Immunocytochemical localization of relaxin in endometrial glands of the pregnant guinea pig.

Relaxin was localized in the cytoplasm of endometrial gland cells in the uterus of day 60 pregnant guinea pigs with the peroxidase-antiperoxidase method. The immunoprecipitate was located over the apical portions of the glandular cells. Stain was not found over non-endometrial portions of the uterus, endometrial surface epithelium or cells of the uterine cervical glands. Agar double-immunodiffusion analyses demonstrated a reaction of identity when extracts of uterus from day 60 pregnant guinea pigs and a porcine relaxin standard were tested with an antiserum produced against highly purified porcine relaxin. An extract of uteri from day 60 pregnant guinea pigs gave a positive response in the mouse uterus bioassay for relaxin.

Effect of relaxin on differentiation of 3T3-L1 preadipocytes.

Relaxin is a member of the insulin-like growth factor family that functions primarily during pregnancy and parturition. Because previous studies have shown that rat adipocytes respond to relaxin, we undertook this study to investigate the effect of relaxin on the differentiation of 3T3-L1 fibroblasts in cell culture. First, relaxin prevented the normal course of cell division during the induction phase, but did not interfere with expression of the adipocyte phenotype, as indicated by lipid accumulation and insulin-sensitive glucose transport. Relaxin-treated cells were 2-3 times larger than cells induced by the normal procedure, as determined by both computer-assisted size analysis and intracellular water volume. These effects on cell division and cell size could be reversed by removal of relaxin during early induction. The concentration of relaxin required to elicit the half-maximal effect was 75 ng/ml (12.5 nM). These studies demonstrate that relaxin affects the proliferative response during the inductive phase of differentiation of the 3T3-L1 cell line without affecting differentiation directly. This effect of relaxin on proliferation is unique and is discussed in relation to the cell cycle. It is our hope that these cells will serve as a model system for investigating the mechanisms by which relaxin functions and allow further studies on receptor structure and function.

Production of antisera against electrophoretically separated relaxin and immunofluorescent localization of relaxin in the porcine corpus luteum.

Antisera to porcine relaxin were produced in rabbits injected with different fractions that had been separated by polyacrylamide gel electrophoresis (PAGE). Analyses by agar double immunodiffusion demonstrated that the different fractions of relaxin separated by PAGE have similar antigenic sites and the individual fractions are indistinguishable from one another by this procedure. Antiserum to porcine relaxin inhibited the interpubic ligament forming ability of the hormone in vivo. Indrict fluorescent antibody studies demonstrated that the hormone was localized only in the corpus luteum of the pregnant sow ovary. Large ovoid or polyhedral cells, assumed to be granulosa lutein cells, exhibited the heaviest fluorescence.

Evidence for relaxin in corpora lutea of late pregnant cows.

Relaxin was isolated from corpora lutea of third trimester pregnant cows. Fractions having relaxin activity eluted in the 6000 and 1400 molecular weight ranges when chromatographed on Bio-Gel P-10. Both fractions were shown to inhibit spontaneous contractions of the mouse uterus, and the 6000 molecular weight fraction promoted formation of the mouse interpubic ligament. Agar diffusion studies with an antiserum to purified porcine relaxin showed a continuous precipitin line with no spurring between the two bovine relaxin fractions and NIHR-P1 porcine relaxin standard. Electrofocusing of the 6000 molecular weight fraction demonstrated three fractions which were capable of inhibiting contractions of the mouse uterus and which cross-reacted with porcine relaxin antiserum. The isoelectric points of these three fractions were 8.8, 10.1, and 11.5. Large luteal cells in corpora lutea from cows in the midtrimester of pregnancy were detected that gave a positive stain for relaxin with the immunoperoxidase method.

De novo synthesis and secretion of prolactin-like protein-B by rat placental explants.

Mid- to late-gestation rat placenta expresses three PRL-related mRNAs, rat placental lactogen-II (rPL-II), rat PRL-like protein-A (rPLP-A), and rat PRL-like protein-B (rPLP-B). The protein product of rPL-II mRNA has been characterized, and the protein products of the rPLP-A mRNA were recently identified. The mol wt of a nonsecreted nonglycosylated rPLP-B protein would be 27,145 based on the mRNA sequence. The present study is the first to report the identification of the rPLP-B protein. Antiserum was generated against a chemically synthesized oligopeptide inferred from a specific region of the rPLP-B cDNA. Three or four distinct proteins synthesized and secreted by rat basal zone explants (day 15 gestation) showed cross-reactivity with the rPLP-B antiserum. The relative mol wt of these immunoreactive proteins is approximately 30,000, with a pI varying from 6.1-6.6. De novo synthesized rPLP-B proteins were not secreted by the explant tissue in the presence of tunicamycin, suggesting that the proteins are glycosylated. These data are consistent with the presence of one potential N-glycosylation site derived from the rPLP-B mRNA sequence. The rPLP-B antiserum showed no cross-reactivity with proteins identified using antisera against rPLP-A, rPL-II, or human pregnancy-specific beta 1-glycoprotein. Immunocytochemical studies were carried out using paraffin sections from placentas of day 14 and 17 pregnant rats which were treated with anti-rPLP-B, followed by avidin-biotin-peroxidase complex. These experiments show perinuclear staining, which was localized in basophilic cytotrophoblast cells, confirming previous in situ mRNA hybridization studies. Although no physiological role has been established for rPLP-B, the synthesis and secretion of this protein by cells in contact only with maternal circulation suggest a hormonal role.

Pregnancy-specific beta 1-glycoprotein messenger ribonucleic acid and immunoreactive protein in the rat testis.

Human pregnancy-specific beta 1-glycoprotein (PS beta G) is synthesized in large quantities by syncytiotrophoblasts of the placenta. Recent studies using a partial cDNA of PS beta G isolated from human term placenta detected the presence of mRNA highly homologous to placental PS beta G in a number of extraplacental tissues, including human and rat testis. The present study determined in the rat that the amount of PS beta G mRNA based on percentage of total tissue RNA was greatest in the testis of mature rats, followed by senescent and prepubertal rats. In experiments using rats with testicular feminization syndrome (Tfm) which exhibit end-organ insensitivity to androgen stimulation, slot blot analysis of testicular RNA showed reduced levels of PS beta G mRNA in Tfm rats compared to normal rats. Northern blot analysis of rat testicular RNA probed with a human placental PS beta G cDNA demonstrated the presence of a single mRNA species of 1.65 kilobases. Subsequent studies investigated whether proteins immunologically similar to PS beta G were present in the testes from normal and Tfm rats. The rat testes were perfused with fixative, and sections from paraffin-embedded tissues were treated with rabbit anti-human PS beta G, followed by the avidin-biotin-peroxidase complex. Testes from the normal rat showed intense immunostaining in late spermatids (steps 16, 17, 18, and 19 of spermiogenesis), residual bodies, as well as cytoplasm of Leydig cells. Spermatozoa within the epididymis also demonstrated intense immunolabeling. Paraffin sections of the testes from the Tfm rat showed light diffuse cytoplasmic immunostaining in cells of the seminiferous tubules. Since spermiogenesis does not proceed normally, and no spermatids were seen, it was not possible to accurately stage the seminiferous tubules of the Tfm rat. The Leydig cells of the Tfm testes stained intensely, however, as was observed in the testes of the normal rat. These data suggest that the rat may provide an animal model for the investigation of the biological function and regulation of PS beta G in the testis.

Identification of proteins immunochemically related to human pregnancy-specific beta 1-glycoprotein in the rat placenta.

Human pregnancy-specific beta 1-glycoprotein (hPS beta G) consists of a set of glycoproteins present in placenta and maternal serum. This study characterized proteins in rat placenta that show immunological cross-reactivity with antisera to hPS beta G. Immunocytochemical studies using two independent preparations of anti-hPS beta G showed intense specific staining within basophilic cytotrophoblast cells of the basal zone of the gestation day 15 rat placenta. In contrast, basophilic cytotrophoblasts located in the labyrinth did not stain. Subsequent experiments used gel electrophoresis and immunoblot analysis to compare PS beta G in human placenta and serum with immunoreactive proteins in rat placenta and serum. A set of two or three proteins was detected in human villous tissue and pregnancy serum with apparent mol wt (Mr) ranging from 54,000-76,000. In contrast rat placenta showed a major immunoreactive protein with 120,000 Mr, while rat serum contained bands of 48,000 64,000 and 69,000 Mr. Explant cultures of rat basal zone tissue secreted two [35S]methionine-labeled proteins that were immunoreactive, a major 120,000 Mr species and a minor 76,000 Mr form, with pI values of 4.6-5.5; tunicamycin inhibited the secretion of both species. Thus, a 120,000 Mr glycoprotein appears to be the major tissue and secreted form of rat PS beta G analog in day 15 placenta. Finally, the cytochemical localization of PS beta G-like proteins in rat placenta showed a progressive gestational shift from giant trophoblast cells in the parietal yolk sac placenta on day 12 to the basal zone cytotrophoblast cells by day 15. Data indicate that the pregnant rat may provide an animal model for investigation of the biological function of PS beta G during late gestation.

Purification and immunohistochemical localization of relaxin in the human term placenta.

Relaxin was purified from human term placentas. A fraction having a molecular weight of approximately 6000 and a specific activity of 11.9 U/mg was eluted from a Bio-Gel P-30 column. This fraction inhibited uterine contractions and produced a linear response to log doses of relaxin in the mouse interpubic ligament bioassay. Electrofocusing of the active Bio-Gel P-30 fraction separated a protein peak with a biological activity of 45 U/mg protein and an isoelectric point of pH 11.4. The electrofocused human relaxin fraction showed cross-reactivity with antiserum produced against highly purified porcine relaxin and formed a single precipitin line with no spurring when compared to purified porcine relaxin in double immunodiffusion assays. Human relaxin was inactivated by the reducing agent dithiothreitol, indicating that disulfide bonds are essential to the biological activity of human placental relaxin. Immunohistochemistry demonstrated that relaxin was located in cells of the basal plate and septae of the placenta.

Immunocytochemical localization of relaxin in human decidua and placenta.

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.

Cytochemical detection of carbohydrate and immunocytochemical detection of relaxin in the same secretory granule.

We administered estradiol and progesterone to spayed guinea pigs, with resultant accumulation of secretory granules in endometrial gland cells. By initially employing protein A-colloidal gold immunolocalization of relaxin, followed by cytochemical staining of carbohydrate with the thiocarbohydrazide-silver proteinate method on the same section, we showed clearly that the secretory granules were composed of a central core containing relaxin and a cortex of carbohydrate-rich material. Use of normal rabbit serum rather than relaxin antiserum, and omission of periodic acid, demonstrated the specificity of the technique.

Enhancement of uterine cervix extensibility in oestrogen-primed mice following administration of relaxin.

The present studies indicated that the extensibility of the mouse uterine cervix was significantly increased by administration of as little as one unit of highly purified preparation of porcine relaxin. No significant change in tensile strength of the cervix was noted when doses were administered that caused increased cervical extensibility and formation of the interpubic ligament. A low coefficient of correlation was found between interpubic ligament formation and increased extensibility of the cervix in oestrogen-primed immature female mice.

Separation of relaxin activities in extracts of ovaries of pregnant sows by polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis has been employed to separate three fractions that have relaxin activity from crude extracts of ovaries of pregnant sows. Bioassays indicated that all fractions had the ability to inhibit spontaneous uterine contractions in vitro and to induce formation of interpubic ligaments in oestrogen-primed mice. The presence of the various fractions in crude extracts of sow ovaries was monitored with the polyacrylamide gel system on days 40, 70 and 100 of pregnancy. Both the total amount and the relative proportions of the fractions were found to change as pregnancy progressed.

Human relaxin inhibits division but not differentiation of 3T3-L1 cells.

For the first time, we demonstrate here the ability of human relaxin to block cell division. During the induction of differentiation of 3T3-L1 fibroblasts to adipocytes, the cells typically undergo two rounds of cell division followed by accumulation of lipid droplets and expression of insulin-stimulated glucose transport as the cells attain the adipocyte phenotype. Human relaxin added during induction had no effect on the development of the adipocyte phenotype or insulin-stimulated glucose transport. However, it blocked cell division at a half-maximal concentration of 1.25 nM, well within physiological range. This could be reversed by the addition of antibodies specific for human relaxin. Thus relaxin joins a select number of hormones with growth inhibitory properties such as transforming growth factor-beta (TGF beta) and mammastatin. Potentially, this is an important but until now unidentified function of relaxin. Unlike other inhibitory polypeptides, like TGF beta, relaxin does not prevent differentiation but rather uncouples it from cell division.

Muscle attachment to the lateral aspect of the articular disk of the human temporomandibular joint.

OBJECTIVE: To investigate the anatomic attachments to the lateral aspect of the anterior band of the human temporomandibular joint articular disk. MATERIAL AND METHODS: Sixteen human cadaver half-heads were dissected and examined macroscopically. RESULTS: No direct attachment was observed between the deep masseter muscle and the temporomandibular joint articular disk. In one specimen, a small band of the anterior temporalis muscle was directly attached to the lateral aspect of the temporomandibular joint disk; whereas, on the same specimen, the attachment of the superior belly of the lateral pterygoid muscle was a comparatively large band. In another specimen, the lateral pterygoid muscle passed in an anterolateral direction. CONCLUSIONS: The masseter muscle has no functional significance in the biomechanics of temporomandibular joint disk displacement. The anterior temporalis muscle may have functional significance when it is accompanied by an anterolaterally divergent lateral pterygoid muscle.

The subepithelial connective tissue graft palatal donor site: anatomic considerations for surgeons.

Surgeons must become completely familiar with the anatomy of the palatal donor site to feel confident in providing the subepithelial connective tissue graft procedure. Variations in the size and shape of the hard palate affect the dimensions of donor tissue harvested, as well as the location of the greater palatine neurovascular bundle. This article classifies palatal vaults according to height as high, average, and shallow. Illustrations and cadaver dissection are utilized to demonstrate that surgeons can gain substantial donor tissue specimens without encountering the neurovascular bundle. Actions to be followed in the unlikely event that the neurovasculature is encountered are reviewed.

Relaxin concentrations in endometrial, placental, and ovarian tissues and in sera from ewes during middle and late pregnancy.

These studies were designed to determine the tissue source of ovine relaxin and to determine the feasibility of using the pregnant ewe for study of relaxin production and secretion. On Day 4 of gestation, ewes were laparotomized, the nonpregnant uterine horn was ligated, and the ovary not containing the corpus luteum was removed. During a second surgery at Day 45 (n = 8) or 140 (n = 9) of gestation, 10-ml blood samples were drawn from a uterine artery, the ovarian vein, and veins draining the pregnant and nonpregnant uterine horns. Endometrial, placental, and luteal tissues were obtained for immunocytochemistry and extraction. Relaxin was detected by a heterologous porcine radioimmunoassay (RIA) in 3 of 54 serum samples (701.3 +/- 25.4 pg/ml, mean +/- SEM). Relaxin was not detected in crude tissue extracts, but low quantities were detected by RIA following Sephadex G-50 column chromatography of tissue extracts. Total relaxin activity for all tissues was equivalent to 0.57 +/- 0.13 ng of porcine relaxin/g tissue (w.w.). Relaxin was not detected immunocytochemically by light or electron microscopy. These data indicate that low quantities of relaxin are present in tissues and sera of pregnant ewes.