Expression of erythropoietin mRNA, protein and receptor in ovine fetal membranes.

Erythropoietin and its receptor have been identified in human, murine and ovine placentas. Based on the common embryonic origin of the placenta and fetal membranes, we postulated that erythropoietin is similarly expressed in the fetal membranes. Using in situ hybridization and immunohistochemistry, we tested the hypothesis that ovine fetal membranes are sites of erythropoietin production and action. At 86, 103 and 138 days gestation, erythropoietin mRNA and protein were present in the amnion localized to the cell layer consisting largely of amniotic epithelium and in the chorion localized to the chorionic columnar cells consisting of cytotrophoblasts. Binucleate cells, differentiated cytotrophoblasts known to produce hormones, were identified in the chorion in the region of erythropoietin expression but were not observed in amniotic tissue. The erythropoietin receptor protein was present in the amnion and chorion at 103 and 138 days gestation but was not observed in either tissue at 86 days. In summary, erythropoietin appears to be produced as well as utilized within the ovine amnion and chorion. Within the amnion, the amniotic epithelial cells express the erythropoietin gene whereas, within the chorion, either the cytotrophoblasts or the binuclear cells may be the source. Due to the presence of the receptor, we speculate that the erythropoietin produced in the membranes may mediate fetal membrane function and/or growth through an autocrine and/or paracrine mechanism. Further, the fetal membranes may be the source of erythropoietin in the amniotic fluid.

Placental expression of erythropoietin mRNA, protein and receptor in sheep.

In ovine placentae at 100 days gestation, we localized the expression of erythropoietin (EPO) mRNA by in situ hybridization and determined the cellular localization of EPO protein and EPO receptor protein by fluorescence immunohistochemistry. Erythropoietin mRNA was observed in maternal tissue at the apical tips of the fetal cytotrophoblastic villi at their interface with the maternal caruncle and was absent from both the centrally located fetal-maternal tissue and the more peripherally located maternal caruncle. An EPO-protein-associated fluorescent signal was observed in the same interface region as the EPO mRNA hybridization signal. An intense fluorescent signal associated with EPO receptor protein was observed in the apical fetal-maternal interface region with a lower density signal dispersed throughout the remainder of the interdigitating fetal-maternal tissue. The predominant cells in the apical fetal-maternal interface were identified as binucleate cells by immunohistochemistry. Thus the localization of the binucleate cells was the same as that for the EPO mRNA and the EPO protein, whereas the EPO receptor had a more generalized distribution. Since the binucleate cells are hormone producing cells, we speculate that the binucleate cells are the source of the EPO that is present in ovine placenta.

Developmental expression of vascular endothelial growth factor (VEGF) receptors and VEGF binding in ovine placenta and fetal membranes.

The receptor tyrosine kinases, kinase-insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1), and their ligand vascular endothelial growth factor (VEGF) are essential for the development and maintenance of placental vascular function during pregnancy. To further understand the role of VEGF in mediating angiogenesis and vascular permeability during development, the cellular localization of KDR and Flt-1 mRNA and protein, and the distribution of(125)I-VEGF binding sites in placenta, chorion and amnion of ovine fetuses were examined at three different gestational ages. In placentae at 62, 103 and 142 days, the predominant site of KDR mRNA and protein, and VEGF binding was the maternal vascular endothelium. In addition, a specific, although weak, signal for KDR mRNA was found in the maternal epithelium. At 103 and 142 days but not 62 days gestation, KDR mRNA and protein as well as VEGF binding sites were abundantly present in the endothelium of villous blood vessels. In the fetal membranes at 62, 103 and 142 days gestation, KDR mRNA and protein were expressed in the amniotic epithelium and intramembranous blood vessel endothelium, where binding of(125)I-VEGF was strong. There was no KDR mRNA or VEGF binding in the chorionic cytotrophoblast. Flt-1 expression was not detectable in placentae or fetal membranes at the three ages studied. In summary, the results demonstrated that VEGF receptors are present in the maternal and fetal vasculatures of the ovine placenta. This expression is consistent with a capillary growth-promoting function of KDR and its ligand VEGF. Further, the presence of KDR and VEGF binding sites in ovine fetal membranes suggests a role for VEGF in promoting intramembranous vascularity and permeability throughout gestation.

Fetal esophageal ligation induces expression of vascular endothelial growth factor messenger ribonucleic acid in fetal membranes.

OBJECTIVE: Obstruction of the fetal esophagus does not always produce the expected polyhydramnios. This is because of increased intramembranous absorption of amniotic fluid into the fetal circulation. A possible mediator for this increased absorption is vascular endothelial growth factor (VEGF). The present objective was to explore whether VEGF gene expression and action would be induced in fetal membranes and placentas of ovine fetuses after esophageal ligation. STUDY DESIGN: Five late-gestation fetal sheep underwent esophageal ligation and 5 served as control animals. On postoperative day 9, amnion, chorion, and placenta were collected for cellular localization and quantitation of VEGF messenger ribonucleic acid by in situ hybridization and Northern blot analysis. Reverse-transcription polymerase chain reaction was used to identify the VEGF molecular forms. Immunostaining with Ki-67 antibody was used to determine the proliferation of vascular endothelium in the fetal membranes and placentas. RESULTS: VEGF messenger ribonucleic acid was localized in amniotic epithelium, chorionic cytotrophoblast, and cytotrophoblast of the placenta. VEGF164 was the major transcript expressed in these tissues. The abundance of VEGF messenger ribonucleic acid in the amnion and chorion, but not in the placenta, was significantly increased in the ligated fetuses in comparison with the control fetuses. The proliferation of the intramembranous blood vessel endothelium was greater in the ligated fetuses than in the control fetuses. CONCLUSION: The levels of VEGF messenger ribonucleic acid and the proliferation of vascular endothelium in the amnion and chorion increased after fetal esophageal ligation. This provides a possible mechanism for the enhanced intramembranous absorption of amniotic fluid through increased vascularity and permeability of the fetal membranes, thus ameliorating the development of polyhydramnios. We speculate that the signal(s) that mediate the increase in VEGF expression is present in either the fetal urine or the fetal lung secretions, or both.

Cellular localization of vascular endothelial growth factor in ovine placenta and fetal membranes.

To further understand the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during development in the sheep placenta and fetal membranes, we examined the localization of VEGF mRNA and protein in placental, chorionic and amniotic tissues by in situ hybridization and immunohistochemistry in ovine fetuses at 62, 102 and 141 days gestation (term=150 days). In the placenta, VEGF mRNA expression and VEGF protein immunostaining were strong in cytotrophoblasts surrounding the villi. In addition, VEGF protein was localized in smooth muscle cells around fetal and maternal blood vessels and in the maternal epithelium. There was no apparent difference in placental VEGF mRNA or protein levels associated with advancing gestation. In the fetal membranes, VEGF mRNA was detected in the amniotic epithelium and the chorionic cytotrophoblastic cell layer. The intensity of the hybridization signals in both amnion and chorion appeared low at 62 days, moderate at 102 days and high at 141 days gestation. VEGF protein was detected in amniotic epithelium and chorionic cytotrophoblasts at all gestational ages studied. The increase in VEGF gene expression in fetal membranes as term approaches suggests that during fetal development VEGF may promote the vascularity and permeability of the microvessels which perfuse the fetal membranes, as well as permeability of the amniotic membrane itself. Thus VEGF may participate in the regulation of amniotic fluid volume.

The LOXL2 gene encodes a new lysyl oxidase-like protein and is expressed at high levels in reproductive tissues.

We have reported in this paper the complete cDNA sequence, gene structure, and tissue-specific expression of LOXL2, a new amine oxidase and a member of an emerging family of human lysyl oxidases. The predicted amino acid sequence, from several overlapping cDNA clones isolated from placenta and spleen cDNA libraries, shared extensive sequence homology with the conserved copper-binding and catalytic domains of both lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) protein. These conserved domains are encoded by five consecutive exons within the LOX, LOXL, and LOXL2 genes that also maintained exon-intron structure conservation. In contrast, six exons encoding the amino-terminal domains diverged both in sequence and structure. Exon 1 of the LOXL2 gene does not encode a signal sequence that is present in LOX and LOXL, suggesting a different processing and intracellular localization for this new protein. Expression of the LOXL2 gene was detected in almost all tissues with the highest steady state mRNA levels in the reproductive tissues, placenta, uterus and prostate. In situ hybridization identified placental syncytial and cytotrophoblasts responsible for the synthesis of LOXL2 mRNA and demonstrated a spatial and temporal expression pattern unique to the LOXL2 gene.

Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term.

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.

Developmental regulation of the human relaxin genes in the decidua and placenta: overexpression in the preterm premature rupture of the fetal membranes.

The decidua and placenta synthesize the human relaxins, termed H1 and H2, believed to be involved in collagen remodeling in the amnion and chorion in an autocrine/paracrine manner. The developmental regulation of the relaxin genes was quantitated in normal pregnancy by in situ hybridization histochemistry with six 48-mer oligonucleotide probes that detect both relaxin genes. A significant increase in relaxin expression occurred in both decidua (p < 0.01) and placenta (p < 0.05) at 12.5-14.4 wk gestation, with the mean peak value in the placenta more than double that of the decidua, suggesting a coordinate regulation of the relaxin genes. At term after spontaneous labor and delivery, a marginal increase in both decidual and placental relaxin gene expression occurred. Given these normal data, three abnormal preterm situations were investigated: 1) premature uterine contractions without prior rupture of the membranes, 2) premature rupture of the fetal membranes (PPROM), 3) cesarean section for medical reasons with intact membranes and no uterine contractions. Tissues showing intrauterine infection were eliminated. Significantly more relaxin was expressed in the preterm decidua from patients with PPROM when compared to patients in group 1 (p < 0.02) or group 3 (p < 0.008). These data were confirmed by Northern analysis with a relaxin cRNA probe. The placental tissues after PPROM also had a significantly higher and a uniform overexpression of relaxin in the placental syncytiotrophoblast. Tissues collected at term, in comparison, showed no such increases in decidua or placenta.

The human prolactin receptor in the fetal membranes, decidua, and placenta.

Human PRL is synthesized and secreted by the maternal decidua, but not by the chorionic cytotrophoblast of the chorion laeve or the placenta. The sites of action for decidual PRL are currently unknown. Accordingly, Northern analysis and in situ hybridization histochemistry have been used respectively to quantitate and localize the expression of the PRL receptor (PRL-R) gene within the uterus during the peripartal period. Immunocytochemistry and Western blot analysis using an anti-PRL-R antibody (U5) localized the translated protein at the cellular level in the same tissues. As judged by the level of expression of the PRL-R gene and its translated products, the chorionic cytotrophoblast has been shown to be a primary site of action. Novel sites were also shown in the decidua, placental trophoblast, and amniotic epithelium. In situ hybridization was not obtained in the latter despite positive Northern analysis and immunostaining. Western analyses with an antibody (U5) to the extracellular domain of the rat PRL-R detected six major molecular species of 95, 85, 63, less than 63, more than 30, and 30 kDa in cytosol from separated amnion, chorion, and decidua. The two bands at 95 and 85 kDa were approximate values only and represent the mature glycosylated forms of the human PRL-R. The other four major bands were partial degradation products from the PRL-R, showing tissue-specific processing and patient to patient variation related to the spectrum of proteases present in these tissues. The 63- and 30-kDa PRL-R-related proteins were detected in both the cytosol and medium from amnion, chorion, and decidua and were also present in amniotic fluid. The 30-kDa species was equal in size to a recently reported PRL-binding protein in human milk. The release of these two PRL-R-related proteins into amniotic fluid suggests possible functions as binding and or/PRL transport proteins in these tissues. The more than 30-kDa species was detected in high amounts in both cytosol and medium from the decidua, but was absent from amniotic fluid. Further work is required to clarify the structural relationships and potential functions of these immunologically PRL-R-related proteins. This study shows that the PRL-R is widely expressed by both fetal and maternal tissues in late pregnancy. Its increased expression during labor and delivery in the chorion, decidua, and placenta supports an autocrine/paracrine role for decidual PRL in the peripartum.

Relaxin gene expression in human reproductive tissues by in situ hybridization.

The expression of the two human relaxin genes termed H1 and H2 in human reproductive tissues ranges from high to very low copy number depending upon the tissue and reproductive state. The aim of this study was to use two approaches to identify total relaxin transcripts (HI and H2) at the cellular level by using a human relaxin H2 riboprobe and a series of six 48-mer synthetic oligoprobes. The results obtained with both methods were similar in all tissues studied; however, a lower background was achieved with the riboprobe. This was especially noticeable after long exposure times, and a better resolution was generally achieved without clustering of the signals. Treatment of the tissues with proteinase-K failed to increase the sensitivity in any tissue with either probe. The relative levels of expression of the total relaxin gene transcripts was estimated from the different exposure times needed to obtain a good hybridization signal. Thus, the order of expression was: corpus luteum of pregnancy > corpus luteum of the cycle > placenta and prostate > decidua parietalis. The results agree well with immunolocalization of the peptide hormone previously performed with both heterologous and homologous relaxin antibodies; the exception was the lack of hybridization signal over the cells of the chorionic cytotrophoblast of the chorion laeve. This suggests that the levels of relaxin gene expression was below the level of detectability with the in situ hybridization technique or that these cells sequester, but do not synthesize, relaxin. Expression in the term placenta varied greatly from tissue to tissue and within any one tissue. A similar variability has been noted for relaxin in this tissue by immunocytochemistry. Methodology for the detection of total relaxin transcripts at the cellular level when expressed in a wide range of copy number will allow the developmental regulation of relaxin gene expression in reproductive and nonreproductive tissues to be visualized.

Estrogenic regulation of proenkephalin mRNA expression in the ventromedial hypothalamus of the adult male rat.

Endogenous opioid peptides and their receptors are regulated by gonadal steroid hormones in the rat hypothalamus. Recent evidence suggests that gonadal steroids are capable of regulating the expression of proenkephalin (PE) mRNA in the ventromedial hypothalamus (VMH) of female, but not male rats. Therefore, we examined the effect of gonadectomy with or without four weeks of hormone treatment on PE mRNA expression in adult male Fisher 344 rats using quantitative in situ hybridization histochemistry. Gonadectomy reduced plasma testosterone and dihydrotestosterone (DHT) levels compared to intact rats, while subsequent estradiol (E2) or DHT treatment increased plasma E2 and DHT levels, respectively. Furthermore, gonadectomy reduced PE mRNA expression in the VMH, but not in the striatum nor the olfactory tubercle compared to intact rats, and this reduction was prevented in the presence of E2, but not DHT. The results suggest that the activation of estrogen receptors normally functions to maintain the level of VMH PE mRNA expression, which is sensitive to hormonal regulation in adult male rats. Thus gonadal steroid hormones might regulate those reproductive functions which are modulated by PE-derived opioid peptides in the male rat brain.

The ontogeny of sex differences in estrogen-induced progesterone receptors in rat brain.

The sex and age-related changes in the concentration of cytosol progesterone receptors (CPR) induced by estrogen (E) treatment in rat brain and pituitary were investigated by a modification of the Palkovits punch procedure using fresh tissue. Young male and female gonadectomized rats, 15, 21, 30, and 42 days of age, were treated for 44 h by a single sc injection of synthetic E. [Moxestrol (Ru2858)]. Adult gonadectomized animals were treated for 72 h by three injections of estradiol benzoate. Cytosol from pituitary and from punches of ventromedial nuclei (VMN), medial preoptic area (mPOA), arcuate nucleus (ARC), and cerebral cortex was labeled with 0.4 nM [3H]promegestrone (Ru5020) to maximize signal to noise and specificity of labeling of CPR. The developmental patterns of CPR differed across brain regions and between males and females. In VMN, females showed higher CPR levels after estrogen priming at 15, 21, and 42 days of age and in the adult; in ARC, females showed higher CPR levels after E priming at 15, 21, and 30 days of age, but not at 42 days or in the adult. In mPOA and pituitary, no consistent sex differences in CPR induction were found. Cortex showed no induction of CPR by E priming. Radioautography of [3H]Ru5020 uptake in VMN of E-primed 15-day-old male and female rats revealed significantly higher labeling in females, thus showing that the CPR levels in vitro reflect a difference in hormone retention in vivo. Female VMN contained more neurons with a higher labeling index than male VMN. Before puberty (approximately = 30 days), there was a decline in CPR levels induced by E priming in both sexes in pituitary, ARC, mPOA, and VMN. At 30 days, the female greater than male sex difference disappeared and tended to reverse in mPOA and VMN, only to be restored again by 42 days of age. Thus, the CPR induction by E priming may reflect underlying changes in E-sensitive brain regions associated with the preparation for puberty, as well as underlying sex differences in response to estrogen programmed by perinatal exposure to testosterone.

Effects of oestradiol and progesterone on stress-induced secretion of prolactin in ovariectomized and/or adrenalectomized female rats.

Prolactin (Prl) secretion in response to an acute stress was studied in ovariectomized (OVX) and/or adrenalectomized (ADX) adult female rats, nontreated or injected sc with a single dose of 5 micrograms oestradiol-17 beta benzoate (OB) or 2 mg progesterone (P). The stress applied consisted of cutting the tip of tail of conscious animals. Radioimmunoassay was used to measure Prl in the serum prepared from blood collected by decapitation 10 min following the stress, i.e., at the point of maximum recorded Prl response. It was found that the capacity of the animals to secrete large quantities of Prl under stress was, when compared to that in intact controls, markedly reduced in OVX or ADX rats and substantially absent in OVX + ADX rats. A 10-fold increase of basal serum Prl, similar in magnitude to the increase in intact controls, was induced by the stress in OVX animals pretreated with OB. On the contrary, pretreatment of OVX animals with P resulted in a complete block of the Prl response to the stress. The stimulative effect of OB was greatly attenuated in stressed OVX + ADX rats. The results suggest that OB potentiates whereas P attenuates the stress-induced secretion of Prl in the female rat, and that the potentiating effect of OB is dependent on functionally intact adrenals.

Effects of aging on prolactin release after stress in female and male rats.

Young adult and elderly male and female intact rats, as well as chronically ovariectomized (OVX) young and elderly female rats, were subjected to an acute stress by cutting the tip of the tail and prolactin (Prl) concentrations were measured in their blood collected by decapitation at various times thereafter. Maximum concentrations of the hormone were markedly lower in all the three groups of elderly rats than those found in the corresponding young animals, and appeared to occur with a delay in the females, but not in the males. In addition, the Prl-response to stress was attenuated in OVX animals regardless of their age. The result of these experiments, performed at two points on the age scale, suggests that in sexually mature rats of both sexes the stress-induced secretion of Prl is inversely related to the age of the animal and that the reverse relationship is retained in OVX females.

[Modern diagnosis and prognosis in isoimmunization in the Rh system]. / Savremena dijagnoza i prognoza izoimunizacije u Rh sistemu.

A total of 118 Rh isoimmunized pregnant women were discovered from 1972 to 1981. In diagnosing the disorder, the author draws attention to the examination of bilirubin in the amnionic fluid as the most reliable indicator, to which the dosing of measurable antibodies and echography are, in his opinion, a precious contribution. The administration of corticosteroids, antihistaminics, capillary protectors, and sedatives proved unreliable. A premature delivery was induced in 79 cases, after the assessment of the fetus maturity. The pregnancy was terminated vaginally in 60 and by cesarean section in 19 cases. Twenty-two near-term children ere delivered without major problems. Failure was recorded in 31 (26.3%) cases: 17 deaths in the uterus and 14 deaths of newborns immediately after birth. Out of 87 surviving children, 36 received exsanguinotransfusion 1-4 times.

Effects of progesterone implants in the mesencephalon of ovariectomized rats on LH and FSH release triggered by exogenous gonadal steroids.

Unilateral implantation of crystalline progesterone into the caudal mesencephalic reticular formation (MRF) of chronically ovariectomized adult rats prevents the triggering effect of exogenous gonadal steroids on LH release and does not affect the release of FSH in the same animal.