Immunoelectron microscopic localization of relaxin in endometrial gland cells of the pregnant guinea pig.код для вставкиСкачать
THE ANATOMICAL RECORD 209:373-379 (1984) lmmunoelectron Microscopic Localization of Relaxin in Endometrial Gland Cells of the Pregnant Guinea Pig RUBE J. PARDO, LYNN H. LARKIN, AND RANDALL H. RENEGAR Department of Anatomy, University of Florida College of Medicine, Gainesville, FL 32610 ABSTRACT Endometrial gland cells in uteri from late-pregnant guinea pigs (day 60 to parturition) resembled typical protein-secreting cells. Extensive rough endoplasmic reticulum and well-developed Golgi complexes were evident. The most striking features of endometrial gland cells were accumulations of large (= 0.5 ym in diameter), dense, membrane-bounded granules. The granules were located in the supranuclear region of the cell and frequently occurred in close proximity to the plasma membrane adjacent to the lumen of the gland. Thin sections of endometrial gland cells treated with relaxin antiserum and either colloidal gold-protein A or colloidal gold-goat antirabbit IgG demonstrated that the granules contained relaxin. These studies provide additional evidence that the uterus of the guinea pig produces relaxin and support the hypothesis that uterin relaxin may play a n important role in pregnancy and parturition in the guinea pig. Previous studies from this as well as other laboratories have demonstrated that the uterus of the guinea pig is a source of relaxin (Zarrow, 1947, 1948; Pardo et al., 1980; Nagao and Bryant-Greenwood, 1981; Pardo and Larkin, 1982). Endometrial (uterine) gland cells (EGC) have been shown to be the source of uterine relaxin in pregnant and nonpregnant guinea pigs by light microscopic immunoperoxidase localization (Pardo et al., 1980; Pardo and Larkin, 1982). While previous studies have established that EGC are sources of relaxin in the guinea pig, several questions remain unanswered. For example: 1)Is relaxin synthesized in EGC or is it produced elsewhere and accumulates in or on the EGC, possibly as receptor-bound hormone? 2) If relaxin is produced by EGC, is it stored in membrane limited granules similar to the relaxin containing granules described in granulosa lutein cells of the pig (Kendall et al., 1978)? 3) How and in which direction is relaxin released from the cellapically into the lumen of the gland, where the hormone would have access to the uterine lumen, or basally into the stroma of the uterus, where it might effect the closely situated uterine musculature, which is a known target organ of relaxin (Porter, 1979)? 0 1984 ALAN R. LISS. INC. The work reported in this communication concerns investigations of the ultrastructure of EGC of the guinea pig with the intent of obtaining answers to the questions just presented. MATERIALS Guinea pigs were obtained from a local vendor and housed in the University of Florida Health Center Animal Resources Department. Animals had access to food and water ad libitum with a photoperiod of 12 hours light and 12 hours dark. Relaxin antiserum (R19) was produced against electrophoretically pure porcine relaxin in New Zealand white rabbits as described by Larkin et al. (1977). The R19 antiserum has been shown to 1)give a single line of identity between partially purified uterine relaxin from guinea pigs and the purified porcine relaxin standard (NIH-RXN-P1) in Ouchterlony plate assays (Pardo and Larkin, 1980),2) inhibit the action of porcine (Larkin et al., 1979) and guinea pig relaxins (Pardo, 1982) in vitro, 3) produce parallel lines of displacement in radioimmunoassay using extracts of guinea pig uteri and purified porcine relaxin (Pardo, Received March 21, 1983; accepted January 30, 1984. 374 R.J. PARDO, L.H. LARKIN. AND R.H. RENEGAR of phosphate-buffered saline (pH 7.4) after each step of the procedure. After imrnunostaining, sections were counterstained with a 2% aqueous uranyl acetate solution for 3 minutes. Both osmicated and nonosmicated immunostained sections were examined in a JEOL 100s electron microscope a t 60 or 80 kV. The following tests for specificity of immunolabeling were carried out: 1)substitution of the relaxin antiserum (R19) with preimmune serum, 2) absorption of R19 antiserum with purified porcine relaxin (NIH-RXN-P1) prior to incubation, 3) omission of the primary antiserum (R19) and substitution with phosphate buffered saline, and 4)dilution of the R19 antiserum until no response was obtained. All of these tests gave results that indicated the procedure specifically detected relaxin. Quantitative evaluation of immunogold METHODS staining was determined for grids treated Adult female guinea pigs were housed with with R19 relaxin antiserum or preimmune a male, and pregnancies were timed from the serum and protein A-gold. A minimum of ten day on which sperm were found in a vaginal fields were photographed for each treatment smear. Three animals on day 60 of pregnancy at a primary magnification of 10,000 or and three animals that demonstrated marked 15,000x and enlarged 2.7 x during the printrelaxation of pelvic ligaments (day 63 to 65) ing process. Granules, randomly chosen by were used in these studies. application of a 1-cm2grid to each print, and Uterine tissues were removed from anes- all nuclei (control area) in each field were thetized guinea pigs and immersed in a 1% analyzed. Surface area for each organelle was solution of glutaraldehyde (0.1 M cacodylate determined with a HIPAD DT-11 digitizer buffer, pH 7.2) for 2 hours. Approximately (Houston Instrument, Austin, TX), interone-half the specimens were postfixed in 1% faced with a TRSSO model 1microcomputer osmium tetroxide for 1hour. Both osmicated (Tandy Corporation, Fort Worth, TX). The and nonosmicated specimens were dehy- number of gold particles present over the drated through a series of cold alcohols and same compartment was counted and the denpropylene oxide and embedded in Araldite sity of labeling calculated (gold particles/ 6005. pm2). Density determinations for each orgaLight gold sections from the osmicated tis- nelle were considered as individual data sues were placed on copper grids and stained points in a 2 x 2 factorial analysis of variwith uranyl acetate and lead citrate (Venable ance with location (granule or nucleus) and and Coggeshall, 1965). Light gold sections treatment (R-19 relaxin antiserum or preimfrom nonosmicated tissues were placed on mune serum) as main effects. nickle grids and processed for immunolabelRESULTS ing after the technique of Roth et al. (1978). Endometrial glands are simple, coiled, tuColloidal gold was prepared following the technique of Frens (1973) and labeled with bular glands that extend from the lumen of protein A as described by Roth et al. (1978). the uterus into the stroma, where their deepGrids containing the nonosmicated sections est portions lie adjacent to the inner circular were first floated on a 10% solution of hydro- muscle layer of the uterus. Sections (1pm) of gen peroxide for 10 minutes. Sections were plastic-embedded specimens viewed with the treated with R19 relaxin antiserum (diluted light microscope revealed the tubular nature 1:lOO) for 1 hour and IgG-gold (diluted 1:10) of the glands and showed dense aggregates or protein A-gold (diluted 150) for 2 hours. of granules within the cytoplasm of the EGC Grids were washed with two 3-minute rinses (Fig. 1).The majority of the EGC exhibited 1982),and 4)produce no cross-reactivity with other growth peptides such as insulin and proinsulin in radioimmunoassay (unpublished observations). Specificity of the R19 antiserum to detect guinea pig uterine reIaxin has been discussed previously in articles concerning localization of the hormone with the light microscope (Pardo et al., 1980; Pardo and Larkin, 1982). Purified porcine relaxin (NIH-RXN-P1, 3,000 U/mg) was obtained from the National Pituitary Agency, National Institute of Arthritis, Metabolism, and Digestive Diseases. Goat antirabbit IgGlabeled colloidal gold (IgG-gold) was purchased from E-Y laboratories (San Mateo, CA). Protein A was obtained from Sigma (St. Louis, MO). Materials used in electron microscopy were purchased from either Ladd Research Industries (Burlington, VT) or Polysciences, Inc. (Warrington, PA). IMMUNOELECTRON LOCALIZATION OF RELAXIN granules, and in most cells the granules were accumulated in the supranuclear regions of the cells near the lumen of the gland. When EGC were examined in the electron microscope, dense cytoplasmic granules were the most striking feature (Fig. 2). The granules (upper range of = 1.0 Fm in diameter) were composed of a dense, homogeneous material and were enclosed by a single membrane. In most sections the limiting membrane was obscured by the granule contents, however, the membrane could be discerned in fortuitous sections of granules or in forming granules (Figs. 2, 3). The Golgi complex appeared to be quite active (Fig. 3) and numerous stacks of Golgi cisternae were noted (Fig. 2). Rough endoplasmic reticulum was present throughout the cytoplasm. Smooth endoplasmic reticulum was noted mainly in the vicinity of the Golgi complex. The luminal surfaces of EGC were characterized by microvilli and well-developed junctional complexes (Fig. 2). Sections stained with either protein A-gold or IgG-gold demonstrated that the granules contained relaxin (Fig. 4). Infrequently, relaxin-containing granules were noted at the luminal surface of the cell (Fig. 412). Since the specimen in Figure 4c was neither osmicated nor stained with lead, the membranes are not well defined, and it is difficult to determine if the granule is actually in the process of being released from the cell into the lumen of the gland. Quantitative evaluation demonstrated that the density of immunolabeling over granules was significantly greater (P < .01) than labeling over nuclei or granules from grids treated with preimmune serum (Table 1). DISCUSSION Work reported in this article supports the contention that relaxin is produced and stored in EGC of the guinea pig uterus. This conclusion is drawn because of the following: TABLE I. Density of immunostaining ouer granules and nuclei in guinea pig endometrial gland cells (protein-A gold technique) R-19 relaxin antiserum Preimmune serum 'Particles/Frn2area *P < .01. Endometrial granules' Nucleus' 258.34* 34.76 23.55 8.39 375 1)The typical endometrial gland cell resembled a protein secreting cell in that it exhibited a n active Golgi complex, significant amounts of rough endoplasmic reticulum, and membrane-bounded dense granules. 2) The granules were shown to contain relaxin with immunolocalization techniques. 3) The presence of a highly active Golgi complex with forming granules indicated that the granule contents most likely were synthesized in EGC. 4) It does not seem likely that relaxin in the EGC was receptor bound, since relaxin was not detected in association with plasma membranes or cytoplasmic vesicles, which would have been expected if relaxin was bound to plasma membrane receptors on the surface of the cell or that had been internalized (Duello et al., 1983). Although profiles demonstrating the actual mechanism of granule release from the cell were not observed in this study, both light and electron microscopic studies indicated that the granules were located in close proximity to the plasma membrane that formed the luminal border of the cell. These observations strongly suggest that the granules are released into the lumen of the gland rather than into the stroma at the base of the EGC. It seems most likely that the secretory process is either very rapid or is of a n episodic nature and has not been arrested and/or detected a t the precise moment of granule release. If relaxin is released into the lumen of the endometrial glands, it would then have free access to the uterine lumen, where it might act locally to influence placental detachment, softening of the uterine cervix, or softening of fetal membranes. The effect relaxin may have on these processes has been discussed previously (Pardo et al., 1980; Nagao and Bryant-Greenwood, 1981; Pardo and Larkin, 1982). Support for a local effect of relaxin was provided by MacLennan et al. (1980) and Perezgrovas and Anderson (19821, who demonstrated that topical application of relaxin caused softening of the uterine cervix in women and cows, respectively. Additional evidence, which indicates relaxin may function as a local hormone, has been L m R S Abbreviations lumen of uterine gland uterine musculature rough endoplasmic reticulum endometrial stroma 376 R.J. PARDO, L.H. LARKIN, AND R.H. RENEGAR IMMUNOELECTRON LOCALIZATION OF RELAXIN 377 Fig. 3. Golgi complex of endometrial gland cell. A limiting membrane can be seen surrounding the forming granules (arrows). Note the accumulation of rough endoplasmic reticulum located around the Golgi complex and in adjacent endometrial gland cells. ~ 2 7 , 6 0 0 . published that demonstrates the presence of relaxin in preovulatory follicles (BryantGreenwood et al., 1980; Matsumoto and Chamley, 1980). Recent studies have indicated that relaxin may act to facilitate rup- Fig. 1. Endometrial gland. The coiled, tubular nature of the gland is demonstrated. These glands extend from the uterine lumen (not shown) through the endometrial stroma to lie adjacent to the uterine musculature. Onepm section stained with toluidine blue. ~ 3 3 0 .Inset. Higher magnification of portion of endometrial gland (indicated by "1. Note accumulations of granules in the supranuclear regions of the cells. Few granules are noted in stromal portions of the gland cells. One-pm section stained with toluidine blue. ~ 8 5 0 . Fig. 2. Endometrial gland cells. Numerous dense granules are seen in the luminal portions of the cytoplasm of the gland cells. Stacks of Golgi cisterne (arrows) are evident. Microvilli and dark-staining junctional complexes are noted at the luminal surfaces of the cells. X 12,600.Inset. Higher magnification of dense granules. Note the homogeneous texture of the granules and the single limiting membrane. ~ 2 4 , 9 0 0 . ture of the follicle wall a t ovulation (Too et al., 1982). Previous observations that relaxin produced by the uterus causes relaxation of pelvic ligaments and can be detected in serum (Zarrow, 1948; Nagao and Bryant-Greenwood, 1981) indicate that relaxin produced by EGC reaches the systemic circulation. Relaxin may reach the systemic circulation by movement from the uterine cavity through the uterine luminal epithelium. However, evidence is not available at this time to support this hypothesis, and immunolabeling studies with the light (Pardo et al., 1980; Pardo and Larkin, 1982) and electron microscopes (unpublished observation) have not detected relaxin in luminal epithelial cells. Relaxin may not have been found because the amount of hormone may be too low to be detected with the techniques employed, or relaxin may be bound to a carrier molecule such that antigenic sites are not exposed to antibodies applied to sectioned material. 378 R.J. PARDO, L.H. LARKIN, AND R.H. RENEGAR Fig. 4. Immunolabeling of granules. a) Incubated with relaxin antiserum (R19) and IgG-gold. While some nonspecific binding is present, the granules are heavily labeled. ~ 2 8 , 0 0 0 .b) Incubated with preimmune serum and IgG-gold. Note diminished labeling over granules. The nucleus is present in the lower portion of the micro- graph. ~ 2 8 , 0 0 0 .c) Incubated with relaxin antiserum (R19) and protein A-gold. Note labeled granule in close proximity to plasma membrane bordering the lumen of the gland. ~ 2 8 , 0 0 0d) . Incubated with preimmune serum and protein A-gold. Note diminished labeling over granules. ~ 2 8 , 0 0 0 . Relaxin-containing granules of EGC are considerably larger than relaxin-containing granules of pig granulosa lutein cells ( = 0.5 pm to 0.2 pm, respectively) (Kendell et al., 1978). The significance of this difference in size of relaxin-containing granules between the two cell types is unclear at this time but most likely relates to the fact that the lutein cell is an endocrine cell, while the endometrial gland cell is an exocrine cell. The findings presented here are consistent with the hypothesis that uterine relaxin may play an important role in pregnancy and/or parturition in the guinea pig. 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