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.

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.

STAT3 in porcine endometrium during early pregnancy induces changes in extracellular matrix components and promotes angiogenesis†.

A molecular interaction between maternal endometrium and implanting conceptus can lead to activation of a variety of transcription factors that regulate expression of several genes necessary for the process of embryo implantation. While, signal transducer and activator of transcription 3 (STAT3) is responsible for decidualization and epithelial remodeling in humans and mice, its role in porcine endometrium has not been explored before. In the present study, we observed a pregnancy dependent increase in gene and protein expression of STAT3. Phosphorylated STAT3 was predominantly present in the endometrium of pregnant animals in luminal and glandular epithelium and in the endothelium of blood vessels with a weak staining in stromal cells. Interleukins, IL-1ß and IL-6, and epidermal growth factor (EGF)-induced STAT3 expression and phosphorylation in endometrial explants collected on Day 13 of the estrous cycle. Biological significance of STAT3 was evaluated by blocking its phosphorylation with STAT3-specific inhibitor, Stattic. Using porcine extracellular matrix (ECM) and adhesion molecule array, EGF was shown to induce changes in gene expression of ECM components: MMP1, MMP3, MMP12, LAMA1, SELL, and ICAM1, which was abrogated in the presence of Stattic. Transcriptional activity of STAT3 was observed in promoter regions of MMP3 and MMP12. Additionally, IL-6-induced STAT3 phosphorylation upregulated VEGF and VCAM1 abundances in endometrial-endothelial cells (EEC). Moreover, IL-6 resulted in an increase in EEC proliferation and capillary formation which was reversed in the presence of Stattic. Results of present study reveal a role for STAT3 phosphorylation in regulating extracellular matrix remodeling and angiogenesis in porcine endometrium to facilitate embryo implantation.

Changes in Porcine Corpus Luteum Proteome Associated with Development, Maintenance, Regression, and Rescue during Estrous Cycle and Early Pregnancy.

Corpus luteum (CL), a transitory gland, undergoes rapid growth in a limited time to produce progesterone (P4) followed by its regression. A complex molecular signaling is involved in controlling luteal P4 production. In the present study, 2D gel electrophoresis-based proteomics and in silico functional analysis were used to identify changes in key proteins and pathways in CL along the different stages of the estrous cycle as its development progresses from early (Day 3) to mid-luteal phase (Day 9), effective functioning (Day 12) followed by regression (Day 15) or, in the case of pregnancy, rescue of function (Day 15). A total of 273 proteins were identified by MALDI-MS/MS analysis that showed significant changes in abundances at different stages of CL development or regression and rescue. Functional annotation of differentially abundant proteins suggested enrichment of several important pathways and functions during CL development and function maintenance including cell survival, endocytosis, oxidative stress response, estradiol metabolism, and angiogenesis. On the other hand, differentially abundant proteins during CL regression were associated with decreased steroid synthesis and metabolism and increased apoptosis, necrosis, and infiltration of immune cells. Establishment of pregnancy rescues CL from regression by maintaining the expression of proteins that support steroidogenesis as pathways such as the super-pathway of cholesterol biosynthesis, RhoA signaling, and functions such as fatty acid metabolism and sterol transport were enriched in CL of pregnancy. In this study, some novel proteins were identified along CL development that advances our understanding of CL survival and steroidogenesis.

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.

Proteomic Analysis of Porcine Pre-ovulatory Follicle Differentiation Into Corpus Luteum.

The luteinization of the follicular cells, following a LH surge, causes extensive molecular and structural changes in preovulatory follicles (POF) that lead to ovulation and ultimate formation of the corpus luteum (CL). The objective of this study was to identify proteins expressed in porcine POF before the LH surge and a new CL formed, 2-3 days after ovulation, and evaluate proteome changes associated with formation of the CL from a follicle. We used 2D-gel electrophoresis-based proteomics and tandem mass spectrometry followed by a functional analysis using Ingenuity Pathway analysis (IPA) to evaluate functional pathways associated with the luteinization process. Protein lysates were prepared from isolated POFs and from the newly formed CL. A total of 422 protein spots were identified in both structures. A total of 15 and 48 proteins or their proteoforms were detected only in the POFs and CL, respectively. An IPA analysis of a POF proteome showed that most of the follicular proteins were involved in cellular infiltration, endoplasmic stress responses, and the protein ubiquitination pathway. Most of the early luteal proteins were associated with steroid metabolism, cell death and survival, free radical scavenging, and the protein ubiquitination pathway. A comparison of a follicular proteome with that of an early luteal proteome revealed that 167 identified proteins or their proteoforms were differentially regulated between POFs and the newly formed CL (p < 0.05 and a fold change of >1.8). Proteins that were significantly more abundant in follicles included cAMP-dependent protein kinase, histone binding protein RBBP4, reticulocalbin, vimentin, and calumenin; more abundant luteal proteins included albumin, farnesyl diphosphate synthase, serine protease inhibitors, elongation factor-1, glutaredoxin, and selenium-binding protein. Proteins that were significantly altered with luteal formation were found to be associated with cholesterol biosynthesis, cell death and survival, and acute phase response. Moreover, upstream regulators of differentially abundant proteins in CL were identified that included insulin growth factor-1, sterol regulatory element-binding transcription factor-1, and nuclear factor erythroid-derived 2. We have identified novel proteins that advance our understanding of (1) processes associated with differentiation of POFs into the CL, (2) possible mechanisms of luteal cell survival, and (3) pathways regulating steroidogenesis in the newly formed CL.

Alterations in the distribution of actin and its binding proteins in the porcine endometrium during early pregnancy: Possible role in epithelial remodeling and embryo adhesion.

During early pregnancy, uterine epithelial cells undergo major transformations in their cytoskeleton that make the endometrium receptive for conceptus attachment. Actin binding proteins (ABPs) such as cofilin, gelsolin, and vinculin are involved in regulating actin polymerization, severing or crosslinking actin to integrins. However, whether ABPs are involved in epithelial remodeling or embryo adhesion in pigs is unknown. Therefore, the expression and distribution of these proteins were investigated in porcine endometrium on Days 10 and 13 (pre-implantation period), and 16 (attachment phase) of the estrous cycle or pregnancy. While day and pregnancy status had no effect on ABP gene expression, the protein abundance of vinculin was significantly higher on Day 13 than on Day 10 (p < 0.05) of the estrous cycle, and its abundance was highest on Day 16 in the pregnant endometrium. Immunofluorescent staining showed alterations in the distribution of these proteins depending on the day of the estrous cycle or early pregnancy examined. Double immunofluorescent staining for the ABPs and actin revealed that while cofilin co-localized with actin in the apical epithelium on Days 13 and 16 of the estrous cycle, in pregnant animals, it was strongly associated with actin in the sub-epithelial stroma of the endometrium. Gelsolin was also co-localized with actin in the apical epithelium on Days 13 and 16 of the estrous cycle, but this association was absent in the pregnant endometrium. Vinculin co-localized with actin in the sub-epithelial stroma on Days 13 and 16 irrespective of the reproductive status, but was additionally associated with actin in the apical epithelium on Day 16 of pregnancy. Vinculin interacted with phosphorylated focal adhesion kinase in the endometrial epithelium, and the interaction was dependent on estradiol-17ß, a conceptus-secreted pregnancy-recognition factor in pigs. Furthermore, silencing vinculin in the endometrial epithelial cells negatively affected trophoblast adhesion to them. In conclusion, the influence of stage and reproductive status on the specific localization of actin and its binding proteins in the porcine endometrium suggests that they play a role in regulating the endometrial cytoskeleton. Moreover, vinculin may facilitate conceptus attachment to the epithelium by interacting with focal adhesion kinase.

Proteomic and network analysis of pregnancy-induced changes in the porcine endometrium on Day 12 of gestation.

Conceptus attachment is a time-sensitive process that requires a synchronized uterine environment created by molecular changes in the endometrium in response to ovarian hormones and conceptus signals. Porcine conceptuses undergo rapid elongation and differentiation, and secrete estrogens that serve as maternal-recognition-of-pregnancy signals during the peri-implantation period (Days 11-12). Pregnancy-induced proteomic changes in the porcine endometrium were measured during this period using two-dimensional differential gel electrophoresis of endometrial protein lysates from Day-12 pregnant versus non-pregnant animals (n = 4 each). Forty-four differentially abundant proteins in the pregnant endometrium were identified by mass spectrometry. The pregnant endometrium was associated with a unique protein profile, revealed by principal component analysis. A pregnancy-dependent increase in the abundance of serpins, cofilin, annexin A2, aldose reductase, cyclophilin, protein disulphide isomerase A3, and peroxiredoxin 1 was observed. Western blotting for some of the selected proteins confirmed their enrichment during pregnancy. Ingenuity pathway analysis identified several functions specifically over-represented among the differentially abundant proteins in the pregnant endometrium, including calcium signaling, angiogenesis, leukocyte migration, and cell movement. Interleukin-1 beta and beta-estradiol were identified as upstream regulators of several high-abundance proteins from pregnancy. Therefore, signals from porcine conceptuses, such as estrogens, interleukin 1B, and epidermal growth factor, either alone or in coordination with other factors, prepare the uterus for implantation. Mol. Reprod. Dev. 83: 827-841, 2016 © 2016 Wiley Periodicals, Inc.

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

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.