Aberrant effects of altrenogest and exposure to exogenous gonadotropins on follicular cysts appearance in gilts.

Research was conducted to determine the effect of altrenogest and exposure to exogenous gonadotropins on ovarian function in prepubertal and mature gilts. Crossbred, presumably sexually mature gilts (n = 51), were fed with altrenogest for 18 consecutive days and the day after the last feeding with altrenogest, gilts were treated with eCG and 72 hours later challenged with hCG. Animals were slaughtered on Days 10 to 13 of their gonadotropins synchronized estrous cycle. Ovaries were examined for the number of CL, number of follicular cysts, and presence of corpora albicantia. Gilts were divided into two groups: those possessing corpora albicantia (group A-mature; n = 36) and those without corpora albicantia (Group W-prepubertal; n = 15) on their ovaries. In addition, each group was divided into two subgroups depending on the presence of follicular cysts (AC and WC) or their absence (AO and WO). There was no difference between the number of CL in group A and group W. Presence of corpora albicantia determined percentage of gilts possessing follicular cysts (13.9% group A vs. 66.7% group W). Gilts without follicular cysts (AO plus WO; n = 36) had higher number of CL (P < 0.01) than gilts bearing cysts (AC plus WC; n = 15). Comparison AO-AC did not show significant difference (P = 0.075) between CL number in mature cyst-free and cysts bearing gilts. A prepubertal gilts not bearing follicular cysts (WO) had higher (P < 0.02) number of CL than gilts bearing cysts. A significant negative correlation between the number of CL and number of follicular cysts was found (r = -0.664; P = 0.007). There were no differences in blood plasma progesterone and estradiol concentration between cyst-free and cyst-bearing gilts. These results indicate: (1) a higher follicular cysts appearance in prepubertal than mature gilts challenged with altrenogest and exposed to exogenous gonadotropins and (2) a negative effect of follicular cysts on the number of CL (ovulations) in prepubertal gilts.

Steroid hormones, prostanoids, and angiogenic systems during rescue of the corpus luteum in pigs.

In order to characterize the transition of the corpora lutea (CL) from acquisition of luteolytic sensitivity to rescue of luteal function: i) the expression of 38 factors associated with steroids, prostanoids, and angiogenic systems and ii) concentrations of the main hormones responsible for maintenance of CL function in cyclic and pregnant pigs were examined. Additionally, the effect of prostaglandin (PG) E2 and F2 α on luteal function during the estrous cycle and pregnancy was evaluated in vitro. Significantly up-regulated gene expression was revealed in CL collected on day 14 of the estrous cycle (CYP19A1, ESR2, PTGS2, HIF1A, and EDN1) and on days 12-14 of pregnancy (SCARB1, PGRMC1, STAR, HSD3B1, NR5A1, PTGFR, PTGER4, and VEGFA). Elevated concentrations of estradiol-17ß and PGE2 occurred in CL on days 12 and 14 of pregnancy respectively, while an increased intraluteal PGF2 α content was noted on day 14 of the estrous cycle. Both PGs increased the synthesis of progesterone by cultured luteal slices obtained on day 14 of pregnancy, in contrast to the action of PGF2 α on the corresponding day of the estrous cycle. PGE2 stimulated cAMP production via PTGER2 and PTGER4, while PGF2 α elevated the content of CREB in cultured luteal slices from CL of pregnant pigs. In silico analysis showed that infiltration of lymphocytes and apoptosis of microvascular endothelium were activated in CL on day 12 of the estrous cycle vs pregnancy. Summarizing, an abundance of E2 and PGE2 during pregnancy regulates specific pathways responsible for steroidogenesis, the prostanoid signaling system and angiogenesis during rescue from luteolysis in porcine CL.

Expression of factors associated with apoptosis in the porcine corpus luteum throughout the luteal phase of the estrous cycle and early pregnancy: their possible involvement in acquisition of luteolytic sensitivity.

The studies on the acquisition of luteolytic sensitivity have been focused mainly on molecular changes induced in the luteal tissue after treatment with exogenous PGF2α or on physiological changes occurring during the estrous cycle. The comparison of changes leading to the acquisition of luteolytic sensitivity after Day 12 of the estrous cycle and corresponding days of pregnancy has not been investigated in the pig. The present study was undertaken to evaluate (1) apoptosis measured as the proportions of early apoptotic, late apoptotic, and viable cells; (2) expression of factors involved in the extrinsic (TNFA/TNFα, TNFRSF1A/TNFR1, TNFRSF1B/TNFR2, FAS/Fas, and FASLG/FasL) and intrinsic (CASP3/Casp3, TP53/p-53, BAX/Bax, and BCL2/Bcl-2) apoptotic pathways, with two components of the activating protein-1 complex, i.e., FOS/Fos and JUN/Jun and IFNG/IFNγ; and (3) concentrations of luteal and blood plasma progesterone (P4) throughout the luteal phase of the estrous cycle and early pregnancy. Corpora lutea (CL) were collected postmortem on Days 8, 10, 12, and 14 of the estrous cycle and the corresponding days of pregnancy. The luteal tissue was subjected to RNA and/or protein isolation and disaggregation of CL cells followed by flow cytometry analysis aimed to determine apoptotic changes. Luteal and blood plasma P4 concentrations decreased on Day 14 of the estrous cycle versus pregnancy (P < 0.05 and P < 0.001, respectively). A significant increase in the number of early apoptotic cells and a decrease in the number of viable cells were observed on Day 14 of the estrous cycle (P < 0.001 and P < 0.05, respectively). Increase (P < 0.05) of TNFA messenger RNA (mRNA) level coincided with that of IFNG on Day 12 of the estrous cycle but not on the corresponding day of pregnancy. The content of FAS mRNA and protein increased on Day 14 of the estrous cycle versus pregnancy (P < 0.05). The mRNA expression of CASP3, BCL-2 and BAX was unchanged in cyclic and pregnant CL, while level of TP53 increased (P < 0.05) on Day 12 of the estrous cycle versus Day 8. The level of FOS and JUN mRNA increased (P < 0.05) on Day 14 of the estrous cycle versus the remaining days. The level of FOS and JUN mRNA was significantly higher (P < 0.001 and P < 0.05, respectively) on Day 14 of the estrous cycle than that on the corresponding day of pregnancy. In summary, the simultaneous increase of TNFA and IFNG transcript in cyclic CL suggests the crucial role of both cytokines in sensitization of porcine CL to further luteolytic action of PGF2α. The upregulated expression of FAS, FOS, and JUN mRNA in the late luteal phase in cyclic CL can indicate their involvement in structural luteolysis. The increased viability of luteal cells and elevated P4 concentrations in pregnant CL confirm the protective role of luteal P4 against apoptosis.

Effect of oestrus synchronization with PGF2α/eCG/hCG on luteal P4 synthesis in early pregnant gilts.

Administration of hormones to synchronize oestrus is a useful tool in animal breeding. However, exogenous ovarian stimulation may be detrimental to reproductive function. This study was aimed to examine whether an oestrus synchronization with PGF2α/eCG/hCG could affect luteal P4 synthesis in early pregnant gilts. Corpora lutea (CLs) were collected on days 9, 12 and 16 of pregnancy from gilts with natural (n = 16) and synchronized (n = 18) oestrus and analysed for (i) the expre-ssion of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A polypeptide (CYP11A1), and 3ß-hydroxysteroid dehydrogenase (3ßHSD); (ii) the concentration of P4 in the luteal tissue and blood; and (iii) the expression of luteinizing hormone receptors (LHR) and oestrogen receptors (ERα and ERß). Additionally, the effect of LH on P4 secretion from CL slices collected from synchronized and naturally ovulated animals has been studied in vitro. PGF2α /eCG/hCG administration increased mRNA expression of StAR, CYP11A1, 3ßHSD, and LHR on day 9 and CYP11A1 and LHR on day 12 of pregnancy compared with the control group (p < 0.05). CYP11A1, 3ßHSD, LHR, ERα and ERß proteins were not affected by synchronization; only StAR protein increased in hormonally treated animals (p = 0.017). The concentration of P4 in luteal tissue was greater on day 9 (p < 0.01), but lower on day 16 (p < 0.05) in gilts with hormonally induced oestrus compared with control animals. Blood serum levels of P4 were lower in synchronized than control gilts (p < 0.001). Synchronization did not affect LH-stimulated P4 secretion from luteal slices; however, greater basal concentration of P4 in incubation medium was detected for CLs collected from synchronized than control gilts (p < 0.05). In conclusion, synchronization of oestrus with PGF2α/eCG/hCG protocol in gilts did not impair the expression of luteal P4 synthesis system, although decreased P4 concentration in the blood.

Effect of vaginal administration of prostaglandin E2 and/or 17beta-estradiol on luteal function and histological characteristics of the cervix in cyclic pigs.

The overall objective of this study was to examine the effect of vaginal administration of prostaglandin E2 (PGE2) and/or 17beta-estradiol (E2) on luteal function maintenance and histological properties of the porcine cervix. For this purpose, crossbred gilts were divided into three groups (n=5 per group) supplied on days 11-16 of the estrous cycle with suppositories containing: (1) placebo (Group I, Control); (2) 0.4 mg of E2 (Group II); (3) 0.4 mg of E2 and 2 mg of PGE2 (Group III). Blood samples were collected on days 11-19 of the estrous cycle to determine the concentration of progesterone (P4). Additionally, to examine local effects of the hormones applied, segments from the uterine and vaginal parts of the cervix and from the ovaries were collected post-mortem. Prolonged luteal function and extended synthesis of P4 were observed in 2 of 5 gilts receiving PGE2 and E2 simultaneously (Group III). Then, these gilts were subdivided into Group IIIA (n = 2; presence of corpora lutea on the ovaries) and Group IIIB (n = 3; lack of corpora lutea). Increased levels of plasma P4 were observed in Group IIIA on days 15-19 compared to Group IIIB and on days 16-19 compared to Group I and Group II (P < 0.05; P < 0.01; P < 0.001, respectively). In the cervix of gilts in Groups II and III, enlarged blood vessels in the lamina propria of both parts of the cervix were observed. Furthermore, in Group II the epithelium of the uterine part of the cervix was thicker (P < 0.001). Our study confirmed the proposed luteotrophic/antiluteolytic actions of E2 and PGE2 applied intravaginally. These results are significant considering that very low doses of E2 were used when compared to previous attempts. Despite the inadequate response to treatments in some of the gilts, the local effects of these hormones on the histological properties of the porcine cervix suggest that further improvements in the vaginal administration route might help to elaborate new methods for enhancing the luteal function in the pig.

The novel effect of hCG administration on luteal function maintenance during the estrous cycle/pregnancy and early embryo development in the pig.

Two independent experiments were performed on cyclic (Experiment I) and pregnant (Experiment II) gilts to examine the effect of human Chorionic Gonadotropin (hCG) administration on day 12 of the estrous cycle/pregnancy on ovarian and endometrial secretory function. Animals were divided into hCG Group (injection of 750 IU hCG) and Control Group (injection of saline). In Experiment I, the prolonged lifespan of the corpus luteum (CL), extended progesterone (P4) production (P < 0.05) and delayed luteolysis were found. In hCG Group increased ratio of PGE2:PGFM during 12 hrs period on day 15 (P < 0.05) of the estrous cycle was observed. In both experiments, higher concentrations of E2 in hCG treated gilts (P < 0.05) on days 14-15 of the estrous cycle/pregnancy were found. In Experiment II, hCG injection did not affect P4, PGE2 and PGFM concentrations in blood plasma, but reduced the number of resorbed embryos on day 30 of pregnancy. In the pregnant hCG treated gilts the immunostaining against von Willebrand Factor (vWF) demonstrated an enhanced (P < 0.05) angiogenesis in CLs and endometrium. Furthermore, the flow cytometry revealed an increased (P < 0.05) viability of cells in CLs of hCG Group. An augmented expression of Steroidogenic Acute Regulatory Protein (STAR; P < 0.05) and LH/hCG receptor mRNA (P < 0.05) in CLs of hCG Group were observed, but an elevated concentration of protein was confirmed only for STAR (P < 0.05). Our studies revealed, for the first time, that administration of hCG affects PGE2:PGFM ratio during the estrous cycle as well as the development of conceptuses through enhanced angiogenesis and decreased luteal apoptosis in early pregnant pigs.

Effect of hCG and eCG treatments on prostaglandins synthesis in the porcine oviduct.

The oviduct plays a crucial role in fertilization, gamete maturation and embryo transport. Prostaglandins are some of the main factors determining its roles. The present study investigated the influence of oestrus synchronization and superovulation on prostaglandins synthesis in the porcine oviduct. Mature cross-bred gilts after exhibiting oestrous cycles were divided into four groups: I, cyclic; II, inseminated; III, synchronized and inseminated; and IV, superovulated and inseminated. Oviducts were collected on the third day of the oestrous cycle or after insemination and divided into isthmus and ampullary parts. This study demonstrated lower mRNA (in the isthmus and ampulla; p < 0.05, p < 0.001, respectively) and protein prostaglandin endoperoxide synthase 2 expression (in the isthmus; p < 0.001) in gilts treated with human chorionic gonadotrophin/equine chorionic gonadotrophin (hCG/eCG) compared with Group II that were inseminated only. In addition, hCG and eCG treatment decreased mPGES-1 mRNA levels in Groups III and IV, in both the isthmus (p < 0.01 in III, p < 0.001 in IV) and ampulla (p < 0.001). The prostaglandin E2 content of oviductal tissues was significantly lower in Groups III (p < 0.05) and IV (p < 0.01 in isthmus, p < 0.0001 in ampulla) compared with Group II. mRNA and protein levels of PGFS in Group IV in the oviductal isthmus were higher (p < 0.01) compared with the non-treated Group II. In effect, the amount of prostaglandin F2α in oviductal tissues of gilts treated with hCG/eCG was significantly elevated (p < 0.001 in isthmus of Groups III and IV; p < 0.05 in ampulla of Group IV). Differential expression of the factors analysed in gilts treated with exogenous gonadotrophins suggests that hCG/eCG stimulation affects prostaglandins synthesis pathway. These changes can alter oviduct functions and in turn affect gamete maturation and fertilization as well as development of embryos and their transport to the uterus.

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs.

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.

Mechanisms for the establishment of pregnancy in the pig.

Establishment of pregnancy in pigs requires continuous function of corpora lutea and endometrial preparation for embryo implantation. Progesterone regulates expression of many proteins necessary for endometrial remodelling and embryo-maternal communications. Attaining the uterine receptivity involves progesterone priming and loss of progesterone receptors in the uterine epithelium before days 10-12 after oestrus. Spermatozoa and oocytes in oviduct alter secretion of specific proteins that exert beneficial effect on gametes and embryos. Moreover, an appropriate leucocyte activation and maintenance of delicate cytokine balance within the oviduct and uterus are important for early pregnancy. This early local immune response is rather mediated by seminal plasma components. These components also influence prostaglandin (PG) synthesis in the oviduct that is important for gamete and embryo transport. Pregnancy establishment requires the biphasic pattern of oestrogen secretion by conceptuses on days 11-12 and 15-30. Conceptus affects lipid signalling system consisting of prostaglandins and lysophosphatic acid. PG synthesis is changed by conceptus signals in favour of luteoprotective PGE(2) . Additionally, existence of PGE(2) positive feedback loop in the endometrium contributes to increased PGE(2) /PGF(2α) ratio during the peri-implantation period. PGE(2) through endometrial PGE(2) receptor (PTGER2) elevates the expression of enzymes involved in PGE(2) synthesis. Higher PGE(2) secretion in uterine lumen coincides with the elevated expression of HOXA10 transcription factor critical for implantation. A stable adhesion between conceptus and endometrium requires reduction in mucin-1 on the apical surface of epithelium and integrin activation by extracellular matrix proteins. Furthermore, growth factors, cytokines and its receptors are involved in embryo-maternal interactions.

Effect of conceptus secretions on HOXA10 and PTGS2 gene expression, and PGE2 release in co-cultured luminal epithelial and stromal cells of the porcine endometrium at the time of early implantation.

Homeobox A10 (HOXA10) gene expression was demonstrated in the endometrium of adult porcine uteri, however there is little information concerning the role of this gene in the pig. Objectives of the present study were to examine: 1) the expression of HOXA10 in the endometrium of cyclic and early pregnant gilts; 2) the effect of estradiol (E(2)) and progesterone (P(4)) on HOXA10 expression in porcine luminal epithelial (LE) and stromal (ST) cells in vitro; 3) the effect of E(2) and conceptus-exposed medium (CEM) on HOXA10 and prostaglandin endoperoxide synthase (PTGS2) gene expression and prostaglandin (PG) E(2) secretion from LE and ST cells in a co-culture model. The abundance of HOXA10 mRNA was increased on day 15 of pregnancy in comparison to day 15 of the estrous cycle. Moreover, increased HOXA10 mRNA level was detected in ST cells after E(2) and P(4) treatment. E(2) stimulated the expression of HOXA10 in LE cells cultured on collagen and pre-treated with steroids, but not in LE on plastic surfaces. Addition of CEM to LE cells cultured in collagen-coated inserts of the co-culture system resulted in elevated HOXA10 and PTGS2 gene expression and PGE(2) secretion in these cells, but not in ST cells cultured in basal compartments. ST cells directly treated with E(2) or CEM showed higher levels of HOXA10 and PTGS2 expression. Blocking of estrogen receptors with ICI-182,780 did not influence the stimulatory effect of CEM. We conclude that HOXA10 expression in the porcine endometrium is closely related to the implantation process and stimulated by conceptus products. Moreover, the co-culture system of LE and ST cells is a promising model for the study of endometrial response to conceptus-derived factors.

Expression of the vascular endothelial growth factor (VEGF-A) and its receptors in the umbilical cord in the course of pregnancy in the pig.

The umbilical cord (UC) and the placenta are important organs through which respiratory gases, nutrients, wastes and biologically active substances are exchanged between the maternal and the foetal system. A rapid placental vascularization observed in the second half of pig pregnancy is positively correlated with the mRNA expression of the vascular endothelial growth factor (VEGF). Based on these findings, we hypothesized that VEGF may have a stimulatory effect in the dynamically growing UC. To further understand the role of the VEGF-VEGFR system during UC development, mRNA and protein expression as well as the cellular localization of VEGF-A, VEGFR-1 and VEGFR-2 in UC were examined on days 40, 60, 75 and 90 of pregnancy and after physiological delivery in the pig (day 114 of pregnancy). Real Time RT-PCR analysis showed an increase in the mRNA levels of VEGF120 and VEGF164 from day 90 of pregnancy. VEGFR-1 mRNA expression was significantly increased on day 75 of pregnancy. No significant changes in VEGFR-2 mRNA expression were detected. In turn, western blot analysis revealed an increase in VEGF-A protein expression on day 40, compared to the later days of pregnancy. A rapid increase in the VEGFR-1 protein level was noted on day 75 and 90 of gestation. No significant changes in VEGFR-2 protein expression were detected on any of the analysed days of pregnancy. Immunohistochemical staining enabled detection of VEGF-VEGFR system, in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium on all analysed days of pregnancy. Positive reactions for VEGF-A and VEGFR-1, but not VEGFR-2, were also observed in myofibroblasts. In conclusion, this data shows that members of the VEGF-VEGFR system are temporally and spatially well localized for playing key roles during umbilical cord formation and its intensive growth observed after day 75 of pregnancy.

Effect of luteinizing hormone and tumour necrosis factor-alpha on VEGF secretion by cultured porcine endometrial stromal cells.

To cope with rising demands for increased blood supply during pregnancy, the vasculature of the uterus undergoes several adaptive changes, including increased permeability, angiogenesis and vasodilatation. Although it is clear that vascular endothelial growth factor (VEGF) plays a paramount role in achieving these adaptations, little is known about regulation of VEGF expression in endometrium during pregnancy. Thus, we have investigated whether luteinizing hormone (LH) and tumour necrosis factor-alpha (TNFalpha) may affect VEGF secretion by stromal cells during early pregnancy in pigs. Real-time reverse transcription/polymerase chain reaction (RT/PCR) of VEGF120 and VEGF164 gene expression revealed significantly higher levels of VEGF164 mRNA in cultured stromal cells (p < 0.0001). The LH-stimulated secretion of VEGF was detected after 24 and 48 h of treatment when doses 50 and 100 ng/ml were used (p < 0.05 and p < 0.01, respectively). The TNFalpha-induced secretion of VEGF by stromal cells was detected only after 24-h treatment with the highest dose used in the experiment (50 ng/ml; p < 0.05). Although the influence of LH on VEGF secretion was more visible compared with TNFalpha, both factors may be considered as potential modulators of adaptive changes in uterine vasculature occurring during pregnancy in the pig.

Effect of estrus induction on prostaglandin content and prostaglandin synthesis enzyme expression in the uterus of early pregnant pigs.

Prostaglandins (PGs) play a pivotal role in maternal recognition of pregnancy and implantation in pigs. In the present study, PGE(2), PGF(2alpha), and PGFM (PGF(2alpha) metabolite) content, as well as PGE(2) synthase (mPGES-1) and PGF(2alpha) synthase (PGFS) expression was investigated in early pregnant gilts with natural (n=21) and PMSG/hCG-stimulated (n=19) estrus. Endometrial tissue samples, uterine luminal flushings (ULFs), and blood serum were collected on days 10-11, 12, and 15 after insemination. Additionally, day 15 conceptuses were collected for mPGES-1 and PGFS protein expression. Effect of estrus induction was observed on day 15 of pregnancy, when the content of PGE(2) in the uterine lumen was fourfold lower in gonadotropin-stimulated gilts in comparison to controls (P<0.001). Decreased PGE(2) content in ULFs of gonadotropin-treated pigs was preceded by lower endometrial mPGES-1 gene expression in hormonally-stimulated animals in comparison to control gilts (P<0.01). On the other hand, estrus induction with PMSG/hCG resulted in higher PGE(2) accumulation in the endometrial tissue on day 15 of pregnancy (P<0.01). Furthermore, PGF(2alpha) content in the endometrium and PGFM levels in blood serum were lower in gonadotropin-treated gilts, especially on day 12 after insemination when compared to control gilts (P<0.01). Finally, PGFS expression in day 15 conceptuses was decreased in animals with hormonally-induced estrus. We conclude that PMSG/hCG stimulation of prepubertal gilts to induce estrus results in changes of PG production and secretion during early pregnancy, which, in turn, may affect conceptus development, implantation, and the course of pregnancy.

mRNA and protein expression of FGF-1, FGF-2 and their receptors in the porcine umbilical cord during pregnancy.

The fibroblast growth factors (FGFs) are multifunctional proteins that, among other roles, regulate structural reorganization of uterine and placental vascular bed during pregnancy. Thus, we analyzed mRNA and protein expression and immunohistochemical localization of FGF-1 and FGF-2, and their receptors (FGFR-1 and FGFR-2) in the developing umbilical cord (UC) on days 40, 60, 75 and 90 of pregnancy and after the physiological delivery in the pig (day 114). qPCR analysis demonstrated an increase in FGF-1 and FGF-2 mRNA levels beginning on day 75 and on day 114 of pregnancy, respectively. In addition, significantly increased FGFR-1IIIc mRNA expression was also found on day 114. On the other hand, no significant changes in FGFR-2IIIb mRNA expression were observed. Western Blot analysis revealed a decrease in FGF-1 and FGFR-2 protein expression after day 40. Beside an increased protein expression of FGF-2 on day 60, no significant changes in FGFR-1 protein expression were detected. Immunohistochemical staining enabled detection of FGF-FGFR system, with different intensity of immunoreaction in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium as well as in myofibroblasts. In conclusion, our results show that members of FGF-FGFR system are expressed specifically in UC structures. Furthermore their day of pregnancy-related expression suggest that they may be an important players during UC formation and development.

Endometrial and conceptus expression of HoxA10, transforming growth factor beta1, leukemia inhibitory factor, and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus.

This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) beta1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72h later. Gilts in the control group (n=23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy (P<0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 (P<0.05) and 15 (P<0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFbeta, LIF, and PGHS-2 on day 12 of pregnancy (P<0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.

Antiluteolytic mechanisms and the establishment of pregnancy in the pig.

Extended exposure of progesterone and conceptus estrogen influences the vascular compartment of the uterus and expression of many factors, such as prostaglandins (PGs), growth factors, extracellular matrix and adhesion molecules, cytokines and transcription factors. One of the supportive mechanisms by which the conceptus inhibits luteolysis is by changing PG synthesis in favor of luteoprotective PGE2. Alteration in PG synthesis may result from increased PGE synthase (mPGES-1) expression in the trophoblast and endometrium on days 10-13 of pregnancy with simultaneous down-regulation of PGF synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1). Conceptus and endometrial, rather than luteal, synthesis of PGE2, is involved in the process of maternal recognition of pregnancy. However, complex (direct and indirect) actions of estrogen on the CL, including decreased luteal VEGF soluble receptor on day 12 of pregnancy, are important for luteal maintenance. Moreover, conceptus signals affect another lipid signaling component - lysophosphatidic acid receptor (LPA3), as well as HoxA10 and Wnt in the endometrium, to create the appropriate uterine environment for establishment of pregnancy and implantation.

Expression of VEGF-receptor system in conceptus during peri-implantation period and endometrial and luteal expression of soluble VEGFR-1 in the pig.

In view of the importance of vascular events observed during gestation, it was hypothesized that the VEGF-receptor system plays a critical role during early pregnancy and maternal recognition of pregnancy in pigs. This hypothesis was tested by examining the expression of the VEGF-receptor system in the porcine conceptus. Additionally, the endometrium, corpus luteum (CL) and embryos were studied for the expression of soluble VEGF receptor 1 (sVEGFR-1), the strong endogenous antagonist of VEGF. The expression patterns show that VEGF164 mRNA levels increase gradually in line with conceptus development, whereas VEGF120 and VEGFR-2 remain unchanged during the peri-implantation period. Interestingly, elevated VEGFR-1 expression was observed in conceptuses on days 15-16 of gestation (P<0.05). Comparison of the endometrial sVEGFR-1 mRNA expression revealed up-regulation on days 12 and 15-16 of pregnancy (P<0.01 and P<0.05, respectively). Furthermore, increased sVEGFR-1 levels were observed on day 12 of the estrous cycle in the CL (P<0.05). Concluding, it seems that conceptus-derived VEGF164 plays crucial role in peri-implantation vascular events in pigs. These results support a potential role of VEGFR-1 in the proper growth and development of porcine conceptus during pregnancy. Moreover, expression patterns of sVEGFR-1 in the endometrium of pregnant pigs suggest that it may participate in vascular remodeling important for successful implantation. Finally, luteal sVEGFR-1 may be involved in the maintenance of CL function whenever pregnancy occurs in pigs.

Conceptus signals for establishment and maintenance of pregnancy in pigs - lipid signaling system.

Establishment of pregnancy in pigs requires estrogen secretion by the conceptus. The developmental changes of embryo before implantation and embryo-uterine cross talk are dependent on various biological molecules secreted by the endometrium and conceptus. An integral part of maternal recognition of pregnancy seems to be also the lipid signaling system consisting of prostaglandin (PG) F2 alpha and E2 and/or lysophosphatic acid (LPA). The downstream enzymes in PG synthesis pathway are: microsomal PGE synthase-1 (mPGES-1), PGF synthase (PGFS) and prostaglandin 9-ketoreductase/carbonyl reductase (CBR1) which catalyzes conversion of PGE 2 into PGF2 alpha. In contrast to mPGES-1, endometrial PGFS is highly increased on days 13-15 similarly as CBR1 on days 16-17 of the estrous cycle. Potential mechanism by which a conceptus inhibits luteolysis is changing the PGE2/PGF2 alpha ratio in favor of PGE2. It may be result of high expression of mPGES-1 in trophoblast and endometrium on days 10-13 of pregnancy and simultaneously the down-regulation of PGFS and CBR1 in conceptuses during this period. The conceptus can alter expression of endometrial CBR1 to modulate the PGE2/PGF2 alpha ratio in the uterus during the maternal recognition of pregnancy. High expression of conceptus and endometrial terminal PG synthases and CBR1 after initiation of blastocyst attachment suggest their involvement in early placentation. The higher LPA3 receptor mRNA expression during the early pregnancy compared to corresponding period of estrous cycle could indicate an important role of LPA and its receptor during the peri-implantation stage of pregnancy in pigs. Above results suggest that the lipid signaling system is an integral part of establishment and maintenance of pregnancy in the pig.

Expression of prostaglandin synthesis pathway enzymes in the porcine corpus luteum during the oestrous cycle and early pregnancy.

Prostaglandins (PGs) of luteal origin may have paracrine and/or autocrine actions on the functions of the corpus luteum (CL). Previously, we have shown that enzymes of PG synthesis pathway such as prostaglandin E synthase (mPGES-1), prostaglandin F synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1) are important in regulation of PG production in the conceptuses and endometrium of cyclic and pregnant pigs. Therefore, localization and expression patterns of these enzymes were determinated in porcine CL. The PGFS protein content was lower in metestrus and higher around luteolysis, and then decreased in late regressing CL. PGFS protein levels were lower on days 5-8 of pregnancy and did not differ between days 10 and 25. Elevated expression of mPGES-1 mRNA was found in early luteal phase. The mPGES-1 protein content, similarly to PGFS, was higher during luteolysis. mPGES-1 mRNA and protein levels were constant between days 5 and 25 of pregnancy. PGFS and mPGES-1 expression was down-regulated on days 16-17 of the oestrous cycle when compared to the corresponding days of pregnancy. Enhanced mPGES-1/PGFS ratio occurred during early luteal phase and days 5-8 of pregnancy. Expression of CBR1 mRNA and protein was constant during the cycle and pregnancy. Our studies revealed higher mPGES-1/PGFS ratios in the CL during early luteal phase and corresponding days of pregnancy that could favor PGE(2) synthesis and may be important in the control of luteal development. However, PG synthesis in the endometrium/conceptus rather than in the CL could be involved in luteolysis and maternal recognition of pregnancy in pigs.

Use of hecate-chorionic gonadotropin beta conjugate in therapy of lutenizing hormone receptor expressing gonadal somatic cell tumors.

Improvement of cancer treatment is a major challenge of medical research. Despite the immense efforts made in the improvement of diagnosis and treatment, cancer remains a major concern and cause of morbidity and mortality. Most of the modern anti-neoplastic therapies have severe side effects, and tumor cells often develop drug resistance. There is promise in the new generation of treatments (gene therapy, immunotherapy, vaccines, etc.) that are under development, but the efficacies and side effects of such therapies have so far been disappointing. Receptor-based therapies are not new, but many normal cells also present the same receptors reducing the specificity of such approaches. Several lytic peptides have been investigated because of they appear to kill cancer cells due to changes of their membrane potential. Thus, linking receptor-specific ligands to lytic peptides is expected to augment the specificity of targeting and decrease the toxicity of lytic peptides on normal cells. One such polypeptide is hecate (an analogue to the bee venom main component, melittin) that preferentially kills cancer cells at low doses. When this peptide is fused with the 81-95 amino acid fragment of chorionic gonadotropin-beta (CGbeta) subunit (hecate-CGbeta), it targets cells expressing luteinizing hormone receptor (LHR), even at very low doses, or when LHR is expressed at low level. Our recent data showed that this peptide conjugate is efficient in destroying LHR-positive cells in xenografts and more importantly in transgenic mouse models developing LHR-positive somatic cell tumors in gonads. The mechanism of action of hecate-CGbeta after binding to LHR is destruction of cell membranes resulting in rapid cell death by necrosis with minimal side effects. This review summarizes our findings on the action of this novel peptide and considers the future potential of this family of targeting peptides in the treatment of neoplasias.