Mesometrial Tissue Research Articles

Abstract P309: Aberrant Corin and PCSK6 in the Placenta of Hyperinsulinemic Dams

Introduction: Corin, a serine protease converts pro-atrial natriuretic peptide (pro-ANP) and pro-brain natriuretic peptide (pro-BNP) to the mature respective peptides. There is evidence that the pregnant uterus produces ANP, where it promotes spiral artery remodelling, essential for adequate blood supply to the developing conceptus. Aim: Here we examine corin and PCSK6, a key enzyme in the conversion of pro-corin to corin, in the placenta of hyperinsulinemic dams (HD) in comparison to normal pregnant dams (NPD). Materials and Methods: Female Wistar rats were rendered hyperinsulinemic by subcutaneous insulin pellet, mated and followed to day 21 of pregnancy. Both groups were then sacrificed and their placentas were dissected together with the implantation site designated mesometrial triangle (MT). Placentas and MT were sectioned from the maternal through the fetal surface to the base of the MT. For western blot (WB) the placentas and MT were separated, snap frozen in liquid nitrogen and stored at –80°C. RNA expression of corin was carried out by in-situ hybridization in 5 NPD and 6 HD with corin probe (RNAscope® Assay 2.5 Detection Reagent, Cat No. 507231) and image analysis was performed. Results: As previously described, HD (n=20) developed hypertension as compared with in NPD (n=16). Moreover, HD exhibited lower placental and foetal weights. Corin and PCSK6 mRNA and immunoreactive peptide were detected in the mesometrial tissues. Corin abundance as determined by western blot was significantly decreased in the placenta/ mesometrial triangle by ~40% (P<0.02), as was PCSK6 immunoreactivity. Corin and PCSK6 mRNAs displayed similar pattern of alterations as protein abundance, where in-situ hybridization for corin mRNA revealed a 50% (P<0.001) expression reduction. Corin mRNA was specifically expressed by cytotrophoblasts in the labyrinth. Concomitant with the downregulation of corin/PCSK6, ANP levels decreased in the MT of HD. Summary and Conclusion: Corin, PCSK6 and ANP are all expressed in the placenta of NPD but declined in dams with chronic hyperinsulinemia, suggesting a role of corin/PCSK6 pathway in the prevention of pregnancy-induced hypertension and intrauterine growth restriction, in this setting.

Porphyromonas gingivalis strain-dependent inhibition of uterine spiral artery remodeling in the pregnant rat.

Porphyromonas gingivalis (Pg) is an important periodontal pathogen that is also implicated in pregnancy complications involving defective deep placentation (DDP). We hypothesized that Pg invasion of the placental bed promotes DDP. Pregnant rats were intravenously inoculated with sterile vehicle, Pg strain W83, or A7436 at gestation day (GD) 14 (acute cohort). Nonpregnant rats received repeated oral inoculations for 3 months before breeding (chronic cohort). Tissues and/or sera were collected at GD18 for analysis. Pg infection status was determined by seroconversion (chronic cohort) and by presence of Pg antigen in utero-placental tissues processed for histology and morphometric assessment of spiral artery remodeling. Mesometrial tissues from seropositive dams were analyzed for expression of interleukin 1β, 6, and 10, TNF, TGF-β, follistatin-related protein 3, and inhibin beta A chain since these genes regulate extravillous trophoblast invasion. The in situ distribution of W83 and A7436 antigen in utero-placental tissues was similar in both cohorts. In the acute cohort, mesometrial stromal necrosis was more common with W83, but arteritis was more common with A7436 infection (P < 0.05). Increased vascular necrosis was seen in mesometrium of chronically infected groups (P < 0.05). Only A7436-infected animals had increased fetal deaths, reduced spiral artery remodeling, reduced inhibin beta A expression, and an increased proportion of FSLT3 positive extravillous trophoblasts within spiral arteries. While infection with both Pg strains produced varying pathology of the deep placental bed, only infection with strain A7436 resulted in impaired spiral artery remodeling.

98 UTERINE INVOLUTION AND VASCULAR PERFUSION DURING EARLY POSTPARTUM IN MARES

In horses, a rapid involution of the uterus occurs right after parturition, allowing the reestablishment of a favourable uterine environment for embryo development. However, limited evidence is found regarding the vascular events during puerperal period in mares. We aimed to evaluate the morphological (size of uterus and uterine fluid content) and haemodynamic (endometrial and mesometrial vascular perfusion) characteristics of the uterine involution process. Mares (n = 10) were evaluated by transrectal ultrasonography from the first day postpartum (d1) to the sixteenth day after first ovulation (D0 = ovulation). For ultrasound exams, a duplex B-mode and Doppler ultrasound instrument (M5 VET; Mindray Medical International Limited, China) equipped with a transrectal transducer was used. The previously pregnant (PH) and nonpregnant (NPH) horns were individually evaluated. Data were analysed for the main effects of horns (PH and NPH), day, and their interaction, using the PROC MIXED procedure of SAS software (9.3 version; SAS Institute Inc., Cary, NC, USA). Discrete variables were analysed by ANOVA. A reduction (P &lt; 0.05) in the uterine diameter was observed during the first 7 days postpartum, but the rate of uterine involution (decrease in uterine size) decreased thereafter. The involution was complete around the d21 for the NPH and around d24 for the PH. Presence of uterine luminal fluid (mm) was increased between d1 (no fluid) and d2 (31.41 ± 2.88) postpartum, followed by a decrease between d4 (30.43 ± 4.52) and d7 (10.20 ± 1.76). No fluid was observed after d16 postpartum or after the third day postovulation (D3). For endometrial and mesometrial vascular perfusion, only a day effect (P &lt; 0.05) was observed. An increase in the endometrial and mesometrial vascularization was detected, respectively, between d1 and d4, and between d1 and d2. Vascular perfusion did not differ after d4 for endometrial tissue, whereas it was reduced (P &lt; 0.05) between d2 and d10 for mesometrium. For the vascular perfusion after ovulation, an increase (P &lt; 0.05) from D0 to D5, followed by a decrease (P &lt; 0.05) between D5 and D11 and an increase (P &lt; 0.05) between D11 and D14 was observed in the endometrial and mesometrial tissues. The profile of the vascular perfusion in endometrium and mesometrium after first ovulation postpartum is similar to that observed during oestrous cycles and early pregnancy, indicating a return of the uterus to the pre-pregnant uterine characteristics in mares.Research was supported by FAPESP process no. 2010/10692-9 and CNPq process no. 135954/2011-8.

FITC localization of renin in rat mesometrial tissue

Renin, a hormone produced primarily by the kidneys in response to low blood pressure, is an immediate downstream gene product of Class I HOX gene regulation in the embryo. Examination of transgenic mice in which renin was linked to gfp reveals renin in renal, adrenal, and gonadal arteries in the embryo, giant trophoblast cells of the placenta, kidney and submandibular gland in both male and female, and uterine artery. In rat, giant trophoblast cells, other cells in fetal and maternal regions of the placenta, and cells of the tunica adventita of uterine artery as well as in the wall of the afferent arteriole of the kidney were labeled with antibody stain. This project examines pregnant and nonpregnant rat mesometrial tissues to identify cells that localize FITC labeled sheep anti‐rat renin antibody. Fluorescent granules were seen in cells resembling fibroblasts throughout the mesometrium and along the walls of small vessels within the mesometrium. Afferent arterioles were labeled and control tissues were not. These results demonstrate that the mesometrium is another site within rat female reproductive tissues in which renin is located, adding support to the hypothesis that extrarenal renin production may play a role in preecclampsia. Work is supported by NSF grant #0132823 and 0726185, and D’Youville College faculty council research funding.

Analysis of cytokine regulators inducing interferon production by mouse uterine natural killer cells.

In mice and women, terminal differentiation of uterine natural killer (uNK) cells commences during endometrial decidualization. Both proliferation and interferon (IFN)-gamma are induced. Uterine NK cell precursors appear to home from secondary lymphoid organs to decidualizing uteri and localize mesometrially to the central decidua basalis, the site of maternal arterial modification at Gestation Days (gd) 9.5-10. In mice, genetic absence of uNK cells results in absence of pregnancy-induced spiral artery modification. Administration of IFN-gamma to uNK-negative pregnant females induces arterial modifications without fetal loss. In this study, we investigated the roles of cytokines, known in other tissues to differentiate and activate NK cells, in induction of IFN-gamma production in normal mouse implantation sites. Fecundity evaluation, implantation site morphometry, and IFN-gamma quantification in interleukin (IL)-12p40(0/0), IL-18(0/0), dual IL-12p40(0/0)/IL-18(0/0) and congenic strains revealed the importance of both IL-12 and IL-18 in the induction of spiral artery modification and IFN-gamma synthesis. Immediately after implantation, IL-18 was localized transiently to decidual cells, but by gd8, IL-18 was produced solely by uNK cells, suggesting that early uNK cells are activated by stroma and lymphocyte-derived signals maintain later uNK cell activation. Mesometrial tissue of C57Bl/6J mice was examined by reverse transcription polymerase chain reaction assay in virgin, early postimplantation, and midgestation females for expression of the heterodimeric cytokines IL-23 (composed of IL-12p40 and a novel alpha chain), IL-27 (composed of two IL-12-related chains) and IL-27R. No expression was detected in virgin uteri. The four genes were induced by gd6, and uNK cells isolated from midgestation transcribed IL-23alpha and IL-27R. This study advances the understanding of uNK cell activation during normal pregnancy.

The decidual prolactin receptor and its regulation by decidua-derived factors.

Decidualization of the endometrial stroma in the rat gives rise to two different cell populations, located either mesometrially or antimesometrially in the uterus. We have previously shown that the rat decidua is the site of production and action of a PRL-like hormone. In this investigation we examined, using reverse transcription-PCR, whether and which type of PRL receptor (PRL-R) messenger RNA (mRNA) is expressed in the decidua, whether the receptor is confined only to one cell population, and whether the PRL-R expression is regulated by decidua-derived factors. The results indicate that the uterus of pseudopregnant rats does not express the PRL-R and that decidualization does not trigger a rapid appearance of PRL-R mRNA. It is only 3 days after the induction of decidualization that the long form of the PRL-R was first expressed. Thereafter, mRNAs for both the short (PRL-RS) and the long (PRL-RL) form became detectable in both antimesometrial and mesometrial decidua, although PRL-RL mRNA was much more abundant than PRL-RS. As development proceeded, PRL-R mRNA decreased and disappeared specifically from the antimesometrial decidua, whereas the mesometrial decidua continued to express this receptor mRNA. Concomitant with down-regulation of the PRL-R in the antimesometrial tissue was a rather abrupt expression of activin A. In contrast, the mesometrial tissue that maintained high levels of PRL-R mRNA expressed little activin A, but produced an activin-binding protein, alpha(2)-macroglobulin (alpha(2)MG). To determine whether activin A and alpha(2)MG regulate PRL-R expression, antimesometrial and mesometrial cells were separated by elutriation and maintained in culture in the presence or absence of activin A, alpha(2)MG, or follistatin. Just after cell separation, both cell populations expressed PRL-R, but not activin A. Within 6 h, activin A mRNA and protein became highly expressed in the mesometrial cells, whereas PRL-RL mRNA became undetectable. In contrast, activin A mRNA was at very low levels in the antimesometrial cells, and no activin A protein could be detected in the medium for at least 12 h. In these cells PRL-RL mRNA remained elevated. Addition of activin A to antimesometrial cells caused a marked down-regulation of PRL-RL mRNA expression, whereas addition of alpha(2)MG and follistatin to mesometrial cells prevented the disappearance of PRL-R. In summary, the results of this investigation 1) indicate that decidualization of the endometrial stroma induces the appearance of both forms of the PRL-R mRNAs; 2) show differential expression of the PRL-R mRNA in the two-cell population forming the decidua; 3) establish that this differential expression is due to two key decidual molecules, activin A and alpha(2) macroglobulin; and 4) demonstrate that activin A can cause the decidual cells to lose the PRL-R and that the disappearance of the decidual PRL-R can be prevented by addition to the culture of two activin binding proteins, follistatin and alpha(2) MG.

Alpha 2-macroglobulin expression in the mesometrial decidua and its regulation by decidual luteotropin and prolactin.

During decidualization, cells of the endometrium grow and differentiate giving rise to two different decidual tissues located in either the antimesometrial or mesometrial site of the uterus in the rat. These tissues have different functions in pregnancy. The antimesometrial decidua is an endocrine gland that secretes hormones, whereas the mesometrial decidua appears to play an important role in limiting trophoblast invasion. Since the decidual tissue of the rat produces alpha 2-macroglobulin (alpha 2MG), we examined whether this potent protease inhibitor is specifically expressed by the mesometrial tissue, the site of trophoblast invasion, and whether the alph 2MG gene is regulated by decidual luteotropin (DLt), the PRL-like hormone secreted by the neighboring antimesometrial cells. To determine the secretory proteins of the rat decidua, antimesometrial and mesometrial tissues were dissected out from pseudopregnant rats and cultured with [35S]methionine. The major protein secreted by the antimesometrial cell was the 29-kilodalton decidual luteotropin, whereas a 180-kilodalton protein was predominantly secreted by the mesometrial tissue. Immunoprecipitation studies of 35S-radiolabeled proteins revealed that this high mol wt protein is alpha 2MG and that it is secreted exclusively by the cells forming the mesometrial tissue. To examine whether the alpha 2MG gene was also expressed specifically in the mesometrial decidua, total RNA was isolated from both mesometrial and antimesometrial tissues of day 9-12 pseudopregnant rats and hybridized with alpha 2MG cDNA. Northern blot analysis revealed a 5.4-kilobase message, which was abundantly expressed in the mesometrial decidua. Little, if any, alpha 2MG mRNA was detected in antimesometrial decidua. The ontogeny of the message in the decidua correlated well with the development of the mesometrial tissue. To examine whether alpha 2MG expression is regulated by DLt and/or PRL, a highly specific polyclonal antibody to DLt was generated and decidual tissues were cultured in the presence or absence of DLt antibodies with or without PRL. Neutralization of DLt caused a marked decrease in alpha 2MG mRNA levels. This down-regulation was totally reversed by the addition of PRL and was not affected by alpha 2MG antibodies. In summary, the results of this investigation revealed a compartmentalized gene expression, synthesis and secretion of alpha 2MG in the decidua. The secretion of this protease inhibitor, specifically by the mesometrial tissue which is the site of trophoblast invasion, may be the reason for the minimal amount of tissue damage that occurs during placentation.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression and secretion of galanin during pregnancy in the rat.

The expression of galanin messenger RNA (mRNA) in the pituitary and conceptus of pregnant rats has been studied at various stages of gestation. Using Northern blot analysis and in situ hybridization we have found that high levels of mRNA coding for galanin were detected in the conceptus during early pregnancy. The level of expression in conceptuses increased until day 11-12 after which the levels decreased rapidly. In contrast, in the pituitary galanin mRNA continued to increase throughout pregnancy, especially in the latter half of pregnancy as serum estradiol levels has been reported to be increased. The size of the galanin transcript was the same in the conceptus and pituitary (0.9 kilobase). Expression of this mRNA was confined to the decidua and was first seen at day 5 of pregnancy at a time when implantation swellings were first observed. Galanin antisense probe hybridized strongly to the decidual cells that surround the implantation site. At day 11 of pregnancy the galanin mRNA was found in both the antimesometrial and the mesometrial tissue with the highest concentration in the region lateral to the antimesometrial cells and continued toward the mesometrial cells. Serum galanin levels measured by an RIA using synthetic rat galanin as standard and antisera raised against porcine galanin, exhibited a temporal pattern similar to the pattern of mRNA expression in decidua, with a 7.1-fold increase at day 12 of pregnancy followed by a decline. In summary, decidual cells may differentiate into an endocrine cell during pregnancy. The galanin secreted by these cells, in addition to acting locally in a paracrine/autocrine fashion, may function as a placental hormone with systemic effects on distal target tissues.

Local transfer of prostaglandin F 2α from the uterine lumen into the venous and arterial blood and into the uterine, mesometrial and ovarian tissue on Day 18 of pregnancy in the pig

Gilts of similar age and body mass were mated with a normal (eight gilts) or vasectomized boar (three gilts). On Day 17 of pregnancy or the oestrous cycle, silastic catheters were implanted into the carotid artery, utero-ovarian vein, uterine artery branch (5–10 cm from the uterine horn) and into the lumen of each uterine horn through the oviduct. The lumens of both uterine horns were ligated close to the cervix. Next day 3H-PGF 2α (10 8 dpm) dissolved in 30 ml of saline was injected into the lumen of the experimental horn and 30 ml of saline into the control horn of conscious animals. Plasma samples were collected through two catheters from the experimental horn and from the carotid artery, and the radioactivity was measured every 10 min for 2 h. In pregnant gilts the radioactivity in the plasma samples collected simultaneously from the uterine artery branch on the experimental side was 50–69% higher than in the carotid artery ( P<0.05) while no significant difference was observed in nonpregnant gilts. The animals were sacrificed at 120 min of the experiment and the radioactivity in different segments of the uterine tissue (endometrium and myometrium) and in different samples of mesometrium and mesovarium was measured. In the experimental horn of pregnant as well as of nonpregnant gilts the radioactivity of the endometrium was much higher than in the myometrium ( P<0.001). The results were opposite in the control horn ( P<0.001). In pregnant, contrary to nonpregnant, gilts, significant differences in the radioactivity of mesometrial tissues taken from the same area 5–10 cm from the uterine horn were found, i.e.: 24.2±9.5, 14.6±3.1 and 7.2± 1.3 (× 10 3dpm/g) in the wall of the vein, wall of the artery and the muscular layer of the mesometrium, respectively ( P<0.01). Labelled prostaglandin was found in the uterine flushing of the control horn (142.3±17.2 × 10 3dpm and 112.9±40.1 × 10 3dpm for pregnant and nonpregnant gilts respectively). This concentration exceeded many fold the concentration in the blood supplying the uterine horn. It is suggested that the back transfer of PGF 2α from the venous blood into the uterus with the arterial blood, and the ability of the uterine vein and artery wall to bind and retain PGF 2α may be a part of the complex corpus luteum protecting mechanism during early pregnancy.

Protein secretion by mesometrial and antimesometrial rat decidual tissue: evidence for differential gene expression.

Our recent finding that decidual luteotropin, a PRL-like hormone secreted by the rat decidua, is found primarily in the antimesometrial cells suggests strongly that the synthesis of this hormone may well be an important function of the antimesometrial tissue. The objective of this investigation was, therefore, 1) to determine whether antimesometrial tissue expresses mRNA for and actively secretes a protein(s) with PRL-like activity, and 2) to examine the pattern of protein production by the antimesometrial and mesometrial zones throughout decidual development. RNA obtained from decidual tissue of day 8 pseudopregnant rats was translated in a cell-free system. The translated products were subjected to PRL receptor affinity chromatography in the presence or absence of ovine PRL to assess binding specificity. The eluted 35S-labeled proteins were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. A major 28K protein bound specifically to and was eluted from PRL receptor-enriched luteal membranes. This protein also cross-reacted with antibodies to human PRL. To determine where the mRNA for this 28K protein is expressed and whether this protein represents a prohormone, RNA isolated from both antimesometrial and mesometrial tissue was translated in the presence or absence of microsomal membranes. The 28K protein was synthesized specifically by RNA isolated from the antimesometrial zone. No apparent change in the relative mol wt of the 28K protein was observed when translation was performed in the presence of microsomal membranes. To determine whether this protein is a secreted product and to investigate the pattern of protein secretion by the mesometrial and antimesometrial decidua, tissue explants obtained from both zones between days 9-13 of pseudopregnancy were cultured in the presence of [35S]methionine. Antimesometrial tissue secreted one major 28K protein which was capable of binding to PRL receptors on luteal membranes and was immunoprecipitated by antibodies to human PRL, whereas the mesometrial tissue primarily secreted an approximately 180K protein. The overall pattern of protein synthesis and release not only differed between the mesometrial and antimesometrial tissues but also differed with each day of pseudopregnancy. The secretion of several proteins decreased with advancing gestational age, while the secretion of other proteins began abruptly after day 11, coincident with regression of the antimesometrial tissue. In summary, results of this investigation have established that rat decidual tissue synthesizes and selectively secretes proteins, and the specificity and rate of production of these distinct