Ultrastructural localization of relaxin in the corpus luteum of the nonpregnant, pseudopregnant, and pregnant pig

Relaxin was localized in luteal cells of ovaries from nonpregnant, pseudopregnant, and pregnant pigs using porcine relaxin antiserum and peroxidase-antiperoxidase light microscopy immunohistochemistry. The number of immunoreactive cells seemed to increase from Days 17 to 106 of gestation. Luteal cells from pseudopregnant (Day 110) and nonpregnant (Day 14 of the estrous cycle) pigs were also positive for relaxin. However, less than 3% of the luteal cells in the nonpregnant animals were immunoreactive. Electron microscopy immunocytochemistry using porcine relaxin antiserum and goat antirabbit immunoglobulin G-colloidal gold demonstrated that relaxin was packaged in the small membrane-bound granules in luteal cells of pregnant as well as pseudopregnant and nonpregnant pigs. The intensity of labeling (number of gold particles) of the granules increased with pregnancy. There was a 10-fold increase in labeling of granules with the 10-nm versus 25-nm diameter gold. The goat antirabbit labeled with the smaller 10-nm gold particles was necessary to demonstrate the apparent low levels of relaxin in the luteal cells of the nonpregnant pigs. These data further indicate that pregnancy is not required for relaxin synthesis. However, physiologic significance of relaxin in corpora lutea of nonpregnant pigs has not been determined.     

Immunocytochemical localization of relaxin in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state

Relaxin is one of the hormones present during pregnancy and it is synthesized primarily by corpora lutea (CL). Other reproductive tissues including CL of the menstrual cycle may also synthesize this hormone. Very little is known, however, about the cellular and subcellular distribution of relaxin in human CL and dependence of luteal relaxin on the reproductive state. The light and electron microscope immunocytochemical studies described here were undertaken to obtain this information using antisera to porcine and human relaxin. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states or in human hepatocytes. Luteal immunostaining was low in early luteal phase; it increased progressively, reaching the highest level in late luteal phase, and then decreased greatly in corpora albicantia. Term pregnancy CL contained similar immunostaining as early luteal phase CL. Mid luteal phase CL contained more immunostained cells than late luteal phase CL, but the late luteal phase CL contained a greater amount of immunostaining per cell than mid luteal phase CL. The immunogold particles due to relaxin were primarily present in secretory granules and to a small extent in rough endoplasmic reticulum. Quantitation revealed that secretory granules contained a much higher number of gold particles than did rough endoplasmic reticulum. These two organelles from late luteal phase CL contained greater numbers of gold particles than those from mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pregnant mouse corpora lutea: immunocytochemical localization of relaxin and ultrastructure

Relaxin was localized in corpora lutea of pregnant mouse ovaries by using the unlabeled antibody peroxidase-antiperoxidase technique and a highly specific rabbit antirat relaxin serum. Relaxin immunostaining was first observed in luteal cells located at the periphery of corpora lutea on Day 10 of gestation. The number of relaxin immunostained cells and the intensity of the stain gradually increased to reach a maximum between Days 16 and 18 of gestation. While a few luteal cells were specifically stained for relaxin on Day 1 postpartum, no luteal cells were stained on Day 2 postpartum. Ultrastructural studies of luteal cells from pregnant mouse ovaries revealed the presence of a distinct electron-dense, membrane-bound granule population, which was first observed on Day 12 of gestation. The granules increased in number to reach a maximum between Days 16 and 18 of gestation, and were absent by Day 2 postpartum. The appearance and disappearance of this granule population closely paralleled the relaxin immunostaining in the luteal cells. We suggest that the granules may be the subcellular sites of relaxin storage in the pregnant mouse ovary.     

Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle

Porcine relaxin has been sought by localization in the corpus luteum of sows on Days 3, 7, 9, 11, 12, 15, 18, 19, and 21 of the estrous cycle, using the avidin-biotin immunoperoxidase method and an antiserum to purified porcine relaxin. Simultaneous localization of relaxin in corpora lutea from sows on Days 108 and 113 of pregnancy was used to compare the intensity of immunostaining with that of corpora lutea of cyclic animals. However, the antiserum dilution necessary for optimal localization differed considerably in these two states (1:10,000 in pregnancy and 1:750 in the cycle), suggesting that lower levels of antigen are present in the luteal cells of the cycle. Relaxin immunostaining was undetectable on Day 3 of the cycle but became evident by Days 7 and 9. At Day 11 staining intensity increased and persisted through Day 15. On Day 18 some stain was still evident, but by Days 19, 20, and 21 there was complete absence of immunostain. Relaxin immunostaining appeared to be located throughout the cytoplasm of the luteal cell, as clear areas in the nuclear region were often observed. The results suggest that relaxin is produced in low amounts by the luteal cells of the cyclic sow and that the levels fluctuate with stage of the cycle. Lack of evidence from radioimmunoassay for a surge of relaxin secretion into the systemic circulation prior to luteolysis in the pig estrous cycle suggests that the relaxin localized in the luteal cells of the cycle may have an intraovarian function.     

Immunochemical and cytochemical studies of relaxin-containing cells in the guinea pig uterus

Uteri and ovaries from cycling, pregnant, and lactating guinea pigs were studied for immunolocalization of relaxin with the light microscope. Endometrial gland cells (EGC) from the same group of animals were examined in the electron microscope for the presence of secretory granules. Those EGC that exhibited high numbers of granules were stained either for relaxin with the protein A colloidal gold method or for carbohydrate with the thiocarbohydrazide technique. Relaxin was found in EGC from middle and late pregnant animals but was not detected in ovaries or uteri from cycling animals. While cytoplasmic granules were noted in most EGC from cycling animals examined, the number of granules was greatest in uteri from estrus and proestrus animals. Granules in EGC from estrus animals contained a carbohydrate-rich material but did not contain relaxin. Endometrial gland cells from animals in early to middle stages of pregnancy (days 15 and 30) contained limited numbers of granules, almost all of which contained carbohydrate. At day 45 of pregnancy, EGC containing many granules were noted. The majority of granules contained relaxin; however, a significant number of EGC contained carbohydrate-rich granules. Infrequently, EGC were noted that contained two populations of granules, and these two populations were assumed to be made up of relaxin-containing and carbohydrate-rich granules. EGC from animals on day 60 of pregnancy typically contained granules, and the majority of these contained relaxin. Carbohydrate-rich granules were observed in EGC of the day 60 animals but were smaller in diameter and were noted in much lower numbers than the relaxin-containing granules. Endometrial gland cells from lactating animals infrequently contained granules. These studies are consistent with the hypothesis that the uterus is the primary source of relaxin in the guinea pig and that relaxin plays an important role in pregnancy and parturition of this species. The observations implicate endometrial glands and their products in the physiology of the cycling animal as well as the pregnant and parturient animal.     

Determination of the source of immunoreactive relaxin in the cat

We have recently reported the secretory profile of relaxin throughout gestation in the cat. Because the appearance of relaxin begins at about Day 20 (Day O = ovulation) and because implantation begins shortly before this at Days 13-14, we hypothesized that relaxin was of feto-placental origin. To test this hypothesis, we used 4 experimental groups: 1) Control (laparotomy-only at Day 23 or 42, n = 4); 2) Early Ovariectomy (Ovx, bilateral ovariectomy between Days 23 and 26, n = 4); 3) Late Ovx (bilateral ovariectomy between Days 40 and 44, n = 4); 4) Tissue Removal (removal of feto-placental units, uterus, and one ovary on Days 16, 21, 28 and 35, n = 1 per day). Pregnancies were maintained in both Ovx groups by progesterone administration. Relaxin secretory patterns in Ovx groups were similar to the Control data. Relaxin was detectable in plasma beginning at about Day 20, with maximum concentrations reached by Day 30. Relaxin concentrations were highest (immunoactivity per mg tissue) in homogenates of placental tissues as compared to luteal, fetal, or uterine tissues. Altogether, these data indicate that the feto-placental unit is the source of relaxin in the cat.     

Characterization of large luteal cells and their secretory granules during the estrous cycle of the cow.

Large steroidogenic cells of the bovine corpora lutea were evaluated for morphological changes on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle. Large cells were readily identified by size (25-50 microns diameter), numerous mitochondria, and the presence of dense secretory granules (150-300 nm in diameter). These granules were found in a discrete cluster and were not dispersed throughout the cytoplasm. Only 3% of the large cells contained a cluster of granules on Day 3. The percentage was highest during midcycle (Day 7, 84%; Day 11, 64%), dropped on Day 14 (26%), and was lowest on Days 17 (16%) and 19 (8%). Electron microscopic immunocytochemistry showed that oxytocin and neurophysin were co-localized in these granules on all days evaluated. As early as Day 14, large cells were observed with characteristics typical of regressing corpora lutea, i.e., a reduction in cells with secretory granules, large cytoplasmic lipid droplets, and swollen mitochondria with dense inclusions. However, since this was a time of the cycle when plasma concentrations of progesterone were very high, this corpus luteum is referred to as involutive rather than regressive. Our results may be summarized as follows: 1) from Day 7 to Day 14 there was a 69% decline in the number of large cells containing oxytocin-laden secretory granules. This occurred prior to the rise in uterine oxytocin receptors and the large luteolytic pulses of prostaglandin that reportedly occur after Day 14. The role of this apparent early release of oxytocin is not known. 2) Large steroidogenic luteal cells of the estrous cycle have morphological characteristics similar to those of large luteal cells during pregnancy. However, large luteal cells of the estrous cycle contain oxytocin whereas those of pregnancy are devoid of oxytocin.     

Fine structure of the corpus luteum of pregnancy in the gerbil (Meriones unguiculatus)

Summary Corpora lutea from gerbils on days 1, 4, 8, 12, 16, 20 and 24 of pregnancy were studied electron microscopically. Similarly, luteal tissue from animals on the day of parturition and one day postpartum was studied (gestation: 24 days ± 8–24h). Agranular endoplasmic reticulum increases in quantity through day 16 and thereafter is somewhat reduced. Granular endoplasmic reticulum and a population of small granules (type I) become abundant during late pregnancy and their possible role in the production and storage of relaxin is discussed. Luteal tissue undergoes a relatively rapid regression which begins on the day of parturition. Conspicuous in the regressing luteal tissue are large (type II) granules (possibly lysosomes), lipid droplets, leucocytic elements and macrophages. Functional correlates of these morphological findings are discussed.The author would like to thank Mrs. Yvis Tablada, Miss Mary Ann Anderson and Mr. Garbis Kerimian for their technical, secretarial and photographic assistance, respectively     

Acute versus chronic effects of human chorionic gonadotrophin on relaxin secretion in rhesus monkeys.

Corpora lutea (CL) were removed from rhesus monkeys (N = 26) at 0 h, 9 h, 3 days, 6 days and 10 days during treatment with hCG to simulate blood concentrations of CG during normal pregnancy. Dispersed luteal cells were incubated in vitro at 37 degrees C for 8 h. Immunoreactive relaxin was measured in incubation medium and in cell extract by radioimmunoassay (RIA). Cellular content and release of relaxin into medium increased as simulated early pregnancy progressed. By 3 days, relaxin content had significantly increased (P less than 0.05) and continued to rise throughout simulated early pregnancy. Significant increases in cellular content and release were observed before the time when relaxin has been detected in the peripheral circulation during this treatment regimen. Within group, total relaxin (cells plus medium) was similar before and after incubation (P greater than 0.05). As such, production of relaxin during the 8-h incubation was not evident. In-vitro exposure of the luteal cells to hCG or dbcAMP had no acute effect on cell content or medium concentration of relaxin at any stage of simulated early pregnancy. Since acute effects of hCG and dbcAMP were not evident in vitro, a sustained gonadotrophic influence may be necessary to augment relaxin production/secretion in the primate CL.     

Serum relaxin levels in prostaglandin E2 induced abortions

Relaxin, a peptide hormone produced only by the corpus luteum of pregnancy, can be used as a marker of luteal function in human pregnancy. Serum immunoreactive relaxin levels were measured serially in six women having second trimester abortions induced with intravaginal prostaglandin E2 (PGE₂) suppositories. All patients aborted within 17 hours of the first suppository. No significant changes were detectable in serum relaxin levels in any of the patients. It is concluded that PGE₂ does not interfere with the corpus luteum's ability to secrete relaxin in the second trimester of human pregnancy.     

Ultrastructural morphology and relaxin immunolocalization in giant trophoblast cells of the golden hamster placenta

Relaxin immunoreactivity was previously demonstrated in three cell types within the hamster placenta; fetal primary and secondary giant trophoblast cells (GTCs) and maternal endometrial granulocytes. The objectives of the present research were to examine the ultrastructure of the GTCs and identify the intracellular relaxin storage site. Primary GTCs, first present on day 8 of gestation, were characterized by numerous polyribosomes and large heterogeneous cytoplasmic inclusions suggesting phagocytic activity. Primary and secondary GTCs from days 10, 14, and 15 of gestation contained numerous polyribosomes, mitochondria with tubular cristae, and extensive Golgi complex, and abundant rough endoplasmic reticulum, all characteristics of a cell actively involved in protein synthesis. Membrane-bound secretory granules were not present. Relaxin was immunolocalized within the Golgi complex of primary and secondary GTCs using the avidin-biotin-peroxidase method. Following differential centrifugation of hamster placental homogenates and radioimmunoassay (RIA) of subcellular fractions, the majority of relaxin immunoactivity was detected in the postmicrosomal fraction; however, the majority of relaxin immunoactivity from similarly treated pig corpora lutea was present in the mitochondrial/granule fraction. These data indicate that hamster placental relaxin is not stored in membrane-bound secretory granules but is contained within the extensive Golgi complex of the GTC.     

Localization and development of chromogranin A and luteinizing hormone immunoreactivities in the secretory-specific cells of the hypophyseal pars tuberalis of the chicken

 The pars tuberalis mainly consists of the secretory cells specific to this portion of the pituitary. We examined the localization and development of luteinizing hormone (LH) and chromogranin A in the chicken pars tuberalis by immunohistochemistry. The vast majority of the chicken pars tuberalis was occupied by cells immunoreactive for both LH and chromogranin A. Furthermore, immunoblot analysis of chicken pars tuberalis extracts with LH antiserum demonstrated that two bands, the large α-subunit and small β-subunit of the LH molecule, were expressed in this tissue as well as in the pars distalis. A band for chromogranin A was also detected in pars tuberalis extracts with chromogranin A antiserum. In contrast to the cells of mammalian species that contain only a few small secretory granules, the specific cells of the chicken pars tuberalis were characterized by the presence of many secretory granules ranging from 90 to 400 nm in diameter. Postembedding immunogold labeling showed that gold particles representing immunoreactivity for LH were densely located on all secretory granules of the secretory-specific cells. Many secretory granules, especially the large ones, of the cells were also loaded with immunogold particles for chromogranin A. Double immunogold labeling confirmed that LH and chromogranin A were colocalized on the same secretory granules. During embryonic development, the primordium of the pars tuberalis was first detected at 8 days of incubation as a small group of cells containing LH- and chromogranin-immunoreactive cells. In the pars distalis, the onset of LH and chromogranin expression occurred earlier, at 6 days of incubation. At 10 days of incubation, the pars tuberalis primordium became large cell masses consisting of LH- and chromogranin-immunoreactive cells, which were located close to the median eminence. Subsequently, the primordium extended along the median eminence progressively with age. At 14 days of incubation, it reached to the rostral end and surrounded the median eminence as slender cell cords. These results indicate that specific cells of the chicken pars tuberalis synthesize a glycoprotein hormone related to the LH molecule, which is stored in the secretory granules together with chromogranin A. The pars tuberalis may be involved in the regulation of gonadal function in a different way from that of the pars distalis. Accepted: 26 August 1997     

Endocrine and ultrasonographic characterization of a successful pregnancy in a Sumatran rhinoceros (Dicerorhinus sumatrensis) supplemented with a synthetic progestin

A Sumatran rhinoceros with a history of early pregnancy loss was supplemented with a synthetic progestin, altrenogest (Regu‐Mate^®), and delivered a healthy, full‐term calf 475 days after mating. Serum hormone concentrations were measured throughout gestation, and ultrasonography was used to monitor embryo/fetal growth and viability. The embryonic vesicle growth curve was characterized by three phases: rapid expansion, plateau, and a final rapid expansion, and was similar to that in the domestic horse. Fetal sex was determined by ultrasound on day 73 of gestation. After day 80 of gestation, transabdominal examinations were more useful than rectal examinations for imaging the fetus. Serum progesterone concentrations remained at luteal levels (1.5±0.5 ng/ml) for the first 2 months of pregnancy, and then they gradually increased. However, progesterone decreased almost to luteal levels during the fifth month before it increased again, and eventually reached peak concentrations (13.3±1.9 ng/ml) shortly before parturition. Relaxin concentrations remained basal (≤0.5 ng/ml) for the first half of the pregnancy, increased to 2.7±1.2 ng/ml and stabilized until 2 weeks before parturition, when relaxin spiked to unusually high concentrations (800–1300 ng/ml). Prolactin concentrations were at baseline (7.2±1.7 ng/ml) throughout most of the gestation, but rose markedly 2 weeks before parturition, reaching concentrations as high as 75 ng/ml. Attempts to measure serum estrogen concentrations were unsuccessful. These data represent the first attempt to characterize pregnancy in the critically endangered Sumatran rhinoceros, a species that heretofore had not successfully reproduced in captivity for 112 years. Zoo Biol 23:219–238, 2004. © 2004 Wiley‐Liss, Inc.     

Development and fine structure of the yolk nucleus of previtellogenic oocytes in the medaka Oryzias latipes

The development and fine structure of yolk nuclei in the cytoplasm of previtellogenic oocytes were examined by electron microscopy during several stages of oogenesis in the medaka, Oryzias latipes. Shortly after oogenesis starts, oocytes 20-30 microm in diameter have much electron-dense (basophilic) cytoplasm, within which a continuous or discontinuous, irregular ring-shaped lower electron-dense area of flocculent appearance (LF) begins to emerge around the nucleus. The yolk nucleus is first recognized within an LF area as a few fragments of dense granular thread measuring 20-25 nm in width. The threads consist of two rows of very dense granules resembling ribosomes or ribonucleoprotein (RNP)-like particles in size and electron density. These thread-like fragments gradually increase in number and length until they assemble into a compact, spherical mass of complicated networks. Analysis of serial sections suggests that the yolk nucleus is a complicated mass of numerous, small deformed vacuoles composed of a single lamella with double layers of ribosomes or RNP-like granules, rather than a mass of granular threads. When oocytes develop to greater than 100 microm in diameter, the yolk nucleus begins to fragment before dispersing throughout the surrounding cytoplasm, concomitantly with the disappearance of LF areas. At this stage of oogenesis, a restricted region of the granulosa cell layer adjacent to the yolk nucleus becomes somewhat columnar in morphology, fixing the vegetal pole region of the oocyte.     

Boolarra virus: Ultrastructure of intracytoplasmic virus formation in cultured Drosophila cells

Boolarra virus (BoV) is a Nodavirus isolated from Oncopera intricoides. Drosophila cell lines 1 (D1) and 2 (D2) were infected with virus and the progession of infection was followed in ultrathin sections viewed by the electron microscope. Viral morphogenesis was restricted to the cytoplasm. Virogenic stroma condensed to electron-dense areas in which were embedded electron-lucent and electron-dense particles. picornaviruslike particles 30 nm in diameter were found in membranous channels and paracrystalline arrays and were scattered throughout the cytoplasm of cells. Virus was released from cisternae and tracts which extended to the cells' periphery. Local disruption of cell membranes also released particles and aggregates of virus into the environment.     

Synergistic effects of insulin-like growth factor I and gonadotrophins on relaxin and progesterone secretion by ageing corpora lutea of pigs.

Insulin-like growth factor I (IGF-I) is involved in paracrine/autocrine regulation of gonadal steroidogenesis and peptide hormone biosynthesis. This study was designed to determine whether IGF-I alone, or an interaction of IGF-I, is involved in augmenting the actions of luteinizing hormone (LH) and prolactin in controlling relaxin and progesterone secretion from ageing corpora lutea of hysterectomized gilts at days 110, 113 and 116 after oestrus. Luteal tissue slices were incubated for 8 h with IGF-I (0, 50, 300 ng ml-1), LH (0, 100, 1000 ng ml-1), and prolactin (0, 100, 1000 ng ml-1) alone or in combination. Progesterone and relaxin concentrations were determined by radioimmunoassay of spent medium and of homogenates from luteal tissue slices before and after incubation. Porcine luteal tissue from day 110 had a net output of 25 ng progesterone and 26 ng relaxin in the control and of 65 ng progesterone and 2125 ng relaxin in the combined IGF-I, LH and prolactin treatment mg-1 of luteal tissue, respectively. IGF-I, LH and prolactin alone or in combination significantly increased (P < 0.01) progesterone production by luteal tissue from day 110, but they were partially effective at day 113 and ineffective at day 116. By contrast, the same hormone treatments increased relaxin production by luteal tissue from days 110 and 113. Even at day 116, prolactin alone or with LH or IGF-I continued to stimulate relaxin production. In conclusion, IGF-I augments the ability of prolactin and LH to increase relaxin production by ageing corpora lutea; however, a decrease in progesterone secretion and an increase in relaxin secretion at day 113 indicate that different mechanisms control progesterone and relaxin secretion in pigs.     

Fine structural and immunohistochemical studies of goat adenohypophysial cells

Summary The fine structure of each type of anterior pituitary cell in the male goat was studied through the application of a superimposition technique in which adjacent thick sections were used to identify individual cells beforehand by light-microscopic immunohistochemistry. A cone of the pars intermedia protrudes into the pars anterior, being surrounded by the narrow pituitary cleft; the immunohistochemical appearances of the cells forming the cone resemble those of the pars anterior. Several follicles appear in the pars anterior. Ultrastructurally GH cells resemble prolactin cells. The secretory granules of both types are spherical; the diameter of the former is about 340 nm, whereas that of the latter is about 440 nm. ACTH cells are polygonal in shape with secretory granules, about 180 nm in diameter, scattered throughout the cytoplasm. TSH cells, which are spherical in shape, contain the smallest secretory granules, 150 nm in diameter. The highly electron-dense LH cells contain numerous secretory granules about 210 nm in diameter. Their nuclei are irregular with incisures. Thus, the anterior pituitary cells of the goat are ultrastructurally characteristic and species-specific.     

Relaxin Treatment in an Ang-II-Based Transgenic Preeclamptic-Rat Model

Relaxin is a peptide related to pregnancy that induces nitric oxide-related and gelatinase-related effects, allowing vasodilation and pregnancy-related adjustments permitting parturition to occur. Relaxin controls the hemodynamic and renovascular adaptive changes that occur during pregnancy. Interest has evolved regarding relaxin and a therapeutic principle in preeclampsia and heart failure. Preeclampsia is a pregnancy disorder, featuring hypertension, proteinuria and placental anomalies. We investigated relaxin in an established transgenic rat model of preeclampsia, where the phenotype is induced by angiotensin (Ang)-II production in mid pregnancy. We gave recombinant relaxin to preeclamtic rats at day 9 of gestation. Hypertension and proteinuria was not ameliorated after relaxin administration. Intrauterine growth retardation of the fetus was unaltered by relaxin. Heart-rate responses and relaxin levels documented drug effects. In this Ang-II-based model of preeclampsia, we could not show a salubrious effect on preeclampsia.     

Ultrastructure of endocrine cells in the stomach of two teleost fish Perca fluviatilis L. and Ameiurus nebulosus L.

Summary In the gastric mucosa of two teleost species, the perch (Perca fluviatilis) and the catfish (Ameiurus nebulosus) three endocrine cell types were found, located predominantly between the mucoid cells of the gastric mucosa. A fourth cell type is present in the gastric glands of catfish. Each cell type was defined by its characteristic secretory granules. Type-I cells were predominant in both fish. These cells contained round or oval granules with a pleomorphic core. The average diameter of granules was 400 nm for the perch and 270 nm for the catfish. Type-II cells of both species displayed small, highly osmiophilic granules about 100 nm in diameter. The secretory granules of type-III cells (260 nm in the perch and 190 nm in the catfish) were round or slightly oval in shape and were filled with a finely particulate electron-dense material. Type-IV cells of the catfish were found in the gastric glands only. Their cytoplasm was filled with homogeneous, moderately electron-dense granules averaging 340 nm in diameter. The physiological significance of these different morphological types of gastric endocrine cells requires further investigation.     

Immunocytochemical studies of relaxin in ovaries of pregnant and cycling mice

Immunocytochemical staining for relaxin in ovarian sections of pregnant mice from day 11 through day 18 of gestation revealed that only corpora lutea (CL) of pregnancy are stained. Evaluation of serial sections of ovaries from a day 16 pregnant mouse revealed that the only luteal structures present are CL of pregnancy. The number of CL present in each ovary equaled the number of implantation sites in each related horn (7 on the right side and 8 on the left side). These large CL varied in shape, being round in some profiles to very elongate in others. All CL were immunochemically stained for relaxin using the peroxidase-antiperoxidase method of L. Sternberger (Immunocytochemistry, 2nd ed. Wiley, New York, 1979). The intensity of the strain varied from cell to cell within each CL. Small luteal structures that were observed to be immunochemically stained for relaxin were demonstrated to represent the periphery of CL of pregnancy. No luteinized follicles were observed and interstitial cells and follicles were not immunochemically stained in any of the day 16 serial ovarian sections or in any of the ovarian sections from pregnant mice on the other days of gestation studied. CL of previous cycles were not observed to be present in the ovaries at days 15, 16, or 18 of gestation. However on day 14 and before, CL of previous cycles were observed and they did not exhibit any relaxin immunostaining. Immunocytochemical studies using the biotin-avidin system revealed that no relaxin immunostaining could be demonstrated in the ovaries of cycling mice at any stage of the estrous cycle. In conclusion, this study revealed that the only ovarian structures demonstrating relaxin immunocytochemical staining in the mouse were CL of pregnancy.