Reproduction Symposium: does grazing on biosolids-treated pasture pose a pathophysiological risk associated with increased exposure to endocrine disrupting compounds?

Biosolids (processed human sewage sludge), which contain low individual concentrations of an array of contaminants including heavy metals and organic pollutants such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), and polychlorinated dibenzodioxins/polychlorinated dibenzofurans known to cause physiological disturbances, are increasingly being used as an agricultural fertilizer. This could pose a health threat to both humans and domestic and wild animal species. This review summarizes results of a unique model, used to determine the effects of exposure to mixtures of environmentally relevant concentrations of pollutants, in sheep grazed on biosolids-treated pastures. Pasture treatment results in nonsignificant increases in environmental chemical (EC) concentrations in soil. Whereas EC concentrations were increased in some tissues of both ewes and their fetuses, concentrations were low and variable and deemed to pose little risk to consumer health. Investigation of the effects of gestational EC exposure on fetal development has highlighted a number of issues. The results indicate that gestational EC exposure can adversely affect gonadal development (males and females) and that these effects can impact testicular morphology, ovarian follicle numbers and health, and the transcriptome and proteome in adult animals. In addition, EC exposure can be associated with altered expression of GnRH, GnRH receptors, galanin receptors, and kisspeptin mRNA within the hypothalamus and pituitary gland, gonadotroph populations within the pituitary gland, and regional aberrations in thyroid morphology. In most cases, these anatomical and functional differences do not result in altered peripheral hormone concentrations or reproductive function (e.g., lambing rate), indicating physiological compensation under the conditions tested. Physiological compensation is also suggested from studies that indicate that EC effects may be greater when exposure occurs either before or during gestation compared with EC exposure throughout life. With regard to human and animal health, this body of work questions the concept of safe individual concentration of EC when EC exposure typically occurs as complex mixtures. It suggests that developmental EC exposure may affect many different physiological systems, with some sex-specific differences in EC sensitivity, and that EC effects may be masked under favorable physiological conditions.

AhR-agonist-induced transcriptional changes of genes involved in thyroid function in primary porcine thyrocytes.

The Ah receptor (AhR) is a ligand transcription factor mediating toxic effects of chemicals such as dioxins. The 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the coplanar polychlorinated biphenyl 126 (PCB 126) are member of the polyhalogenated aromatic hydrocarbons family exerting a variety of toxic effects in a tissue-specific and species-specific manner including thyroid function. In the present study, we aimed to investigate the effects of TCDD (1 and 10 nM) and dioxin-like PCB 126 (306 nM) on the AhR signaling pathway and on the gene expression profiles of key factors involved in thyroid function, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium iodide symporter (NIS), TSH receptor (TSHR), and cathepsins (Cat B and L), using a primary porcine thyrocyte culture as the experimental model. AhR and ARNT expression was detected both as mRNA and on the protein level. Expression did not vary upon treatment with either TCDD or PCB 126. However, treatment with TCDD and PCB 126 induced an AhR signaling response, as indicated by the expression of the AhR-target gene cytochrome P-450 1A1 (CYP1A1). Both 10 nM TCDD and PCB 126 treatment induced a significant downregulation in the expression of NIS and cathepsin B without affecting any of the other parameters investigated. In conclusion, these data indicate that (a) thyrocytes are targets of TCDD and TCDD-like compounds and (b) there is evidence for two independent most likely AhR-mediated molecular mechanisms, by which these compounds negatively interfere with thyroid function.

Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells.

Information on the regulation of steroid hormone receptors and their distinct functions within the human endometrial epithelium is largely unavailable. We have immortalized human primary endometrial epithelial cells (EECs) isolated from a normal proliferative phase endometrium by stably transfecting the catalytic subunit (hTERT) of the human telomerase complex and cultured these hTERT-EECs now for over 350 population doublings. Active hTERT was detected in hTERT-EECs employing the telomerase repeat amplification assay protocol. hTERT-EECs revealed a polarized, non-invasive epithelial phenotype with apical microvilli and production of a basal lamina when grown on a three-dimensional collagen-fibroblast lattice. Employing atomic force microscopy, living hTERT-EECs were shown to produce extracellular matrix (ECM) components and ECM secretion was modified by estrogen and progesterone (P4). hTERT-EECs expressed inducible and functional endogenous estrogen receptor-alpha (ER-alpha) as demonstrated by estrogen response element reporter assays and induction of P4 receptor (PR). P4 treatment down-regulated PR expression, induced MUC-1 gene activity and resulted in increased ER-beta transcriptional activity. Gene activities of cytokines and their receptors interleukin (IL)-6, leukemia inhibitory factor (LIF), IL-11 and IL-6 receptor (IL6-R), LIF receptor and gp130 relevant to implantation revealed a 17 beta-estradiol (E2)-mediated up-regulation of IL-6 and an E2- and P4-mediated up-regulation of IL6-R in hTERT-EECs. Thus, hTERT-EECs may be regarded as a novel in vitro model to investigate the role of human EECs in steroid hormone-dependent normal physiology and pathologies, including implantation failure, endometriosis and endometrial cancer.

Molecular actions of polyhalogenated arylhydrocarbons (PAHs) in female reproduction.

Polyhalogenated aromatic arylhydrocarbons (PAHs) such as polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), the polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent lipophilic pollutants, which affect female fertility resulting in severe reproductive dysregulation, including anovulation, reduced conception rates, abortion, menstrual abnormalities and developmental defects of female reproductive tissues. Many PAHs exert their effects by activating a family of basic helix-loop-helix (bHLH) transcription factors, the arylhydrocarbon receptor (AhR) and the arylhydrocarbon receptor nuclear translocator (ARNT), which result in the expression of AhR target molecules. Complex interactions between PAH-mediated AhR activation and ER signalling pathways have been discovered which may contribute to the developmental malformations, impact on reproductive dysfunctions and promote carcinogenic dedifferentiation of tissues within the female reproductive tract. This review will focus on the multifaceted roles of PAHs in key organs of the female reproductive tract, the ovary, uterus/ endometrium and the mammary gland. The complexity and diversity of actions unleashed by PAHs in these female reproductive tissues identify these environmental pollutants as important endocrine disrupting toxicants impacting on female fertility.

Molecular interactions of the aryl hydrocarbon receptor and its biological and toxicological relevance for reproduction.

The dioxin/aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsive to both natural and man-made environmental compounds. AhR and its nuclear partner ARNT are expressed in the female reproductive tract in a variety of species and several indications suggest that the AhR might play a pivotal role in the physiology of reproduction. Furthermore, it appears to be the mediator of most, if not all, the adverse effects on reproduction of a group of highly potent environmental pollutants collectively called aryl hydrocarbons (AHs), including the highly toxic compound 2,3,7,8-tetrachlor-odibenzo-p-dioxin (TCDD). Although a large body of recent literature has implicated AhR in multiple signal transduction pathways, the mechanisms of action resulting in a wide spectrum of effects on female reproduction are largely unknown. Here we summarize the major types of molecular cross-talks that have been identified for the AhR and linked cell signaling pathways and that are relevant for the understanding of the role of this transcription factor in female reproduction.

Epidermal growth factor-like ligands and erbB genes in the peri-implantation rabbit uterus and blastocyst.

Molecular cloning of the partial cDNA coding sequences of the four erbB receptors and the epidermal growth factor (EGF)-like ligands EGF, transforming growth factor alpha (TGF), and heparin-binding EGF (HB-EGF) has provided the basis for a comprehensive analysis of the spatiotemporal expression pattern of the EGF receptor/ligand system during the peri-implantation period in the rabbit. Employing nonradioactive in situ hybridization and immunolocalization, we observed differential expression of erbB1-erbB3 within the trophectoderm of the blastocyst. ErbB1 was strongly expressed in the cytotrophoblast but was downregulated upon syncytium formation. ErbB3 was a product of both the cyto- and syncytiotrophoblast. Despite the expression of erbB2 mRNA, the trophectoderm was devoid of immunoreactive ErbB2. ErbB4 gene activity was exclusively detected in the trophoblast at midpregnancy. The luminal and glandular epithelium and stroma of the nonpregnant, pseudopregnant, and pregnant rabbit uterus at Day 6 of gestation also expressed ErbB1-ErbB3. In the peri-implantation period, gene activities of erbB1-erbB3 were upregulated upon decidualization. At the site of implantation, uterine luminal epithelial cells apposing the preimplantation blastocyst displayed a distinct membrane immunolocalization of ErbB2, identifying the uterine epithelium as target for EGF, TGFalpha, and HB-EGF derived from both the embryonic trophectoderm and the uterine epithelium. In the luminal epithelium at the antimesometrial uterine site, HB-EGF gene activity was upregulated at the time of blastocyst attachment, but this upregulation was not reflected in an increase in immunoreactive HB-EGF. The detection of tyrosine phosphorylated ErbB2 in the rabbit placenta indicated the presence of a functional ErbB/EGF-like system in the pregnant rabbit uterus. This study provides strong evidence for a role of the ErbB/EGF-like system in embryo/maternal interactions during the peri-implantation period in the rabbit.

Molecular remodeling of members of the relaxin family during primate evolution.

Employing comparative analysis of the cDNA-coding sequences of the unique preprorelaxin of the Afro-lorisiform Galago crassicaudatus and the Malagasy lemur Varecia variegata and the relaxin-like factor (RLF) of G. crassicaudatus, we demonstrated distinct differences in the dynamics of molecular remodeling of both hormones during primate evolution. The lorisiform and lemuriform preprorelaxin sequences encoded identical hormones, providing the first endocrinological evidence for the monophyletic origin of all Strepsirrhini. Structural analysis revealed the lemuriform members of the relaxin family to be potentially bioactive single-gene products. In contrast to the "two-prong" relaxin receptor-binding motif (RELVR) present within the B-domains of other primate relaxins, strepsirrhine relaxin contained a unique "three-prong" motif (RRLIR) with highest sequence homology to the receptor-binding motif of the evolutionarily much older skate relaxin. In contrast to relaxin, the RLF molecule was highly conserved during primate evolution and contained within its B-domain the putative relaxin receptor-binding motif and a pentameric sequence implicated in binding to specific RLF receptors. Mutually exclusive expression of strepsirrhine preprorelaxin and RLF were observed in the fetal villous trophoblast cells of the strepsirrhine placenta and postpubertal testicular Leydig cells, respectively, reflecting distinct functional roles for both hormones within the reproductive tract of Strepsirrhini.

Cellular localization of human relaxin-like factor in the cyclic endometrium and placenta.

We have studied the cellular localization of the relaxin-like factor (RLF) in the histologically normal cyclic endometrium collected from days 3--26 of the menstrual cycle. RLF transcripts and protein were detected in the luminal and glandular epithelium and in stromal cells at all stages of the cyclic endometrium. Increased expression of RLF was observed in endometrial tissues in the proliferative as compared to the secretory phase, suggesting that oestrogens affect RLF gene activity in the human endometrium. The cellular localization of RLF transcripts and protein was also determined in first trimester placental tissues obtained from normal and ectopic tubal implantation sites and in third trimester placentae of normal and pre-eclamptic pregnancies. In first trimester placenta, weaker expression of RLF was observed in the syncytiotrophoblast as compared to the underlying cytotrophoblast. Extravillous trophoblast cells constitutively expressed RLF. Trophoblast cells were the main source of RLF in the human placenta and trophoblastic RLF gene activity was unaffected by either the site of implantation or the invasive properties of the cytotrophoblast as demonstrated by samples from patients with tubal implantation and pre-eclampsia respectively. Decidual cells weakly expressed RLF. The presence of unprocessed and cleaved immunoreactive RLF in term placenta was determined by Western analysis. The above results suggest a functional role for both RLF isoforms within normal placental tissue.

Canine relaxin-like factor: unique molecular structure and differential expression within reproductive tissues of the dog.

Employing postpubertal testicular tissue, we determined the cDNA coding sequence of a truncated canine relaxin-like factor (RLF) consisting of a signal peptide of 28 amino acids (aa), a B-domain of 23 aa, a truncated C-domain of 34 aa, and an A domain of 26 aa, respectively. Within the B-domain of canine RLF, the putative relaxin receptor binding motif contained a single substitution with the C-terminal arginine replaced by a serine residue, and the putative RLF receptor binding motif was truncated. Leydig cells specifically expressed RLF in the normal postpubertal and cryptochid testis as well as in testicular Leydig cell adenoma. The epididymis was an additional source of RLF in the dog. In the female reproductive tract, expression of immunoreactive RLF and relaxin were compared. Within the ovary, RLF, but not relaxin, was detected in follicular theca interna and granulosa cells and the corpus luteum. In the nonpregnant uterus, luminal and glandular epithelium coexpressed RLF and relaxin. Uteroplacental tissue at early stages of gestation revealed RLF expression in the proliferative fetal villous cytotrophoblast and in maternal uterine cells. In the mature canine placenta, the trophoblast surrounding the maternal blood vessels and the hemophagous cytotrophoblast of the paraplacental zone expressed RLF. Canine relaxin was absent in the paraplacental areas. Western analysis of placental tissue extracts revealed the presence of specific immunoreactive bands likely resembling unprocessed and enzymatically cleaved RLF. Differential expression of RLF and relaxin appears to reflect distinct autocrine and paracrine functions of RLF in canine reproductive tissues.

Targeting lipopolysaccharides by the nontoxic polymyxin B nonapeptide sensitizes resistant Escherichia coli to the bactericidal effect of human neutrophils.

The nonapeptide of polymyxin B (PMBN) has been reported to sensitize various pathogenic gram-negative bacteria to the direct bactericidal effect of human serum. To investigate the impact of PMBN on human neutrophil-effected killing of the serum- and phagocytosis-resistant Escherichia coli strains C14 and O111, serum was coapplied with PMBN or with neutrophils, but this did not result in decreased numbers of viable bacteria. In contrast, the most potent bacterial killing occurred in the presence of neutrophils plus serum components plus PMBN. The effect of this on E. coli C14 was the appearance of inositol phosphates, diacylglycerol, respiratory burst, elastase liberation, and generation of lipid mediators (leukotriene B(4), 5-HETE, and platelet-activating factor). Strong neutrophil activation required early, but not late, complement components and was blocked by inhibition of phagocytosis with cytochalasin D. PMBN seems to cause dramatic support of natural host defense by complement-dependent sensitization of E. coli to the bactericidal efficacy of human neutrophils.

Ruminant relaxin in the pregnant one-humped camel (Camelus dromedarius).

We have determined the cDNA sequence of preprorelaxin in the pregnant one-humped camel by employing reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Camel preprorelaxin consisted of 600 base pairs (bp) encoding a protein of 199 amino acids (aa) with a signal peptide of 25 aa (75 bp), a B domain of 28 aa (84 bp), a C domain of 121 aa (366 bp), and an A domain of 24 aa (72 bp). The N terminus of the C domain of camel prorelaxin contained the unique proline-rich repetitive sequence (-RPAP)(3)-(-K/RPAL-)(2), and within the B domain the classical -GRELVR- receptor binding motif was found. Camel preprorelaxin showed highest homology with porcine (74.6%) and equine (65.4%) relaxin. The ovary and the uteroplacental unit were a dual source of relaxin in the pregnant dromedary. Within the ovary, weak expression of relaxin was detected in large luteal cells of the mature corpus luteum. In the ovarian follicles, immunoreactive relaxin, but not relaxin mRNA, was detected in the granulosa and theca interna cell layer. Beginning at around Day 93 of gestation and coinciding with increasing interdigitation of the fetal villus with the underlying maternal endometrium, uterine luminal epithelial cells in the uteroplacental tissue expressed relaxin. Weak expression of immunoreactive relaxin, but not relaxin mRNA, was observed in villous trophoblast cells. Pseudostratified trophoblast cells at the base of the placental villi and multinucleate giant cells did not express relaxin.

Relaxin-like factor (RLF) mRNA expression in the fallow deer.

Employing RT- and RACE-PCR on RNA isolated from testicular tissue, we have cloned the coding cDNA sequence for the RLF, also known as Insl3, of the fallow deer. The RLF coding sequence consisted of 396 bp encoding a peptide of 131 amino acids and shared highest homology with bovine, sheep and goat RLF. Northern analysis revealed a single 0.9 kb transcript in the deer testis. There is only one RLF gene in the deer genome. Nonradioactive in situ hybridization revealed the Leydig cells to be the sole source for RLF mRNA in the deer testis. In the non-pregnant uterus, RLF transcripts were located in the luminal and glandular epithelium of the endometrium. Within the ovary of the pregnant doe, follicular theca interna cells and the corpus luteum expressed RLF transcripts. In uteroplacental tissues, luminal and glandular epithelium, fetal uninucleate and binucleate trophoblast cells (BNC) of the basic villous trophoblast layer expressed RLF mRNA. BNC located at the apical trophoblast layer or the tip of the fetal villus were devoid of RLF transcripts. Pseudostratified trophoblast cells at the base of fetal villi coexpressed RLF mRNA and immunoreactive MHC class Ib molecules.

Relaxin-like factor (RLF) is differentially expressed in the normal and neoplastic human mammary gland.

BACKGROUND: Expression of relaxin-like factor (RLF), a member of the relaxin family, was studied in normal, benign, and malignant neoplastic human breast tissue. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and nonradioactive in situ hybridization were employed to detect RLF transcripts. RLF epitopes were detected with a rabbit polyclonal antiserum generated against the putative receptor binding domain of human RLF. The RLF antiserum was characterized by Western blot analysis on human testicular and placental tissues, recombinant glutathione S-transferase-RLF fusion protein, and baculovirus-derived recombinant marmoset-RLF and marmoset-relaxin. RESULTS: RT-PCR analysis revealed RLF amplicons in a cDNA library of normal human breast tissue and in malignant neoplastic breast tissue. RLF hybridization signals were localized exclusively in the tubuloalveolar and ductal breast epithelium but were absent in stromal cells. Benign breast disease displayed weaker RLF hybridization signals compared with normal tubuloalveolar breast tissue. Malignant transformation of breast epithelial tissues resulted in down-regulation of RLF gene expression. The weakest expression of RLF mRNA was observed in lymph node metastases of corresponding primary ductal carcinomas. Immunoreactive RLF was exclusively expressed in breast epithelial cells. Despite strong RLF hybridization signals, the tubuloalveolar epithelial cells of normal breast tissue displayed only very weak immunoreactive RLF. Benign breast disease showed clearly detectable levels of both RLF mRNA and immunoreactive protein. In contrast, epithelial cells in breast carcinoma and lymph node metastases displayed strong expression of immunoreactive RLF, although expression of RLF transcripts was weak. CONCLUSIONS: Results demonstrated that transcriptional and posttranscriptional mechanisms affected human RLF gene expression in normal and neoplastic epithelial breast cells.

Expression of the arylhydrocarbon receptor and the arylhydrocarbon receptor nuclear translocator during early gestation in the rabbit uterus.

The arylhydrocarbon receptor (AhR) and the arylhydrocarbon receptor nuclear translocator (ARNT) are members of the PAS gene family mediating toxic effects of xenobiotics such as dioxin and polychlorinated biphenyls. We have analyzed the expression and cellular distribution of rabbit AhR and ARNT mRNA and protein level in the nonpregnant uterus and the pregnant and pseudopregnant uterus at Days 6 to 12 of gestation. In the preimplantation uterus at Day 6 of gestation and in the interimplantation and pseudopregnant uterus at Days 7, 8, 9, and 12 of gestation, low levels of AhR transcripts were detected in the glandular uterine epithelium. Upon attachment of the blastocyst at Day 7 of gestation, a strong expression of AhR and ARNT mRNA was observed in the luminal and glandular epithelium of the antimesometrial uterine compartment. In contrast, AhR and ARNT expression was low in the luminal epithelium of the paraplacental and the mesometrial placental fold. AhR mRNA was also detected in the trophoblast cells. During early placentation at Day 9 of gestation, expression of AhR and ARNT was first observed in the perivascular decidualized stromal cells and, at Day 12, extended to the decidualized stromal cells of the placental bed. Within the placenta, the syncytiotrophoblast expressed only low levels of AhR and ARNT mRNA and no protein. The specific expression patterns of AhR and ARNT during early gestation suggest functional roles for both transcription factors during feto-maternal interactions in the rabbit.

Relaxin-like factor expression in a human ovarian Sertoli-Leydig cell tumor.

Relaxin-like factor (RLF), a new member of the insulin-like growth factor family, is a reliable marker for normal Leydig cells in the human postpubertal testis (1). Expression of the RLF gene appears to be developmentally regulated, given that only during puberty is RLF expression up-regulated. We recently demonstrated down-regulation of the human RLF gene in testicular Leydig cell tumors indicating dedifferentiation of the Leydig cells within the tumor (2). Ovarian Sertoli-Leydig cell tumors (SLCTs), histologically typed as androblastomas, are rare, potentially malignant sex-cord stromal tumors exhibiting testicular-like structure and differentiation of various degrees. In the present study, we investigated the expression of RLF, 17alpha-hydroxylase, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), Ki-67, and cytokeratin 18 in a human ovarian SLCT of low differentiation.

Molecular cloning and localization of caprine relaxin-like factor (RLF) mRNA within the goat testis.

The relaxin-like factor (RLF) is one of the insulin-like molecules, which also includes insulin, insulin-like growth factor I and II, placentin, and relaxin. Employing RT- and RACE-PCR on RNA isolated from goat testicular tissue, we report the cloning and nucleic acid sequence of goat RLF. The caprine RLF cDNA coding sequence consisted of 396 base pairs encoding a peptide of 131 amino acids. Caprine RLF showed the highest homology in nucleic acid and amino acid sequence with bovine and sheep RLF, suggesting conservation of the RLF gene among ruminants. Northern blot analysis revealed a single 0.9 kb RLF transcript expressed in the goat testis but not in the epididymis, liver, or muscle tissue. Only a single goat RLF gene is present in the goat genome as determined by Southern blot analysis. Employing nonradioactive in situ hybridization for goat RLF mRNA and immunohistochemistry for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17alpha-hydroxylase, we identified the Leydig cells as the sole source of RLF mRNA in the goat testis.

Canine preprorelaxin: nucleic acid sequence and localization within the canine placenta.

Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E-->D and L-->Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta.

Nucleic acid sequence of feline preprorelaxin and its localization within the feline placenta.

The cat placenta is known to secrete large amounts of relaxin. We employed uteroplacental tissue at approximately Day 35 of gestation to determine the nucleic acid sequence of feline preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Feline preprorelaxin cDNA was found to consist of 540 base pairs encoding a protein of 180 amino acids (aa). We identified a signal peptide of 25 aa, a B domain of 33 aa, a C domain of 98 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the B domain contained one substitution from the classical GRELVR motif (L-->F). Feline preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a specific 1-kilobase transcript present in total RNA of feline uteroplacental tissue but not of liver tissue. Nonradioactive in situ hybridization was used to localize relaxin mRNA, and immunohistochemistry was used to localize the relaxin hormone and cytokeratin, in tissues of the feto-maternal interface recovered from two queens at Day 35 of gestation. Specific hybridization signals for relaxin mRNA were exclusively detected in cells located in the lamellar placental labyrinth but were absent from other placental and nonplacental uterine parts. The cells expressing relaxin mRNA also displayed immunoreactivity for cytokeratin and were, therefore, identified as trophoblast cells. Immunoreactive relaxin colocalized in those placental areas expressing relaxin mRNA. Trophoblast cells located at the villous chorioallantoic tips invading the endometrium and extravillous trophoblast cells in the junctional placental zone were devoid of relaxin.

Placental localization of relaxin in the pregnant mare.

In situ hybridization employing a cRNA probe derived from a 428-bp fragment of equine relaxin was used to localize relaxin mRNA, and immunocytochemistry was used to localize relaxin itself, in tissues of the placenta-endometrium interface recovered between 33 and 153 days of gestation from mares carrying intraspecific horse, interspecific mule and extraspecific donkey conceptuses. Immunocytochemical staining was also used to localize trophoblast-specific and class I major histocompatibility complex (MHC) antigens on some specimens. Relaxin mRNA and relaxin were both present in the single-cell non-invasive trophoblast layer of the allantochorion between 45 and 153 days of gestation in all three types of equine pregnancy examined. Both, however, were absent from the invasive trophoblast cells of the progenitor chorionic girdle and the differentiated trophoblast cells of the endometrial cups throughout the latters' 60-80-day period of development and regression. Discrete and irregularly spaced clusters of elongated pseudostratified trophoblast cells on the allantochorion remained negative for relaxin mRNA and ligand, but stained strongly for equine trophoblast-specific antigens. These areolae-like structures of the mature horse placenta overlie the mouths of endometrial glands between adjacent microcotyledons and they are clearly involved with the uptake of uterine milk for fetal sustenance. It is speculated that their loose attachment to the endometrium and weak expression of class 1 MHC antigens may serve to tolerize the mother to the paternally-inherited histocompatibility antigens of the fetus.