Atretic preovulatory follicles could be precursors of ovarian lutein cysts in the pig.

Ovarian cysts contribute to reduced reproductive performance in pigs. Unfortunately, the mechanism of lutein cysts formation remains unknown. Here, we compared the endocrine and molecular milieus of intact, healthy preovulatory follicles (PF), gonadotropin (eCG/hCG)-induced healthy and atretic-like PF, as well as gonadotropin-provoked and spontaneous ovarian cysts in gilts. Several endocrine and molecular indicators and microRNA were compared in walls of PF and cysts. Intact and healthy PF, showed high estradiol/androstendione and low progesterone levels associated with CYP17A1, HSD17B1, and CYP19A1 elevation and reduced StAR/HSD3B1 protein expression. In contrast, low estradiol/androstendione and high progesterone concentrations, accompanied by decreased CYP17A1, HSD17B1, CYP19A1 and increased HSD3B1 protein abundance, appeared in atretic-like PF, gonadotropin-induced and spontaneous cysts. High progesterone receptor (PGR) protein abundance was maintained in intact and healthy PF, while it dropped in atretic-like PF, gonadotropins-induced and spontaneous cysts. The atretic PF showed high level of TNFα compared to healthy PF. In conclusion, follicular lutein cysts could be recruited from atretic-like PF with lost estrogenic milieu and inability to ovulate. Ovulatory cascade was presumably disrupted by a low PGR and high TNFα levels associated with earlier luteinization of follicular walls. These results suggest a novel mechanism of lutein ovarian cysts development in pigs and, perhaps, other species.

The Role of Reduced Oxygen Supply and Transcription Factors cJUN and CREB in Progesterone Production during the Corpus Luteum Rescue in Gilts.

The corpus luteum plays a fundamental role in regulating reproduction via progesterone production. Still, there is little data on factors regulating the maintenance of luteal function during early pregnancy in gilts. Previous studies emphasize the role of hypoxia and HIF-1 in the regulation of steroidogenic and angiogenic genes expression and progesterone production by ovarian cells. Using the corpus luteum of cyclic and early pregnant gilts we analyzed: (1) the in vitro effects of reduced oxygen tension on progesterone production and mRNA expression of HIF1A and luteal function regulators, STAR and VEGFA; (2) the ex vivo profiles of mRNA and protein expression of HIF-1α, STAR, VEGFA and transcription factors-cJUN and CREB, regulating STAR expression, in the corpus luteum of cyclic and pregnant gilts. The synthesis of progesterone was gradually inhibited in cyclic or pregnant gilt luteal tissue (on day 13 of cycle or pregnancy) incubated in a decreasing concentration−20%, 10%, and 3% of oxygen (O2). Luteal tissues of pregnant gilts produced trace amounts of progesterone in 10% O2, which was similar to cyclic gilts in 3% O2. HIF1A expression increased after 24 h of incubation in tissues of cyclic gilts in 3% vs. 20% O2 (p < 0.01), whereas levels of STAR and VEGFA increased significantly in cyclic and pregnant gilt tissues incubated in 10% and 3% vs. 20% O2. The ex vivo mRNA expression of HIF1A and VEGFA was elevated (p < 0.001) on day 14 vs. day 12 of pregnancy. The protein expression of HIF-1 and VEGFA increased (p < 0.001), whereas the level of STAR (mRNA and protein) and progesterone dropped (p < 0.001) on day 14 of the estrous cycle vs. a parallel day of pregnancy and/or day 12 of the estrous cycle. The content of phosphorylated cJUN and CREB was elevated (p < 0.01) in the luteal tissue on day 12 or 14 of pregnancy vs. parallel days of the estrous cycle. These increases of phosphorylated cJUN and CREB may be involved in STAR expression in the luteal tissue during early pregnancy in gilts.

Attainment of Sexual Maturity and Gonadotropin Priming in Gilts Determine Follicular Development, Endocrine Milieu and Response to Ovulatory Triggers.

The routine procedure of estrous cycle synchronization in pigs allows for the use of gonadotropins to stimulate ovarian activity. The applied protocols of eCG and hFSH priming similarly affected development of ovarian follicles in two classes 3−6 mm and >6 mm of diameter, however, the number of small follicles (<3 mm) was 2-fold higher in hFSH- than in eCG-primed prepubertal gilts. The attainment of sexual maturity increased concentration of estradiol, testosterone and androstenedione in the follicular fluid of hFSH/eCG-primed gilts, however, prostaglandin E2 and F2α metabolite increased in mature hFSH- and eCG-primed gilts, respectively. The maturity increased mRNA and/or protein expression of key steroidogenic enzymes, prostaglandin synthases or luteinizing hormone receptors in follicular walls. Both hormonal primers played a moderate role in affecting expression of steroidogenic enzymes in follicular walls. In vitro studies showed higher estradiol production in r-hLH (p = 0.04)- and r-hCG (p = 0.049)-stimulated follicular walls of mature gilts than in prepubertal hFSH-primed gilts. Both ovulatory triggers decreased the abundance of LHCG/FSH mRNA receptors in follicular walls, which mimic downregulation of these receptors by a preovulatory LH surge, confirmed in vivo. These data revealed the importance of sexual maturity in the protection of the estrogenic environment, and the selective, moderate role of eCG and FSH in the activation of steroidogenic enzymes in preovulatory follicles.

Endocrine and molecular milieus of ovarian follicles are diversely affected by human chorionic gonadotropin and gonadotropin-releasing hormone in prepubertal and mature gilts.

Different strategies are used to meet optimal reproductive performance or manage reproductive health. Although exogenous human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH) agonists (A) are commonly used to trigger ovulation in estrous cycle synchronization, little is known about their effect on the ovarian follicle. Here, we explored whether hCG- and GnRH-A-induced native luteinizing hormone (LH) can affect the endocrine and molecular milieus of ovarian preovulatory follicles in pigs at different stages of sexual development. We collected ovaries 30 h after hCG/GnRH-A administration from altrenogest and pregnant mare serum gonadotropin (eCG)-primed prepubertal and sexually mature gilts. Several endocrine and molecular alternations were indicated, including broad hormonal trigger-induced changes in follicular fluid steroid hormones and prostaglandin levels. However, sexual maturity affected only estradiol levels. Trigger- and/or maturity-dependent changes in the abundance of hormone receptors (FSHR and LHCGR) and proteins associated with lipid metabolism and steroidogenesis (e.g., STAR, HSD3B1, and CYP11A1), prostaglandin synthesis (PTGS2 and PTGFS), extracellular matrix remodeling (MMP1 and TIMP1), protein folding (HSPs), molecular transport (TF), and cell function and survival (e.g., VIM) were observed. These data revealed different endocrine properties of exogenous and endogenous gonadotropins, with a potent progestational/androgenic role of hCG and estrogenic/pro-developmental function of LH.

Altrenogest affects the development and endocrine milieu of ovarian follicles in prepubertal and mature gilts†.

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared with controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, luteinizing hormone (LH)-stimulated cyclic adenosine monophosphate (cAMP) generation, and LH receptor messenger RNA (mRNA) expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E2 in prepubertal gilts and PGF2alpha metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as human chorionic gonadotropin- and forskolin-induced cAMP were also affected. In addition, altrenogest downregulated CYP17A1 mRNA in the prepubertal theca layer and PGF2alpha synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

The presence of CC chemokines and their aberrant role in the porcine corpus luteum.

The process of luteal regression is tightly regulated by the immune system and chemokines-small cytokines responsible mostly for the activation and migration of immune cells. The role of chemokines in porcine corpus luteum (CL) function is still not well understood. The aim of this study was to investigate the expression profile and distribution of CC chemokines in the porcine CL during the natural oestrous cycle and early pregnancy. Additionally, the effect of PGF2α on the expression of selected chemokines and their luteotropic and apoptotic influence on CL cells were studied in vitro. The expression levels of the chemokines CCL2, CCL4, and CCL5 and the chemokine receptor CCR5 were time-dependent (low on Days 8-10 and high on Days 12-14 of the oestrous cycle). Moreover, CCL8 and CCR2 transcript levels were also elevated during the period of luteolysis. The immunolocalization of CCL2, CCL4, CCL5, CCR1, CCR2 and CCR5 was determined using CL sections obtained from cycling and pregnant pigs. The immunofluorescence signals were localized mainly in luteal cells. PGF2α treatment of CL cells caused increased mRNA expression of CCL2 and CCR1. CCL2 treatment alone upregulated the expression of genes BAX, BCL2 and StAR in CL cells in vitro, but additional experiments showed that the chemokines CCL2, CCL4 and CCL5 alone do not cause apoptosis in a mixed population of CL cells. The chemokine CCL4 increased the transcript levels of StAR and HSD3-ß1. Additionally, CCL5 led to the inhibition of BAX gene expression. The differential spatiotemporal expression of CCL2, CCL4, CCL5 and CCR5 throughout the oestrous cycle and the direct but aberrant effect of these three chemokines on genes associated with apoptosis and progesterone synthesis indicate the complicated involvement of these factors in the regulation of luteolysis in pigs.

Regulation of the porcine corpus luteum during pregnancy.

The new corpora lutea (CLs) in pigs are formed from the preovulatory follicles after the luteinizing hormone (LH) surge. However, total autonomy and independence of CLs from LH up to Day 12 of cycle has recently been questioned. Transformation of estrous cycle CL to CL of pregnancy initiated by embryonic signals requires not only the cessation of prostaglandin F2 (PGF2α) supply to the luteal tissue but also needs the CL to overcome luteolytic acquisition and/or changing its sensitivity to PGF2α during Days 12-14 of pregnancy. The luteolytic cascade is prevented by inhibition of lymphocyte infiltration and leucocyte recruitment, limitation of cell apoptosis, upregulation of pregnancy-associated genes and an enhanced antiluteolytic role of PGE2 Our 'two-signal switch hypothesis' highlights the importance of post PGF2α and PGE2 receptor signaling pathways activation in CLs during luteolysis and rescue. The 'luteolytic switch' involves increased expression of many regression mediators and activation of the post PTGFR signaling pathway. The 'rescue switch' initiated by embryonic signals - estradiol 17ß and PGE2 - induces post PTGER2/4 pathway, turning the 'luteolytic switch' off and triggering activity of genes responsible for CL maintenance. In mid and late pregnancy, CLs are maintained by LH and the synergistic action of metabolic hormones. This paper provides an outline of recent views on CL regression, rescue and maintenance during pregnancy in pigs that conflict with previous paradigms and highlights new findings regarding the actions of prostaglandins, role of microRNAs (miRNA) and immune system and signaling pathways governing the life cycle of porcine CL.

Embryo-maternal dialogue during pregnancy establishment and implantation in the pig.

Porcine conceptuses secrete pregnancy-recognition signals (estrogens, including estradiol-17ß) that inhibit luteolysis, thereby prolonging progesterone production by corpora lutea. The supportive mechanism by which the conceptus also inhibits luteolysis is by shifting endometrial prostaglandin (PG) synthesis to luteoprotective PGE2. Progesterone stimulates endometrial production of factors that are essential for conceptus development. Priming the uterus by progesterone and loss of progesterone receptors from the uterine epithelium by D1ay 10-12 after estrus are key for achieving endometrial receptivity for implantation. Conceptus implantation involves a series of events, many resembling the inflammatory reaction, that are greatly influenced by cytokines, growth factors, and prostaglandins. We herein present a novel, dual role for PGF2α in corpora lutea that depends on the acquisition of luteolytic sensitivity, based on the knowledge that PGF2α triggers pathways involved in luteolysis during the estrous cycle or/and may have an alternative function in maintaining progesterone synthesis during pregnancy. We also point out a new role for PGF2α that, together with PGE2, can act as embryonic signal mediators. PGF2α, which until recently was considered undesirable for promoting pregnancy, is now known to stimulate conceptus-maternal interactions and angiogenesis in the endometrium. This function is in line with other important prostaglandin functions, such as stimulating adhesion of trophoblasts (PGE2, PGI2) as well as endometrial vascular functions and trophoblast cell proliferation (PGI2). Finally, microRNAs have emerged as important post-transcriptional regulators of gene function, adding a new area of investigation that may enhance understanding of conceptus-endometrial interactions.

Seasonal infertility in gilts and sows: Aetiology, clinical implications and treatments.

In gilts and sows, the summer-autumn period often is characterized by reduced fertility. Heat stress and long photoperiods during the warm season can cause a reduction in feed intake and an imbalance of the hypothalamic-hypophysial-ovarian axis. The increased variability in the interval between oestrus onset and ovulation results in an increased number of poorly timed inseminations. The altered endocrine activity compromises follicular and corpora luteal development, reduces oocyte quality and increases embryo mortality. This paper reviews current knowledge on the metabolic and endocrine mechanisms associated with seasonal infertility in gilts and sows and describes some pharmacological approaches that can be utilized to counter this infertility.

Functional consequences of knocking down porcine prostaglandin synthases in SK-6 swine kidney cell line.

We hypothesized that in vitro knock-down of the previously cloned genes of prostaglandin synthases will result in a reduction of synthesis, and thus secretion of their respective products, i.e., prostaglandin (PG) E2 or PGF2α. For this purpose, we designed short hairpin RNA (shRNA)-encoding constructs to knock down porcine mPGES-1 and PGFS (also named as AKR1CL1 in GenBank) and used them to transfect swine kidney SK-6 cells. Knocking down PGFS or mPGES-1 transcripts resulted in at least 50% inhibition of protein expression of each respective enzyme, as well as a reduction in the production of their respective prostaglandin, PGF(2α) or PGE(2). These results confirmed the identities of PGFS and mPGES-1. Moreover, they illustrate a unique opportunity to use the gene knock-down constructs in primary endometrial cells in order to study their biological roles in the porcine endometrium, particularly during the establishment of pregnancy.

Evidence for the presence of stem/progenitor cells in porcine endometrium.

A population of adult stem cells responsible for cyclic reconstructing and remodeling has been proposed to reside in the highly regenerative mammalian endometrium. Recently, stem/progenitor cells have been identified in the human and mouse endometrium, but less is known about these cells in livestock animals. Using Hoechst 33342 fluorescent dye staining and flow cytometry, we identified an emerging cell side population that may be responsible for the regeneration process of the porcine endometrium. The percentage of side-population cells on Day 19 of the estrous cycle was significantly higher than that on Days 2-4. Moreover, single cells were able to seed clones that could differentiate into three independent mesenchymal-cell lineages. We also demonstrated the expression of specific markers of self-renewal cells on these side-population cells and the presence of a population of cells among the stromal cells that possess markers for mesenchymal stem cells. These results indicate that the porcine endometrium contains a population of cells with the capacity for self-renewal and a high rate of proliferation, which depend on the phase of the estrous cycle. These cells could potentially be involved in the cyclic reconstruction of the porcine endometrium.

Precision-cut luteal slices: a promising approach for studying luteal function in pigs.

The study was aimed to validate the precision-cut luteal slices to investigate porcine luteal function. Corpora lutea (CLs) were cut into 180-µm thick slices using Krumdick Tissue Slicer. The viability, tissue structure and steroidogenic acute regulatory protein (STAR) expression in the luteal slices did not differ between the beginning and the end of the 24-h incubation period. The luteal progesterone secretion showed a time- and dose-dependent response to porcine luteinizing hormone. The effects of prostaglandin F(2α) and 17ß-estradiol on progesterone secretion by porcine luteal slices were comparable to the previously reported in vivo results of the CL microdialysis system in the pig.

Global gene expression profiling of porcine endometria on Days 12 and 16 of the estrous cycle and pregnancy.

The objective of the study was to investigate transcriptomic profile of pig endometrium on Days 12 and 16 of pregnancy in comparison with the respective days of the estrous cycle. Labeled complementary DNA was hybridized to Porcine Long Oligo microarray containing 13,297 oligonucleotide probes, which represented complementary DNA and expressed sequence tags. Statistical analysis revealed 110 differentially expressed genes (DEGs) on Day 12 of pregnancy and 179 DEGs on Day 16 of pregnancy. In silico analysis of gene function and functionality networks revealed links between genes implicated in cell death and survival, protein synthesis, lipid metabolism, cellular movement, tissue development, and cell-to-cell signaling. On Day 12 of pregnancy, estrogen, transforming growth factor (TGF) ß1, and fibroblast growth factor (FGF) 2, and on Day 16 of pregnancy, epidermal growth factor (EGF), insulin, interleukin 11 (IL-11), and FGF family members were indicated as possible upstream regulators of several DEGs. Obtained results showed changes in global endometrial gene expression at the time of maternal recognition of pregnancy and embryo implantation. Additionally, these data revealed signaling molecules, which together with E2, may evoke molecular changes in the uterus, leading to successful pregnancy establishment.

Transgenic mice expressing inhibin α-subunit promoter (inhα)/Simian Virus 40 T-antigen (Tag) transgene as a model for the therapy of granulosa cell-derived ovarian cancer.

Granulosa cell tumors are rare, 3-7.6% of primary ovarian tumors, although with poor prognosis as the tumor-related mortality rate is 37.3%, with 80% of deaths occurring on recurrence. We have created a transgenic (TG) murine model for gonadal somatic cell tumors by expressing the powerful viral oncogene, Simian Virus 40 T-antigen (Tag), under the regulation of murine inhibin α-subunit 6 kb promoter (inhα/Tag). Gonadotropin dependent ovarian granulosa cell tumors were formed in females by the age of 5-6 months, with a 100% penetrance. We have successfully used the inhα/Tag model to test different treatment strategies for ovarian tumors. With a gene therapy trial in inhα/Tag mice crossbred with inhα/HSV-TK (herpes simplex virus thymidine kinase) mice (double TG), we proved the principle that targeted expression of HSV-TK gene in gonadal somatic cell tumors enabled tumor ablation by anti-herpes treatment. When we aimed at targeted destruction of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) expressing inhα/Tag tumor cells in vivo by a lytic peptide Hecate-CGß conjugate, we could successfully kill the tumor cells, sparing the normal cells. We recently found high zona pellucida glycoprotein 3 (ZP3) expression in inhα/Tag granulosa cell tumors, as well as in human granulosa cell tumors. We tested the concept of treating the ovarian tumors of inhα/Tag mice by vaccination against the ectopically expressed ZP3. Immunotherapy with recombinant human (rh) ZP3 was highly successful with no objective side effects in inhα/Tag females, suggesting rhZP3 immunization as a novel strategy for the immunotherapy of ovarian granulosa cell tumors.

Estrus synchronization affects WNT signaling in the porcine reproductive tract and embryos.

The purpose of the study was to investigate an effect of estrus synchronization with prostaglandin (PG) F(2α) and PMSG/hCG on WNT4, WNT5A, WNT7A, ß-catenin (CTNNB1) and E-cadherin (CDH1) gene expression. The weight of the uterus, morphometrical parameters of the endometrium and the number of CL were recorded. The analysis of estradiol (E(2)), prostaglandin (PG) F(2α) and E(2) content in the uterine luminal flushings (ULFs) and progesterone (P(4)) level in the blood serum were conducted. RNA was isolated from endometrial, luteal and embryonic tissue of pregnant non-synchronized (Control; n = 15) and pregnant synchronized (PGF(2α)/PMSG/hCG; n = 15) pigs. Whereas there was no change in uterine weight, differences in height of endometrial surface and glandular epithelium were found. However, height of the endometrium, number of the glands and capillaries were unaffected. The total number of the CLs was higher (P < 0.05) in animals treated with PGF(2α)/PMSG/hCG. The amount of E(2) and P(4) was lower (P < 0.05, P < 0.001, respectively) in pregnant gilts administrated with PGF(2α)/PMSG/hCG. The concentration of PGF(2α) in ULFs was not affected by hormonal management, while PGE(2) was higher (P < 0.01) in hormonally in comparison to non-hormonally treated pigs. The content of WNT4 mRNA in conceptuses increased on particular Days studied in Control and PGF(2α)/PMSG/hCG administered animals. WNT7A and CTNNB1 were affected by PGF(2α)/PMSG/hCG treatment in both conceptuses (P < 0.001, P < 0.05) and endometrial tissue (P < 0.001, P < 0.01). The PGF(2α)/PMSG/hCG treatment resulted in elevated expression of WNT4 (P < 0.001) and CTNNB1 (P < 0.05) in luteal tissue in comparison to the Control gilts. Moreover, luteal amount of WNT5A mRNA was higher in PGF(2α)/PMSG/hCG animals in comparison to the Control group (P < 0.05). Presented data show that exogenous hormones administration can affect gene expression in the porcine reproductive tract and embryo.

Immortalization of swine umbilical vein endothelial cells (SUVECs) with the simian virus 40 large-T antigen.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis.

Gene expression of WNTs, ß-catenin and E-cadherin during the periimplantation period of pregnancy in pigs--involvement of steroid hormones.

WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. ß-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, ß-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17ß-estradiol (E(2)) and progesterone (P(4)) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, ß-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E(2) on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E(2) and P(4) level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, ß-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.

Effect of steroids on HOXA10 mRNA and protein expression and prostaglandin production in the porcine endometrium.

The homeobox A (HOXA) family of genes is responsible for segmental development of the female reproductive tract during embryogenesis. However, HOXA10 has been shown to be essential not only for uterus development, but also for implantation. Persistent expression and steroid-dependent regulation of this gene has been demonstrated in adult human, primate, murine and canine uteri. Moreover, HOXA10-dependent expression of prostaglandin H synthase-2 (PGHS-2), a key enzyme in prostaglandin production, has been previously detected. The role of the HOXA10 gene in the porcine uterus is not well established. Therefore, the present studies were undertaken to 1) examine the effect of E(2) and P(4) on HOXA10 mRNA and protein content in the endometrium collected on day 9 of the estrous cycle and 2) determine the PGHS-2 protein expression and PGE(2) and PGF(2α) secretion from endometrial tissue in response to steroid treatment. Endometrial explants collected from mature gilts on day 9 of the estrous cycle were incubated with E(2) (1-100 nM), P(4) (10-1000 nM) or E(2) (10 nM) and P(4) (100 nM) for 24 h. E(2) alone or E(2) in the presence of P(4) increased HOXA10 mRNA expression in the endometrium (P<0.05). The HOXA10 protein level was upregulated in response to E(2), P(4) and both steroids administered simultaneously (P<0.05). Moreover, E(2) and P(4) stimulated PGHS-2 protein expression in cultured endometrial explants. PGE(2), but not PGF(2α), secretion increased in the presence of E(2) (P<0.05). However, the release of both prostaglandins was decreased after treatment of endometrial explants with the highest dose of P(4) (P<0.01). These results demonstrate that E(2) and P(4) are important regulators of HOXA10 gene expression in the adult porcine endometrium during the mid-luteal phase of the estrous cycle. Additionally, the similar profiles of endometrial HOXA10 and PGHS-2 expression in the presence of E(2) and P(4) indicate that both genes are simultaneously regulated by steroids in the porcine uterus.

Seminal plasma affects prostaglandin synthesis in the porcine oviduct.

Seminal fluids introduced to the female reproductive tract at mating can affect subsequent events, such as ovulation, fertilization, conception, and pregnancy. Bioactive molecules present in seminal plasma can modify the cellular composition, structure, and function of local tissues and of tissues distal to the tract. The oviduct plays a decisive role in reproduction providing a beneficial milieu for gamete maturation, fertilization, and early embryonic development. Therefore we have investigated whether intrauterine infusion of seminal plasma can modulate prostaglandin (PG) synthesis in the porcine oviduct through regulation of gene and protein expression of enzymes of prostaglandin synthesis pathway. Among several enzymes involved in the prostaglandin synthesis pathway tested in the present study PGF(2α) synthase (PTGFS) and prostaglandin 9-ketoreductase (CBR1), which convert PGE(2) to PGF(2α), expression were significantly down-regulated in the oviducts on Day 1 after seminal plasma infusion into the uterine horns. The effects of the treatment were transient and by Day 5 levels of PTGFS and CBR1 were comparable in seminal plasma-treated and control animals. Additionally, increased PGE(2) to PGF(2α) and PGFM to PGF(2α) ratios in the oviductal tissues were indicated. Our results clearly demonstrate that seminal plasma affects prostaglandin synthesis in the porcine oviduct. Altered PTGFS and CBR1 expression in consequence changed PGE(2) to PGF(2α) and PGFM to PGF(2α) ratios in the porcine oviduct.

Oxytocin and tumor necrosis factor alpha stimulate expression of prostaglandin E2 synthase and secretion of prostaglandin E2 by luminal epithelial cells of the porcine endometrium during early pregnancy.

Oxytocin (OXT) and tumor necrosis factor α (TNF) have been implicated in the control of luteolysis by stimulating endometrial secretion of luteolytic prostaglandin F(2α) (PGF(2α)). Nevertheless, OXT concentration in porcine uterine lumen increases markedly on days 11-12 of pregnancy, and TNF is expressed in endometrium during pregnancy. The objective of the study was to determine the effect of OXT and TNF on expression of the enzymes involved in PG synthesis: PG-endoperoxide synthase 2 (PTGS2), PGE(2) synthase (mPGES-1) and PGF synthase, and PGE(2) receptor (PTGER2), as well as on PG secretion by endometrial luminal epithelial cells (LECs) on days 11-12 of the estrous cycle and pregnancy. LECs isolated from gilts on days 11-12 of the estrous cycle (n=8) and pregnancy (n=7) were treated with OXT (100  nmol/l) and TNF (0.6  nmol/l) for 24  h. OXT increased PTGS2 mRNA and mPGES-1 protein contents, as well as PGE(2) secretion but only on days 11-12 of pregnancy. TNF stimulated PTGS2 and mPGES-1 mRNA, as well as mPGES-1 protein expression and PGE(2) release on days 11-12 of pregnancy and the estrous cycle. In addition, expressions of PTGER2 and PTGER4 were determined in corpus luteum (CL). Abundance of PTGER2 mRNA and PTGER4 protein in CL was upregulated on day 14 of pregnancy versus day 14 of the estrous cycle. This study indicates that TNF and OXT regulate PGE(2) synthesis in LECs during early pregnancy. PGE(2) secreted by LECs, after reaching ovaries, could have a luteoprotective effect through luteal PTGER2 and PTGER4, or may directly promote uterine function and conceptus development.