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A novel member of the insulin superfamily has previously been shown to be expressed only in porcine pre and postnatal Leydig cells and its human analogue demonstrated in the human testes but not in other organs and hence has been tentatively termed Leydig insulin-like peptide (Ley I-L). However, we have detected hLey I-L gene expression in the cyclic human corpus luteum and trophoblast by the reverse transcriptase-polymerase chain reaction (RT-PCR), with primers selected from the published human Ley I-L sequence. Normal and neoplastic breast tissue and fetal membranes with adhering decidua did not express the gene. The overall sequence of the trophoblast gene was in agreement with that reported with minor changes only in the putative connecting peptide, confirmed by restricted enzyme digestion. A 290 bp RT-PCR product was cloned and used as a cDNA probe in Northern analyses; hybridization was readily shown with cyclic corpora lutea but not with other tissues. The broader spectrum of the expression of this gene will warrant a new nomenclature when its biological activities are known. The different intensity of expression in the corpus luteum and trophoblast suggest endocrine and autocrine/paracrine roles respectively in these tissues in which H2 relaxin and H1/H2 relaxins coexist respectively and at similar levels of expression. Operationally the amino acid sequence homologies between the processed H1 and H2 relaxins and hLey I-L may qualify the specificity claimed for immunostaining the human relaxins in the corpus luteum and trophoblast.

Relaxin is a peptide hormone whose A- and B-chains are derived by posttranslational cleavage from a single 185-amino acid preprorelaxin. Two genes in the human genome (H1 and H2) code for two polypeptides significantly different in amino acid sequence. The full spectrum of biological activities of these two polypeptides has not been examined, but transcription appears to be limited to the H2 relaxin gene in the human corpus luteum. Relaxin is also synthesized by the human decidua, placental trophoblast, and prostate gland; therefore, the expression of the human relaxin genes in these tissues has been examined using the reverse transcription polymerase chain reaction. The mRNA from decidua, placental trophoblast, and prostate was reverse transcribed and then amplified by polymerase chain reaction, using a series of oligonucleotide primers that were specific for but would not distinguish between human H1 and H2 relaxins. Using mRNA from these tissues, two amplified cDNA species were detected, whose identities were confirmed by Southern blots, HpaI and HpaII restriction enzyme analysis, and dideoxy sequencing. We have confirmed that the corpus luteum does not contain detectable H1 relaxin mRNA. However, we demonstrated for the first time relaxin H1 gene expression in the decidua, placental trophoblast, and prostate, and we have also shown that there are marked tissue differences in the relative amounts of expression of the H1 and H2 relaxin mRNA forms. The functional significance is unknown, but if both mRNAs are translated, differential expression of the two genes may result in tissue-specific differences in the production of these relaxins as well as in their binding and actions.

Two monoclonal antibodies (Mab 5B4 and K24) were used for the immunocytochemical localization of human GH (hGH) and human placental GH in the human pituitary and placenta. On the basis of prior selection by RIA, Mab 5B4 was known to be directed to the N-terminal of hGH and was able to detect both pituitary and placental hGH, since their primary amino acid sequences are identical in this domain. Mab K24 was directed to an epitope present in hGH, but not in placental hGH, and was able to detect pituitary hGH only; neither the 5B4 or K24 Mab detected human placental lactogen. Five placentas were studied from women at term after elective cesarean section without labor. The cells of the anterior pituitary gland and the syncytiotrophoblast of the placental villi were stained with Mab 5B4, whereas K24 only stained the pituitary cells; results consistent with the RIA screen. Mab 5B4 specifically stained two different cell types in the placental basal plate. Neither antiserum stained any cell in the amniotic or chorionic membranes or the adherent decidua. The results demonstrate that pituitary hGH and placental hGH are expressed uniquely in the pituitary and fetal placenta, respectively. In addition, the placental hGH gene is also expressed in the placental basal plate, a region of mixed fetal and maternal cells.

A crude relaxin preparation has been obtained from the basal plate region of electric cesarean section human placentae, as well as normal term placentae. The relaxin isolated has different charge properties by ion exchange chromatography from porcine relaxin, although it is of approximately the same molecular size. The most potent fraction obtained has approximately 0.7% of the immunoactivity of purified porcine relaxin when compared with porcine relaxin in a RIA based on porcine material, suggesting significant amino acid sequence differences between the putative human relaxin and its porcine counterpart.

Specimens from 20 human term placentas were stained with 4 different antisera produced against porcine relaxin (Rlx) using the avidin-biotin immunoperoxidase procedure. Cells of the parietal decidua adherent to the fetal membranes, cells of the chorionic cytotrophoblast, as well as cells of the placental basal plate consistently stained with all 4 anti-Rlx sera. Occasionally, Rlx was detected in epithelial cells lining the amniotic membrane. The syncytiotrophoblast stained for Rlx in 2 specimens only. This response was seen only in syncytiotrophoblast that lined villi in close proximity to the basal plate. Syncytiotrophoblast of the chorionic villi either did not stain at all or gave very weak positive immunostaining with the anti-Rlx sera in all specimens. No difference was noted in staining patterns among placentas delivered by elective cesarean section or vaginal delivery.

The production of PRL by the human decidua is generally accepted, but the production of relaxin by this tissue is not. The two hormones were localized in decidual tissue using the avidin-biotin immunoperoxidase procedure with antisera to human PRL and to a synthetic 14-amino acid sequence of the connecting peptide of human relaxin (hCp14). The object of using the hCp14 antiserum was to verify relaxin production by the detection of C-peptide and/or prorelaxin. Cells of the parietal decidua adherent to the fetal membranes stained with both antisera, and immunostaining for both hormones in the same cell was seen. Also, the decidua-like cells of the placental basal plate stained with both antisera. The chorionic cytotrophoblast stained with the antiserum to hCp14, but not the antiserum to human PRL, whereas the placental syncytiotrophoblast stained for PRL and/or human placental lactogen (hPL), but not hCp14. The PRL staining in all tissues was lost when anti-PRL serum absorbed with human placental lactogen (hPL) was used. This finding suggests that the antiserum to PRL could not distinguish between PRL and hPL. It appears, therefore, that the parietal decidua cells and the decidua-like cells of the placental basal plate may be capable of producing both relaxin and PRL, while the syncytiotrophoblast produces hPL and possibly PRL.

Nine women were studied for one menstrual cycle prior to the insertion of an intrauterine progesterone contraceptive system (IPCS) delivering 65 microng progesterone/day into the uterus and again at 1 month after its insertion. Eight of these women were again studied between 6-8 months after the insertion of the IPCS. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17beta, progesterone, prolactin and relaxin were measured in each plasma sample. The data from each study were combined according to the day of the LH peak. Ovulation occurred in all the cycles studied in spite of an elevation in plasma estradiol-17beta and a depression of prolactin and relaxin immunoactivities at the 6-8 month follow up. Menstruations noted at the 6-8 month of use occurred while levels of estradiol-17beta and progesterone were elevated.Plasma levels of luteinizing hormone, follicle stimulating hormone, estradiol-17beta, progesterone, prolactin, and relaxin were studied in 9 women before and after insertion of an Alza, progesterone-releasing, IUD. The IUD releases approximately 65 mcg/day of progesterone into the uterus. The lengths of the preovulatory and postovulatory phases of the menstrual cycle were not affected by the device. Ovulation occurred in all the cycles, even though plasma levels of estradiol-17beta were significantly (p less than .05) increased and plasma levels of prolactin and relaxin were significantly (p less than .05) decreased as late as 6-8 months after insertion. During menstruation at 6-8 months after insertion, plasma levels of estradiol-17beta and progesterone were high. The results are discussed.