Prolonged exposure to dexamethasone alters the proteome and cellular phenotype of human testicular peritubular cells.

Human testicular peritubular cells (HTPCs) are smooth muscle cells, which in the testis form a small compartment surrounding the seminiferous tubules. Contractions of HTPCs are responsible for sperm transport, HTPCs contribute to spermatogenesis, have immunological roles and are a site of glucocorticoid receptor expression. Importantly, HTPCs maintain their characteristics in vitro, and thus can serve as an experimental window into the male gonad. Previously we reported consequences of 3-day treatment with Dexamethasone (Dex), a synthetic glucocorticoid and multi-purpose anti-inflammatory drug. However, as glucocorticoid therapies in man often last longer, we now studied consequences of a prolonged 7-day exposure to 1 µM Dex. Combining live cell imaging with quantative proteomics of samples taken from men, we confirmed our recent findings but more importantly, found numerous novel proteomic alterations induced by prolonged Dex treatment. The comparison of the 7-day treatment with the 3-day treatment dataset revealed that extracellular matrix- and focal adhesion-related proteins become more prominent after 7 days of treatment. In contrast, extended stimulation is, for example, associated with a decrease of proteins related to cholesterol and steroid metabolism. Our dataset, which describes phenotypic and proteomic alterations, is a valuable resource for further research projects investigating effects of Dex on human testicular cells.

Reduced oxygen concentrations regulate the phenotype and function of human granulosa cells in vitro and cause a diminished steroidogenic but increased inflammatory cellular reaction.

Oxygen (O2) concentrations have recently been discussed as important regulators of ovarian cells. Human IVF-derived granulosa cells (human GCs) can be maintained in vitro and are a widely used cellular model for the human ovary. Typically, GCs are cultured at atmospheric O2 levels (approximately around 20%), yet the O2 conditions in vivo, especially in the preovulatory follicle, are estimated to be much lower. Therefore, we comprehensively evaluated the consequences of atmospheric versus hypoxic (1% O2) conditions for 4 days on human GCs. We found lower cellular RNA and protein levels but unchanged cell numbers at 1% O2, indicating reduced transcriptional and/or translational activity. A proteomic analysis showed that 391 proteins were indeed decreased, yet 133 proteins were increased under hypoxic conditions. According to gene ontology (GO) enrichment analysis, pathways associated with metabolic processes, for example amino acid-catabolic-processes, mitochondrial protein biosynthesis, and steroid biosynthesis, were downregulated. Pathways associated with glycolysis, chemical homeostasis, cellular response to hypoxia, and actin filament bundle assembly were upregulated. In accordance with lower CYP11A1 (a cholesterol side-chain cleavage enzyme) levels, progesterone release was decreased. A proteome profiler, as well as IL-6 and IL-8 ELISA assays, revealed that hypoxia led to increased secretion of pro-inflammatory and angiogenic factors. Immunofluorescence studies showed nuclear localization of hypoxia-inducible factor 1α (HIF1α) in human GCs upon acute (2 h) exposure to 1% O2 but not in cells exposed to 1% O2 for 4 days. Hence, the role of HIF1α may be restricted to initiation of the hypoxic response in human GCs. The results provide a detailed picture of hypoxia-induced phenotypic changes in human GCs and reveal that chronically low O2 conditions inhibit the steroidogenic but promote the inflammatory phenotype of these cells.

The Molecular Signature of Human Testicular Peritubular Cells Revealed by Single-Cell Analysis.

Peritubular cells of the human testis form a small compartment surrounding the seminiferous tubules. They are crucial for sperm transport, and they emerge as contributors to the spermatogonial stem cell niche. They are among the least known cell types of the human body. We employed single-cell RNA sequencing of cultured human testicular peritubular cells (HTPCs), which had been isolated from testicular samples of donors with normal spermatogenesis. The significant overlap between our results and recently published ex vivo data indicates that HTPCs are a highly adequate cellular model to define and study these cells. Thus, based on the expression of several markers, HTPCs can be classified as testicular smooth muscle cells. Small differences between the in vivo/in vitro expressed genes may be due to cellular plasticity. Plasticity was also shown upon addition of FCS to the culture medium. Based on transcriptome similarities, four cellular states were identified. Further analyses confirmed the presence of known stem cell niche-relevant factors (e.g., GDNF) and identified unknown functions, e.g., the ability to produce retinoic acid. Therefore, HTPCs allow us to define the signature(s) and delineate the functions of human testicular peritubular cells. The data may also serve as a resource for future studies to better understand male (in)fertility.

Profound Effects of Dexamethasone on the Immunological State, Synthesis and Secretion Capacity of Human Testicular Peritubular Cells.

The functions of human testicular peritubular cells (HTPCs), forming a small compartment located between the seminiferous epithelium and the interstitial areas of the testis, are not fully known but go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR) indicates that they may be regulated by glucocorticoids (GCs). Herein, we studied the consequences of the GC dexamethasone (Dex) in cultured HTPCs, which serves as a unique window into the human testis. We examined changes in cytokines, mainly by qPCR and ELISA. A holistic mass-spectrometry-based proteome analysis of cellular and secreted proteins was also performed. Dex, used in a therapeutic concentration, decreased the transcript level of proinflammatory cytokines, e.g., IL6, IL8 and MCP1. An siRNA-mediated knockdown of GR reduced the actions on IL6. Changes in IL6 were confirmed by ELISA measurements. Of note, Dex also lowered GR levels. The proteomic results revealed strong responses after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h of Dex exposure (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were extracellular matrix (ECM) and basement membrane components (e.g., FBLN2, COL1A2 and COL3A1), as well as PTX3 and StAR. These results pinpoint novel, profound effects of Dex in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may occur in men upon treatment with Dex for medical reasons.

Filamin A Orchestrates Cytoskeletal Structure, Cell Migration and Stem Cell Characteristics in Human Seminoma TCam-2 Cells.

Filamins are large dimeric F-actin cross-linking proteins, crucial for the mechanosensitive properties of a number of cell types. Due to their interaction with a variety of different proteins, they exert important regulatory functions. However, in the human testis the role of filamins has been insufficiently explored. Immunohistochemical staining of human testis samples identified filamin A (FLNA) in spermatogonia and peritubular myoid cells. Investigation of different testicular tumor samples indicated that seminoma also express FLNA. Moreover, mass spectrometric analyses identified FLNA as one of the most abundant proteins in human seminoma TCam-2 cells. We therefore focused on FLNA in TCam-2 cells, and identified by co-immunoprecipitation LAD1, RUVBL1 and DAZAP1, in addition to several cytoskeletal proteins, as interactors of FLNA. To study the role of FLNA in TCam-2 cells, we generated FLNA-deficient cells using the CRISPR/Cas9 system. Loss of FLNA causes an irregular arrangement of the actin cytoskeleton and mechanical instability, impaired adhesive properties and disturbed migratory behavior. Furthermore, transcriptional activity of typical stem cell factors is increased in the absence of FLNA. In summary, our data suggest that FLNA is crucially involved in balancing stem cell characteristics and invasive properties in human seminoma cells and possibly human testicular germ cells.

Palmitic Acid Targets Human Testicular Peritubular Cells and Causes a Pro-Inflammatory Response.

Palmitic acid (PA) is a major fatty acid, derived from diet and endogenous production, which is being linked to inflammation. While such actions of PA at the level of the testis remain difficult to examine, we reasoned that studies in human testicular cells may be instructive. Human testicular peritubular cells (HTPCs) can be isolated from men and cultured. They have contractile properties but also produce Interleukin 6 (IL6), express the inflammasome member NLRP3, and via glia cell line derived neurotrophic factor (GDNF), they contribute to the spermatogonial stem cell niche. We found that PA at 100 µM significantly increased the levels of IL6, while NLRP3 or the related Interleukin 1 beta (IL1beta) were not affected. The contractility marker calponin (CNN1) and the growth factor GDNF were likewise not affected. ELISA studies confirmed the stimulatory PA actions on IL6. Hence, PA derived from diet and/or endogenous sources may be able to foster a pro-inflammatory milieu in the testis. A possible link of these results to diet and high fat intake and obesity is indicated by the about 12-fold elevated testicular levels of IL6 in testes of obese rhesus monkeys (n = 3), fed with a Western Style diet. They had elevated 2-5-fold increased body fat and increased circulating triglyceride levels. Further consequences of PA and obesity for testicular functions remain to be evaluated.

The Glucocorticoid Receptor NR3C1 in Testicular Peritubular Cells is Developmentally Regulated and Linked to the Smooth Muscle-Like Cellular Phenotype.

Whether glucocorticoids (GC) can directly affect human testicular functions is not well understood. A predominant site of GC receptor (GR; NR3C1) expression in the adult testis are peritubular smooth muscle-like cells, which express smooth muscle actin (ACTA2), contract and thereby are involved in sperm transport. In contrast to the adult, neither GR nor ACTA2, or elastin (ELN) were detected in the peritubular compartment before puberty in non-human primate testes. In isolated human testicular peritubular cells (HTPCs), activation of GR by dexamethasone (Dex) caused the translocation of GR to the nucleus and stimulated expression of ACTA2 and ELN, without affecting the expression of collagens. Cytoskeletal ACTA2-rearrangements were observed and were associated with an increased ability to contract. Our results indicate post-pubertal testicular roles of GC in the maintenance of the contractile, smooth muscle-like phenotype of peritubular cells.

Human testicular peritubular cells, mast cells and testicular inflammation.

In man, the wall of seminiferous tubules forms a testicular compartment, which contains several layers of smooth muscle-like, "myoid", peritubular cells and extracellular matrix. Its architecture and its cellular composition change in male infertility associated with impaired spermatogenesis. Increased deposits of extracellular matrix, changes in the smooth muscle-like phenotype of peritubular cells and accumulation of immune cells, especially mast cells, are among the striking alterations. Taken together, the changes indicate that inflammatory events take place in particular within this compartment. This short review summarises recent studies, which pinpoint possible mechanisms of the interplay between peritubular cells and mast cells, which may contribute to sterile inflammation and impairments of testicular function. These insights are based mainly on cellular studies, for which we used isolated human testicular peritubular cells (HTPCs), and on the examination of human testicular sections. Recent data on immunological properties of peritubular cells, unexpected roles of the extracellular matrix factor, biglycan, which is secreted by peritubular cells and functions of mast cell products (chymase, tryptase and ATP) are presented. We believe that the results may foster a better understanding of peritubular cells, their roles in the human testis and specifically their involvement in infertility.

Alpha 1 adrenergic receptor-mediated inflammatory responses in human testicular peritubular cells.

Stress activates the sympathetic nervous system and is linked to impaired fertility in man. We hypothesized that catecholamines by acting on testicular cells have a role in these events, possibly by fostering an inflammatory environment. The cells of the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), express adrenergic receptors (ADRs) α1B, α1D, ß1 and ß2. A selective α1-ADR agonist, phenylephrine, increased intracellular Ca2+-levels in cultured HTPCs and induced COX-2, IL-6 and MCP-1 mRNA expression without affecting IL-1ß mRNA. These changes were paralleled by a significant increase in the secretion of IL-6 and MCP-1. Epinephrine was also effective, but salbutamol, a selective ß2-ADR agonist was not. Our results suggest that stress-associated elevation of catecholamines may be able to promote inflammatory events by targeting peritubular cells in the human testis. Blockage of α1-ADRs may therefore be a novel way to interfere with stress-related impairment of male reproductive functions.

Prostaglandin E2 (PGE2) is a testicular peritubular cell-derived factor involved in human testicular homeostasis.

In man, blockage of prostaglandin (PG)-production e.g. by non-steroidal anti-inflammatory drug (NSAIDs) may have negative testicular side effects, implying beneficial actions of PGs in the testis. We examined human testicular samples and isolated human testicular peritubular cells (HTPCs) to explore sites of PG-synthesis and targets. HTPCs express cyclooxygenase 1 (COX1) and secrete PGE2. Receptors (EP1, 2, 4) were specifically identified in peritubular cells. In HTPCs PGE2 significantly increased mRNA levels of the contractility protein calponin, but did not induce contractions. PGE2, as well as EP1 and EP4 receptor agonists, significantly increased glia cell line derived neurotrophic factor (GDNF) mRNA and/or protein levels. Importantly, the NSAID ibuprofen reduced PGE2 and this action also lowered SMA and calponin mRNA levels and levels of secreted GDNF protein. The results reveal an unknown PGE2 system in the human testis, in involving peritubular cells, which may be prone to interference by NSAIDs.

Mast cells in human testicular biopsies from patients with mixed atrophy: increased numbers, heterogeneity, and expression of cyclooxygenase 2 and prostaglandin D2 synthase.

OBJECTIVE: To determine intratesticular abundance and distribution of tryptase-positive mast cells (MCs) and to examine the expression of key enzymes of prostaglandin (PG) synthesis, cyclooxygenase 2 (COX2), and PGD2 synthase in the testes of men with mixed atrophy (MA) syndrome and in normal samples. DESIGN: Retrospective study. SETTING: Academic research institute and andrology practice. PATIENT(S): Nineteen men. INTERVENTION(S): Testicular biopsies. MAIN OUTCOME MEASURE(S): Immunohistochemistry and evaluation of COX2 and tryptase-positive MCs, laser microdissection of immunoreactive cells followed by reverse transcriptase polymerase chain reaction for COX2 and PGDS-H mRNA, and transmission electron microscopy. RESULT(S): In line with previous studies, few tryptase-positive MCs, but no COX2-positive cells, were observed in testes with normal spermatogenesis. In MA samples, the number of tryptase-positive MCs was significantly increased and the cells accumulated in the walls of the seminiferous tubules. In 11 of 13 MA samples, COX2 protein was detected. In 2 cases, Leydig cells were positive; however, in all 11 of 13 cases, COX2 was localized to MCs, coexpressing tryptase. The proportion of MCs coexpressing COX2 varied from 4% to 35%. Laser microdissection of tryptase/COX2-positive MCs followed by reverse transcriptase polymerase chain reaction revealed PGDS-H mRNA. Transmission electron microscopy identified typical MCs with abundant granules and another subtype with only a few granules, implying that MCs may differentiate in the testes. CONCLUSION(S): In patients with MA, testicular MC numbers and phenotypes change with respect to the ability to express COX2 and synthesize PGs. MCs and PGs have emerged as players in spermatogenic dysfunction.

Relationships between uterine blood flow, peripheral sex steroids, expression of endometrial estrogen receptors and nitric oxide synthases during the estrous cycle in mares.

The objective of this study was to investigate the relationships between uterine perfusion and estrogen, progesterone and the uterine nitric oxide synthase (NOS) system in five trotter mares during the estrous cycle. Color Doppler sonography for measurement of uterine blood flow and collection of blood for determination of plasma estrogen and progesterone concentrations were performed on days 0 (= ovulation), 1, 5, 11 and 15 and daily during estrus (days -1 to -4) of one estrous cycle; endometrial biopsy collection for mRNA expression analysis of NOS and estrogen receptors was performed on days 0, 1, 5, 11, 15 and -3. Blood flow in each uterine artery was assessed by calculating the mean time-averaged maximum velocity (TAMV) and the pulsatility index (PI). Plasma concentrations of estrogen and progesterone were determined using specific enzyme immunoassays. The mRNA expressions of endothelial NOS (eNOS), inducible NOS (iNOS) as well as estrogen receptors α (ERα) and ß (ERß) were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The TAMV and PI had a biphasic pattern during the estrous cycle (P<0.05), with maximum and minimum, respectively, values on days 5 and -4. Estrogen receptor mRNA concentrations increased significantly during days 15 (ERα) and -3 (ERß). Transcript expression of eNOS, but not iNOS, had a biphasic pattern during the cycle (P<0.05) with maximum levels on days 5 and -3 and correlated positively with TAMV (r=0.81, P=0.05). We infer that the uterine NOS system, especially eNOS, plays an important role in the regulation of uterine blood flow during the estrous cycle in mares.

Biosensor analyses of serum autoantibodies: application to antiphospholipid syndrome and systemic lupus erythematosus.

Autoimmune disorders are rare human diseases characterized by the presence of circulating autoantibodies that bind the body's own structural compounds as target antigens. The detection of autoantibodies is important for the diagnostic process. Immunofluorescence and immunoassay methods do not allow a reliable characterization of binding characteristics. Therefore, novel analytical techniques should be considered. This review describes the application of surface plasmon resonance biosensor systems for the diagnosis of autoimmune disorders. The covalent attachment of native antigens to the sensor chip is a suitable method for obtaining highly reproducible analyses of autoantibodies, allowing the evaluation of kinetic rate and affinity constants, and it may enable the identification of disease-relevant autoantibodies linked to disease progression. The autoantibody microarray is another future-oriented technique. Patterns of differential antigen recognition should allow early diagnosis. This is due to the fact that a broad range of autoreactive B cell responses in autoimmune disorders can only be mirrored by including a sufficient number of antigens in a microarray format.

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine.

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.

Effect of the luteinising hormone surge on regulation of vascular endothelial growth factor and extracellular matrix-degrading proteinases and their inhibitors in bovine follicles.

The aim of the present study was to evaluate the pattern of regulation of vascular endothelial growth factor (VEGF)-A (isoforms 121, 165, 189), VEGF receptor tyrosine kinases (VEGF-R1 and VEGF-R2), matrix metalloproteinase (MMP)-1, MMP-2, MMP-14, MMP-19, tissue-specific inhibitor of metalloproteinases (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in time-defined follicle classes before (0 h) and after the application of gonadotrophin-releasing hormone (GnRH). Bovine ovaries containing periovulatory follicles or new corpora lutea (CL; Days 1-2) were collected 0, 4, 10, 20 and 25 h (follicles) or 60 h (CL) after the injection of GnRH. Transcripts of VEGF isoforms (VEGF(121), VEGF(165), VEGF(189)) were upregulated 4 h after GnRH injection (during the luteinising hormone (LH) surge) and decreased thereafter to lowest levels around ovulation. All VEGF isoforms and their receptors were upregulated again after ovulation. The VEGF peptide concentration in follicular fluid decreased 20 h after GnRH injection, followed by an increase in follicles 25 h after GnRH. Expression of MMP-1 mRNA increased rapidly 4 h after GnRH injection and remained high during the entire experimental period. In contrast, MMP-19 mRNA increased significantly only after ovulation. Expression of TIMP-1 mRNA increased 4 h after GnRH and again after ovulation. Expression of tPA mRNA increased 4 h after GnRH and remained high during the entire experimental period, whereas expression of uPA transcripts increased significantly only after ovulation. Both uPAR and PAI-1 mRNA levels increased in follicles 4 h after GnRH and again after ovulation. The amount of MMP-1 protein (immunolocalisation) increased in follicles 10 h after GnRH: additional staining was observed in the granulosa cell layer. In conclusion, the temporal and spatial pattern of regulation of VEGF and extracellular matrix-degrading proteinases during periovulation suggests they are important mediators of the LH-dependent rupture of bovine follicles and for early CL formation (angiogenesis).

Expression of 11beta-hydroxysteroid-dehydrogenase 2 in Sertoli cells of boar testes.

11beta-Hydroxysteroid-dehydrogenase 2 (11beta-HSD2) activity occurs in boar testes but it is not known which cell types express 11beta-HSD2 mRNA and protein. Therefore, testes samples were taken from mature boars. For immunocytochemistry and Western blot pig-specific antibodies were raised against a 10-amino acid peptide corresponding to amino acids 391-400 of the coding sequence. Quantitative PCR was performed in testis homogenates and additionally RT-PCR in samples collected by UV-single cell microdissection. Data show that in interstitial tissue 11beta-HSD2 is expressed in Leydig cells and additionally in blood capillaries. In tubuli, 11beta-HSD2 primarily is formed in Sertoli cells whereas occurrence in spermatogonia could not be definitely proven. Because glucocorticoid receptors were never found in boar Leydig cells, it is concluded that the expression of 11beta-HSD2 in several types of cells forms consecutive lines of defense to protect spermatogonia against glucocorticoid-induced apoptosis.

Changes of testicular aromatase expression during fetal development in male pigs (Sus scrofa).

Male pig fetuses secrete considerable amounts of estrogens, but the location of aromatase activity within the fetal testis is not known. The location of aromatase expression was investigated by immunocytochemistry in fetal testes from week 6 (n = 5), weeks 10, 13, and 15 (each: n = 6) of gestation and additionally in neonates (n = 4). Blood was sampled from the umbilical artery of fetuses and jugular vein of neonates. Histological evaluation of testes involved morphological criteria and counting of Leydig cells, Sertoli cells, and gonocytes. Aromatase activity was localized immunocytochemically and quantified by the percentage of positive stained cells within the same cell type. Aromatase expression was further characterized by quantitative RT-PCR. Concentrations of estrogens, testosterone, FSH, and LH were measured in blood plasma. Total estrogens increased from week 10 to a maximum of 31.03 nmol/l in week 15. Increased testosterone concentrations were only measured at week 6 and were paralleled by slightly elevated estrogens. Thereafter, testosterone dropped and was low throughout. The increase of estrogens was not paralleled by a similar increase of FSH and LH but was related to the increase of the total number of Leydig cells. This increase was also found for mRNA expression. Both Leydig cells and gonocytes were identified as contributors to estrogen formation. Gonocytes were the main source of aromatase at week 10, when gene expression by Leydig cells is low due to the preparation of a wave of Leydig cell mitosis.

Expression of fibroblast growth factor 1 (FGF1) and FGF7 in mature follicles during the periovulatory period after GnRH in the cow.

The aim of this study was to evaluate the expression pattern of mRNA for fibroblast growth factor 1 (FGF1), FGF7, and their receptor variants (FGFR2IIIb) in time-defined follicle classes before LH surge, between LH surge and ovulation, and in the early corpus luteum (CL) in the cow. The ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (before LH surge); 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (periovulation), and early CL (Days 2-3). The mRNA expression was analyzed by quantitative real-time PCR (RotorGene 3000). The mRNA expression of FGF1 showed no significant differences in the follicle groups examined, but increased significantly at the early CL phase. A transient increase in FGF7 mRNA expression was observed 3-5 h after GnRH and again in the early CL phase. In contrast, the expression of FGFR2IIIb was constant throughout the period from the final growth of the follicle to early CL formation. The results of this study suggest that FGF1 and FGF7 may be involved differently in the process of follicle maturation and CL formation, which is strongly dependent on angiogenesis.

Regulation of endometrial fibroblast growth factor 7 (FGF-7) and its receptor FGFR2IIIb in gilts after sex steroid replacements, and during the estrous cycle and early gestation.

The aim of this study was to characterize the effect of ovarian steroids and early gestation on the expression of fibroblast growth factor 7 (FGF-7) and its receptor (FGFR2IIIb) in the porcine endometrium. In Experiment 1, gilts were ovariectomized (OVX) on day 10 of the estrous cycle and treated thereafter with vehicle (VEH), progesterone (P4), estradiol benzoate (EB), or P4+EB. Days 12 and 20 cyclic gilts (C12 and C20) were used to determine the influence of physiologically low and high plasma estradiol and progesterone concentrations on their expression. In Experiment 2, the expression of FGF-7 and FGFR2IIIB was characterized on days 1 (G 1) and 12 (G 12) of gestation. FGF-7 and FGFR2IIIb mRNA were quantified by quantitative real-time RT-PCR, and localization of FGF-7 protein in steroid-treated and early pregnant gilts was performed by immunohistochemistry. VEH-gilts expressed both FGF-7 and FGFR2IIIB mRNA. We found a significant effect of EB, but no effects of P4 or P4+EB on the mRNA expression of FGF-7. FGFR2IIIb mRNA significantly decreased after the EB and combined P4+EB treatments, compared to P4 only substituted animals. Day 12 cyclic gilts showed significantly higher FGF-7 and FGFR2IIIb mRNA expression compared with day 20 gilts. Between day 1 and 12 of gestation, FGF-7 mRNA expression differed highly while FGFR2IIIb transcripts only varied significantly. FGF-7 protein was localized in endometrial epithelia, vascular smooth muscle, and the endothelium of different types of blood vessels. Staining was weak in VEH and P4 treated gilts, whereas it was prominent following EB and P4+EB. FGF-7 antibody strongly stained the luminal epithelium on day 12 of gestation. In summary, FGF-7 and FGFR2IIIb mRNA expression is regulated differently by exogenous ovarian steroids, assuming progesterone in connection with a specific amount of 17beta-estradiol, whereas the receptor seems to be inhibited by estradiol. Both transcripts coordinately increased during the progesterone dominated phase on day 12 both in cyclic and early pregnant gilts. We conclude that estradiol and progesterone are involved in the regulation of this ligand-receptor system, which might have an important role in preparing endometrial tissue for implantation in gilts.

Expression of epidermal growth factor receptor (EGF-R), vascular endothelial growth factor receptor (VEGF-R) and fibroblast growth factor receptor (FGF-R) systems in porcine oviduct and endometrium during the time of implantation.

The oviduct and uterus provide the environment for the establishment of pregnancy. Among others, growth factor systems are involved in functional signaling interactions at the pre- and peri-implantation maternal-conceptus interface in pigs. Distinct regulation of epidermal growth factor Receptor (EGF-R), vascular endothelial growth factor receptor (VEGF-R) and fibroblast growth factor receptor (FGF-R) systems and of bioactivation of EGF-R in porcine oviduct and endometrium during the estrous cycle, early pregnancy and during steroid replacement in ovariectomized gilts is summarized. Remarkable influences of ovarian steroids and EGF on the expression of specific markers of transcription and translation in these tissues are discussed. Known biological effects of the EGF, VEGF and FGF are related to cellular differentiation and angiogenesis. This suggests their involvement in the transformation of the endometrium into a decidua subsequently leading towards successful establishment of pregnancy. Peripheral steroids may exert their effects on epithelial cells both in a direct genomic manner or through mediators such as growth factors. The aim of our study was to draw specific attention to the paracrine regulation in the porcine endometrium especially during the implantation window.