non-stimulated gilts. The expression of BCL-2 and BAX mRNA, and BCL-2/BAX mRNA ratio was significantly higher in COCs derived from PMSG/hCG-treated when compared to PMSG/hCG + PGF2alpha-treated or non-stimulated subjects. The level of P4 in serum was similar in animals from all experimental groups, while its concentration in FF was greater in gilts subjected to PMSG/hCG treatment than in PMSG/hCG + PGF2alpha-stimulated and non-stimulated gilts. The concentration of E2 did not differ in the serum or FF between the control group and the hormonally stimulated groups.ConclusionsHormonal induction of estrus affected maternal effect gene transcripts levels in COCs and and oocyte nuclear maturation. The inclusion of PGF2alpha into the stimulation protocol enabled maintaining of physiological concentration of P4 in FF. Additionally, both hormonal treatments seem to be beneficial for apoptosis prevention through increasing BCL-2/BAX transcript ratio.

Transcript abundance and apoptosis in day-7 porcine blastocyst cultured with exogenous insulin-like growth factor-I

Exogenous growth factors may increase the efficiency of embryo development in vitro. The aim of the present study was to examine the effects of insulin-like growth factor (IGF)-I on porcine embryo development. Porcine embryos obtained by in vitro fertilization were cultured for seven days in the presence of IGF-I (50, 100 or 150ng/ml). Subsequently, relative transcript abundance (RA) of IGF-related genes (IGFR1, IGFBP2, and IGFBP3), glucose transporter genes (SLC2A4 and SLC2A8), and apoptosis-related genes (BAX and BCL-XL) was analyzed. No differences were observed in the cleavage rate on day 2 post insemination (pi) and blastocysts rate on day 7pi between IGF-treated and control embryos. IGF-I treatment did not affect RA of IGFR1, IGFBP3, and SLC2A4 genes, but decreased RA of IGFBP2 and SLC2A8 genes. The percentage of TUNEL-positive nuclei in blastocysts did not differ between the experimental groups. However, RA of BAX and BCL-XL genes decreased in response to all IGF-I concentrations, whereas the BCL-XL/BAX RA ratio was enhanced when embryos were cultured in medium containing 150ng/ml of IGF-I. These results indicate that IGF-I did not stimulate in vitro development of porcine embryos through the IGF signaling system, nor did IGF-I stimulate RA of glucose transporter genes. However, IGF-I at the highest dose was able to increase the BCL-XL/BAX transcript expression ratio. This may indicate that the primary role of IGF-I during the first days of embryo development in the pig is associated with anti-apoptotic actions rather than with growth stimulation.

Effects of seminal plasma and the presence of a conceptus on regulation of lymphocyte-cytokine network in porcine endometrium

Infusion of seminal plasma in the uterus is known to elicit an instant inflammatory response in the porcine uterus, but whether or not it prepares a uterine immunological response to the presence of conceptuses is not well understood. Seminal plasma induced long‐term modulatory effects and conceptus‐induced immune changes in leukocyte populations were measured by flow cytometry and mRNAs for various cytokines by quantitative reverse‐transcriptase PCR in porcine endometrium collected on Days 6 and 13 from cycling and pregnant animals or from animals given seminal plasma infusions. Seminal plasma infusion induced long‐term modulatory effects, resulting in significantly more endometrial FoxP3‐positive T‐regulatory and T‐helper cells 6 days after infusion as compared to cycling and pregnant animals. The number of T‐cytotoxic and T‐null cells did not change between the studied groups. The early molecular effects of seminal plasma were not observed at 13‐days post‐infusion, although animals on Day 13 of pregnancy did show significantly more T‐cells (of any type investigated). Seminal plasma also showed a delayed effect on cytokine expression, specifically exhibiting a significant increase in interleukin 10 (IL10) and a decrease in granulocyte macrophage colony‐stimulating factor (GMCSF) gene expression on Day 13 as compared to Day 6 of cycling or pregnant gilts. The results indicate a delayed regulatory effect of seminal plasma on immune responses in the porcine uterus, which are similar to immune changes generated by implanting conceptuses. Mol. Reprod. Dev. 2014.

Effect of the conceptus on uterine prostaglandin-F2α and prostaglandin-E2 release and synthesis during the periimplantation period in the pig

The present study was conducted to evaluate the effect of the conceptus on uterine prostaglandin-F2alpha (PGF2alpha) and prostaglandin-E2 (PGE2) release and the expression of prostaglandin synthase enzymes during the periimplantation period in the pig. A surgically generated model with conceptuses developing in only one of the uterine horns was created. The highest concentration of PGF2alpha and PGE2 was found in the gravid uterine horn, compared with the non-gravid horn and the intact horn of cyclic gilts. Endometrial concentration of both PGs in pregnant gilts was elevated regardless of the conceptus in the uterine horn, whereas only myometrial PGE2 concentration increased during pregnancy. Expression of prostaglandin-E2 synthase (mPGES-1) mRNA in the endometrium was upregulated during the oestrous cycle, while protein expression presented a similar pattern to that of PGE2 concentration in the uterine flushings. Prostaglandin-F2alpha synthase (PGFS) mRNA and protein expression in the endometrium did not differ between pregnancy and oestrous cycle but PGFS mRNA in the myometrium increased during pregnancy both in the gravid and the non-gravid uterine horns. We suggest a local effect of the conceptus on PG release pathways but also a more systemic effect within the whole uterus with regard to PG synthesis and accumulation in uterine tissues.

The effect of embryo presence on the expression of peroxisome proliferator activated receptor (PPAR) genes in the porcine reproductive system during periimplantation

This study was undertaken to determine the effect of the presence of embryos in the uterine horn on peroxisome proliferator activated receptors (PPARs; A, D, G) gene expression in the reproductive tissues of gilts subjected to a surgical procedure. The uterus consisted of one intact horn connected to the uterine corpus and the second horn detached from the uterine corpus but connected with the contiguous ovary. The gilts were hormonally stimulated and divided into two groups: the first group, inseminated (pregnant) and the second group (cyclic), with surgical procedure but not inseminated. The animals of both groups were slaughtered on day 14 of pregnancy or on day 14 of the oestrous cycle, respectively. PPARs mRNA abundance in the endometrium and the corpus luteum (CL) was analysed by quantitative real-time PCR. During pregnancy, PPARA and PPARD μmRNA abundance in the porcine endometrium was significantly higher in the horn containing embryos than in the contralateral horn, where embryos were absent. The endometrial PPARG1 mRNA abundance did not differ between the two horns during pregnancy and the oestrous cycle, but a higher level of the transcript was observed during pregnancy when compared to the oestrous cycle. In the CL, there were no significant differences in PPARA and PPARDμ mRNA abundance between horns in pregnant or cyclic sows. However, there was a significant increase of PPARA and PPARD transcript level in the CL from cyclic compared with pregnant sows. The results of our study suggest that PPARA and PPARD have regulatory functions in early pregnancy, and they indicate that increased levels of endometrial gene expression are correlated with the presence of embryos in the uterine horn. Higher levels of PPARA and PPARD expression in the porcine CL on day 14 of the oestrous cycle than on day 14 of pregnancy suggest that both forms are involved in the regulation of CL functions.

Maternal effect gene expression in porcine metaphase II oocytes and embryos in vitro: effect of epidermal growth factor, interleukin-1β and leukemia inhibitory factor

Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1β or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1β 10 ng/ml or LIF 50 ng/ml. After maturation for 44-46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1β did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1β decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1β in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.