Inhibition of postpartum uterine involution in the rat by relaxin

The possible contribution of relaxin to the support of uterine accommodation during late gestation by retarding tissue lysis was examined using the involuting postpartum uteri of unilaterally pregnant rats. In otherwise intact animals, twice-daily administration of 0.1 mg of relaxin (porcine fraction B) significantly retarded the regression of both gravid and, to a greater extent, nongravid tissue during the first 4 days postpartum, and collagenolysis was similarly delayed. Immediate postpartum ovariectomy had little effect on the uterus, although 5 micrograms estradiol benzoate daily suppressed uterine involution in the gravid tissue to about 50% and was even more effective in the nongravid uterus. Relaxin alone had little effect on the gravid uterus following ovariectomy, but augmented estrogen to the extent that less than half of the tissue and its collagen were lost during 4 days. The effect on nongravid tissue was even more striking in that the combination of estrogen and relaxin prevented any degradation of tissue in general or of collagen. Although we have reported that relaxin can stimulate uterine collagen synthesis as well as uterine growth, the magnitude of its postpartum effect in the presence of estrogen suggests a stabilizing or anticatabolic effect upon the uterus.     

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.     

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.     

The competitive ability of stem cells from mice with Hertwig's anemia

Mice homozygous for the gene, an, have a macrocytic, normochromic anemia. In this report, attempts have been made to cure Hertwig's anemia (an/an) by injecting genetically normal (+/+) stem cells. The anemia of unirradiated an/an mice was alleviated but not completely cured by injection of as many as 3 X 10(7) +/+ bone marrow cells. Lethal irradiation of the an/an recipients was necessary before injections of 10(7) +/+ marrow cells were effective in normalizing the blood parameters. The inability to achieve normal blood values without first destroying the host's own stem cells suggested that the indigenous an/an cells compete effectively with injected +/+ cells. This hypothesis was tested by injecting varying numbers of stem cells from C57BL/6J-+/+ mice, together with stem cells from either WBB6F1-an/an or, as controls, from their WBB6F1-+/+ littermates, into lethally irradiated hosts. The C57BL/6J and WBB6F1 mice have electrophoretically distinguishable hemoglobins. The an/an cells are able to compete in the repopulation of the host hematopoietic tissue as shown by the presence of WBB6F1 hemoglobin in the recipients. The cells from mice with Hertwig's anemia, however, do not compete as effectively as do the same number of cells from the +/+ littermates. These results indicate that the pluripotent hematopoietic stem cells of an/an mice are reduced in number, seeding capacity, or proliferative potential.     

Stimulation of rat uterine collagen synthesis by relaxin

Immature, ovariectomized, estrogen-primed rats respond to the administration of porcine relaxin by an increase in the incorporation of labeled amino acids ([14C]leucine, [14C]phenylalanine, [3H]proline) into uterine proteins in vitro. The maximum response occurs about 12 hr after a single injection of 0.1 mg relaxin in benzopurpurine 4B solution; subsequently, the relaxin effect declines but is still apparent after 24 hr. Smaller, but still significant increases in incorporation rates can be induced by relaxin in the absence of estrogen priming. Uterine collagen synthesis, as indicated by the incorporation of [3H]proline and its conversion to hydroxyproline, appears to be a primary target of the relaxin stimulus, since the effect of relaxin upon proline incorporation into uterine collagen is significantly greater than its effect upon labeling of noncollagen protein.     

Uterine growth-promoting effects of relaxin: a morphometric and histological analysis

The hormone relaxin has been implicated in the regulation of several processes in the reproductive tract during pregnancy and parturition. This study investigated the uterine effects of relaxin in immature and mature ovariectomized, estrogen-primed rats using morphometric and histochemical analysis. Rats were sprayed at 30 or 70 days of age and given estrogen (5 micrograms) 7 days later. After a week, they received an injection of porcine relaxin (100 micrograms) and were killed 6 h later; controls received vehicle alone. Histological sections were obtained from 7 levels of each uterine horn, and the volumes of endometrium and myometrium were calculated by use of a Zeiss Videoplan Computer Image Analyzer. In immature animals, relaxin treatment doubled uterine weights during the treatment period, and cross sections from relaxin-treated animals exhibited significant increases in the areas of both the myometrium and endometrium, 150% and 130% respectively. Mature animals were less responsive to relaxin although they also exhibited significant increases in uterine weight (31%), myometrial volume (29%), and endometrial volume (22%). With the use of Masson's Trichrome stain for collagen, we observed that relaxin alters the connective tissue framework of both endometrium and myometrium; control uterine collagen appears highly organized and dense with compact collagen fibers, whereas the collagen of relaxin-treated uteri is loosely arranged and disorganized with widely separated collagen fibers. Relaxin-stimulated uteri exhibited significantly greater vascularization, as evidenced by the size of arteries and veins in the vascular region between the circular and longitudinal muscle layers. Increased vascularization and uterine blood flow may be one mechanism involved in relaxin's uterotropic effect and is being investigated further.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabbit relaxin: the influence of pregnancy and ovariectomy during pregnancy on the plasma profile.

A porcine relaxin radioimmunoassay was developed to evaluate the profile of immunoreactive relaxin in rabbit plasma. Relaxin was nondetectable in pseudopregnant (Days 1, 4, 5-8, 12, and 16), nonpregnant, and male rabbits. However, in pregnant rabbits, relaxin was detected during the peri-implantation period (Days 4-9). Peak concentrations were reached on Day 15 and were maintained until parturition (Day 32). Relaxin concentrations abruptly decreased on Day 1 postpartum to low but detectable concentrations that were unchanged during the first week postpartum. In contrast, progesterone concentrations peaked earlier (Day 13), decreased after Day 25, and were not detectable on Day 1 postpartum. The effect of ovariectomy on the profile of plasma relaxin was evaluated. Four pregnant rabbits were ovariectomized (Day 13) and treated with medroxyprogesterone acetate to maintain pregnancy. As in normal pregnant rabbits, relaxin was observed initially during the peri-implantation period (Days 4-9) and increased to peak concentrations by Day 16. These concentrations were maintained until parturition and abruptly decreased on Day 1 postpartum to low yet detectable concentrations during the first week postpartum. The concentrations of relaxin in the plasma of ovariectomized medroxyprogesterone-treated rabbits were not different from those in three sham controls. These results indicate that the ovary is not a significant source of relaxin in pregnant rabbits. The unique observation of the presence of relaxin during the peri-implantation period suggests that this hormone has a role in preparing the rabbit uterus for implantation. The continued presence of relaxin during the first week postpartum may represent residual hormone, or it may suggest a physiological role during the early postpartum period.     

Collagen studies in late pregnant relaxin null mice

The relaxin knockout (rlx -/-) mouse was used to assess the effect, during pregnancy, of relaxin with regard to water, collagen content, growth, and morphology of the nipple (N), vagina (V), uterus, cervix (C), pubic symphysis (PS), and mammary gland (MG). The results presented here indicate that during pregnancy, relaxin increases the growth of the N, C, V, and PS. Large increases in water content in the PS (20%) occurred in pregnant (Day 18.5) wild-type (rlx +/+) mice but not in rlx -/- animals. This indicates that in the PS, relaxin might increase the concentration of a water-retaining extracellular matrix component (hyaluronate). In the pregnant rlx +/+ mouse, collagen content decreased significantly in the N and V but not in other tissues. There were no significant changes in the rlx -/- mouse. This contrasts with findings in the rat, in which relaxin has been found to cause decreases in collagen concentrations in the V, C, and PS. Histological analysis showed that the collagen stain was more condensed in the tissues (V, C, PS, N, and MG) of rlx -/- mice than in those of rlx +/+ mice. This phenomenon indicates that the failure of collagen degradation and lack of growth in the N underlie the inability of the rlx -/- mice to feed their young, as reported previously. Vaginal and cervical luminal epithelia, which proliferated markedly in the rlx +/+ pregnant mice, remained relatively atrophic in the rlx -/- mice. As proliferation and differentiation of uterine and vaginal epithelia are thought to be induced by a paracrine stromal factor that acts upon estrogen stimulation, our results indicate that relaxin may be this paracrine factor.     

Concentrations of progesterone, prolactin and relaxin in the luteal phase and pregnancy in normal and short-cycling German Shepherd dogs

Twenty-two nonpregnant and 19 pregnant German Shepherd dogs were assigned to either a control group or a suspected short-cycling group, based on the interestrous interval (> or = 6 month and < 5 month, respectively) and data from previous pregnancies. Blood serum concentrations of progesterone and prolactin were determined from days 5 to 60 (day 0 = ovulation) for characterization of luteal function. In pregnant bitches, placental integrity was additionally assessed by relaxin concentrations. The nonpregnant, suspected short-cycling bitches had significantly lower progesterone concentrations than the controls, indicating decreased luteal activity both in the autonomous and prolactin-dependent period. In the pregnant suspected short-cycling bitches, unavoidable progesterone supplementation prevented assessment of luteal function; it may have suppressed prolactin secretion (significantly lower prolactin concentrations from days 20 to 60, compared with the pregnant control group), but deficient prolactin secretion affecting luteal function cannot be excluded. The significantly lower relaxin concentrations, together with a high incidence of embryonic death found in the pregnant, suspected short-cycling group, may indicate loss of placental integrity and may have caused decreased prolactin concentrations.     

Pregnancy-stimulated growth of vascular smooth muscle cells: Importance of protein kinase C-dependent synergy between estrogen and platelet-derived growth factor

Dramatic smooth muscle cell (SMC) growth occurs in the uterine artery during pregnancy. The potential for pregnancy-associated growth may also exist at other vascular sites. We tested the hypothesis that increased growth of uterine artery SMC isolated from pregnant (vs. nonpregnant) guinea pigs would be detectable in culture, that pregnancy-associated phenotypic changes would also be found in nonuterine vascular cells (aortic SMC), and that the enhanced growth would be dependent on estrogen, peptide growth factors like platelet-derived growth factor (PDGF), and protein kinase C (PKC). Growth responses were measured by [³H]-thymidine incorporation and cell counts. Uterine artery SMC from pregnant guinea pigs grew to a higher plateau density with serum stimulation, had increased spontaneous DNA synthesis and persistent growth following serum withdrawal, and were more responsive to 3–30 ng/ml PDGF-BB than nonpregnant cells. Aortic SMC from pregnant animals also grew to a higher plateau density and had enhanced responsiveness to PDGF-BB. This increased response to PDGF-BB by pregnant uterine artery and aortic SMC (40–233% increase over nonpregnant PDGF result) was reproduced in nonpregnant cells by pretreatment for 1–24 h with 17-beta(β)-estradiol (30–100 nM). Neither the pregnancy-induced difference nor the estradiol pretreatment was associated with increased PDGF-BB binding activity. The synergistic effect of 17β-estradiol was partially (62%) reproduced with 17-alpha(α)-estradiol, an isomer which does not bind the estrogen receptor. This suggested that 17β-estradiol modulates the PDGF-BB response by both estrogen-receptor- and nonreceptor-mediated mechanisms. To test if the estrogen effects were dependent on PKC, two different antagonist strategies (3 μM dihydrosphingosine and phorbol-ester-induced downregulation) were applied prior to 17α- or β-estradiol and blocked the enhanced responses to PDGF. The synergistic effect of 17β-estradiol on PDGF was then reproduced by 1 h pretreatment with the cell-permeable PKC activator, 10 nM PMA. We conclude that pregnancy stimulates increased growth of uterine and aortic SMC in vitro which is dependent on estrogen, PDGF, and PKC and may be important in vascular remodeling during pregnancy. © 1996 Wiley-Liss, Inc.     

Immunocytochemical localization of relaxin in the golden hamster (Mesocricetus auratus) during the last half of gestation

The objective of this study was to determine the tissue source of relaxin in pregnant hamsters by immunocytochemical techniques. Ovarian, uterine, and placental tissues were recovered from hamsters on Days 8, 10, 12, 14, and 15 of gestation and processed for light microscopy. Relaxin immunoreactivity was localized in tissue sections by the avidin-biotin-peroxidase technique using antiserum to porcine relaxin. On Day 8 of gestation, relaxin immunoreactivity was localized in primary giant trophoblast cells (GTC-1s) adjacent to the uterine decidua. On Day 10, relaxin immunoreactivity was localized in GTC-1s, secondary giant trophoblast cells (GTC-2s) adjacent to the ectoplacental cone, and endometrial granulocytes in the wall of sheathed arteries. On Day 12, relaxin immunoreactivity was observed primarily in GTC-2s interspersed among cells of the placental trophospongium but not in cells of the placental labyrinth. The intensity of staining and number of relaxin immunoreactive GTCs increased between Days 12 and 14 but was decreased by Day 15 PM. Relaxin was not localized in uterine glands or corpora lutea. These observations suggest that the placenta is the tissue source of relaxin in pregnant hamsters.     

Serum immunoreactive relaxin in women during a 24-h period

Serum relaxin concentrations were measured every 30 min during a 24-h period in nonpregnant and pregnant women. Relaxin was undetectable in all serum samples obtained from 3 nonpregnant women. Relaxin was detectable in all serum samples obtained from 2 pregnant women. However, neither episodic secretion of relaxin nor a 24-h rhythm in relaxin secretion was discernible in these women.     

Effect of estradiol and relaxin on growth of fibroblastic cells isolated from guinea pig mammary glands

Fibroblasts were isolated from the mammary glands of guinea pigs and grown in 96-well culture plates. They were treated with a factorial arrangement of porcine relaxin (0.0, 0.5, 1.0 or 1.5 micrograms/ml) and estradiol-17 beta (0, 200, 400 or 600 pg/ml). Tritiated thymidine or uridine was added to a final activity of 25 nCi per well and the cells incubated at 37 degrees C for 48 h. Cells were then harvested onto filter paper and counted for tritium. Controls (0.0 micrograms/ml relaxin and 0 pg/ml estradiol) incorporated 3.7 nCi of tritiated thymidine and 4.8 nCi tritiated uridine. Both relaxin and estradiol altered the incorporation of thymidine and uridine. There was also an interaction between the two hormones. Thymidine incorporation with no estradiol and 1.5 micrograms/ml relaxin was 129% of controls. The optimum incorporation of thymidine occurred with 0.5 micrograms/ml relaxin and 400 pg/ml estradiol. This combination of hormones gave a response of 145% of controls. Uridine incorporation followed a different pattern. Relaxin alone at a concentration of 1.5 micrograms/ml gave a near-optimum response of 141% of control. The optimum combination of relaxin and estradiol for uridine incorporation was 1.5 micrograms/ml relaxin and 400 pg/ml estradiol, which gave a response of 156% of controls. These data indicated that relaxin and estradiol alter DNA and RNA synthesis in mammary fibroblasts and thus may be important in controlling the growth of the mammary gland stroma.     

Morphological changes induced in mouse mammary gland by porcine and human relaxin

The effects of pure porcine relaxin and of human decidual extracts with relaxin-like activity on the mammary gland of virgin mice primed with estrogen have been studied by the light microscope. Porcine relaxin enhanced the changes induced by estrogen alone; the effect was different in the various mammary tissues. In the stroma, relaxin only slightly increased the loosening of connective tissue, the extent of the adipose tissue and of the capillary bed, as well as the degranulation of the mast cells. The changes in the parenchyma, such as elongation and branching of ducts, are strikingly enhanced. Moreover, relaxin seems to promote differentiation of the cells forming the walls of distal ducts, and of the myoepithelial cells. Tissue extracts of human decidua with relaxin-like activity induce changes in the mammary gland similar to those due to porcine relaxin. Such data indicate that relaxin synergizes with estrogen to cause growth of ducts of the mammary gland and that tissue extracts of human decidua have a similar effect, thus providing further evidence that decidua may be a source of relaxin in humans.     

Abnormal relaxin secretion during pregnancy in women with type 1 diabetes

To test the hypothesis that relaxin may play a role in the fetal abnormalities associated with pregnancy in type 1 diabetic women, we previously compared gestational relaxin concentrations in diabetic and clinically normal women using a porcine relaxin radioimmunoassay (RIA): Serum immunoactive relaxin was significantly (P < 0.001) elevated in the diabetic women. To confirm and extend this work in a larger group of subjects, we have now used an enzyme-linked immunosorbent assay (ELISA) specific for human H2 relaxin (the normal human gene product) to determine immunoactive serum relaxin concentrations in serial samples from 61 Type 1 diabetic and 21 normal pregnant women. Samples from 22 of the diabetic and nine of the normal women were also directly compared in the porcine relaxin RIA. ELISA-determined serum relaxin was higher (P < 0.001) at 24 and 36 weeks of pregnancy in type 1 diabetic women than in controls, confirming previous findings. However, the geometric mean increase in immunoactive relaxin concentration in identical samples from pregnant diabetic women over that of controls was significantly greater with the RIA than with the ELISA (271% vs 44%; P < 0.001). To investigate this discrepancy, the specificity and epitope selectivity of the RIA and the ELISA were compared using several synthetic polypeptides, including human relaxins H1 and H2, and relaxin and insulin derivatives. Both assays showed great specificity, but the porcine RIA selectively identified the epitopes of the receptor-binding domain of the relaxin B chain and cross-reacted strongly with H1 and H2 relaxins. In contrast, only the H2 peptide was detected by the ELISA antiserum. Therefore, the marked discrepancy between the RIA and the ELISA could be due to the presence in the diabetic samples of another relaxin-like molecule in addition to the normal H2 relaxin. The biological consequences of elevated serum relaxin in diabetic pregnancy remain to be elucidated.     

Effects of estradiol and progesterone on accumulation of relaxin- and carbohydrate-containing granules in endometrial gland cells of the guinea pig

Guinea pigs were spayed and given various regimens of injections of estradiol and progesterone. The following were monitored in each animal: pubic separation (relaxin stimulation), resorption of the vaginal membrane (estrogen priming), and the presence of PAS-positive granules and/or relaxin in endometrial gland cells (EGC). Injections of estradiol alone resulted in resorption of the vaginal membrane, accumulation of PAS-positive granules in EGC of all animals, and accumulation of relaxin in EGC of two of four animals; but they did not cause pubic separation. Progesterone injections did not result in resorption of the vaginal membrane, separation of pubes, or accumulation of PAS-positive granules; however, one of three animals demonstrated a few EGC that contained relaxin. Animals that received both estradiol and progesterone exhibited PAS-positive granules and relaxin in EGC as well as separated pubes, but did not have resorbed vaginal membranes. Upon examination with the electron microscope, EGC from animals that received estradiol alone exhibited remarkable numbers of secretory granules that contained a carbohydrate-rich material. Secretory granules were not prominent in EGC from animals that received progesterone alone. Estradiol and progesterone injections resulted in accumulation of secretory granules in EGC that contained relaxin and a carbohydrate-rich material. The observations that estradiol and progesterone induce relaxin production in EGC support the hypothesis that uterine relaxin plays an important role in pregnancy and/or parturition in the guinea pig.     

Estrogen-induced disruption of neonatal porcine uterine development alters adult uterine function

In the pig, estradiol-17beta valerate (EV) exposure from birth (Postnatal Day [PND] 0) disrupts estrogen receptor-alpha (ER)-dependent uterine development and increases embryo mortality in adults. To determine effects of neonatal EV exposure on adult uterine morphology and function, 36 gilts received corn oil (CO) or EV from PND 0 to PND 13. Cyclic and pregnant (PX) adults from each treatment group were hysterectomized on Day 12 after estrus/mating. Treatment and pregnancy effects were determined for uterine weight and horn volume, uterine luminal fluid (ULF) protein and estradiol content, endometrial incorporation of 3H-leucine (3H-Leu) into nondialyzable product, and endometrial mRNA levels for ER, progesterone receptor (PR), uteroferrin (UF), retinol-binding protein (RBP), and keratinocyte growth factor (KGF). Adults cycled normally and had similar numbers of corpora lutea. Uteri of PX gilts contained tubular/filamentous conceptuses, and ULF estradiol content was unaffected by treatment. However, pregnancy increased uterine weight and size only in CO gilts (Treatment x Status, P < 0.01). Treatment reduced ULF protein content (P < 0.01), endometrial 3H-Leu incorporation (P < 0.05), and the pregnancy-associated increase in ULF protein (Treatment x Status, P < 0.01). Treatment did not affect endometrial ER or PR mRNA levels but attenuated the pregnancy-associated increase in UF mRNA (Treatment x Status; P < 0.01), increased RBP (P < 0.10), and decreased KGF mRNA levels (P < 0.05). These results establish that transient postnatal estrogen exposure affects porcine uterine responsiveness to potentially embryotrophic signals and that estrogen-sensitive postnatal uterine organizational events are determinants of uterine size and functionality.     

Steroid regulation of cell specific secreted phosphoprotein 1 (osteopontin) expression in the pregnant porcine uterus

Secreted phosphoprotein 1 (SPP1, commonly referred to as osteopontin and formerly known as bone sialoprotein 1, early T-lymphocyte activation 1) is an extracellular matrix/adhesion molecule that is upregulated in the pregnant uterus of all mammals examined to date. This study focused on the pig, which has true epitheliochorial placentation and exhibits induction of SPP1 mRNA in luminal epithelium (LE) just before conceptus attachment and in glandular epithelium (GE) after Day 30 of pregnancy. The objective of this study was to determine steroid regulation of SPP1 mRNA and protein in porcine uterine epithelium. To examine the effect of estrogen, cyclic gilts were treated daily (Days 11-14) with 5 mg estradiol benzoate (i.m.) and hysterectomized on Day 15. To evaluate the long-term effect of pseudopregnancy, cyclic gilts were given daily injections (Days 11-15) with steroid as above and hysterectomized on Day 90. In situ hybridization showed high expression of SPP1 mRNA only in LE contiguous with apposing conceptus tissue on Day 15 of pregnancy. In contrast, estrogen injection resulted in moderate but uniform SPP1 mRNA in all LE of Day 15 nonpregnant gilts, with expression maintained through Day 90 of pseudopregnancy. SPP1 mRNA also localized to the GE of Day 90 pseudopregnant gilts, similar to expression in late gestation. Consistent with in situ hybridization results, SPP1 protein localized to the apical surface of LE in all estrogen-treated gilts and in the GE on Day 90 of pseudopregnancy. We conclude that, in pregnant pigs, SPP1 is induced by conceptus estrogen in uterine LE and is regulated in GE in a manner coincident with CL/placental progesterone production.     

Detection of relaxin release by porcine luteal cells using a reverse hemolytic plaque assay: effect of prostaglandins E2 and F2 alpha, human chorionic gonadotropin, and oxytocin

The release of relaxin from cultured porcine luteal cells derived from pregnant sows was detected by a reverse hemolytic plaque assay. In this assay, luteal cells are cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis--a plaque--develops around relaxin-releasing luteal cells. Treatment with prostaglandin E2 (10(-8) and 10(-6) M) significantly accelerated the rate of plaque formation; in contrast, human chorionic gonadotropin (10-1,000 IU/ml) inhibited the rate of plaque formation. Oxytocin (10(-8) to 10(-4) M) had no detectable effect on relaxin release. However, none of these treatments or long-term preexposure to prostaglandin F2 alpha increased the total proportion of large luteal cells that released relaxin, which remained at about 50%. These results are consistent with the idea that prostaglandins of uterine and/or luteal origin and pituitary luteinizing hormone may contribute, alone or perhaps in combination, to the overall regulation of ovarian relaxin release during pregnancy in the sow. In addition, the results indicate that the effects of prostaglandins are restricted to a subpopulation of large luteal cells that release detectable amounts of relaxin in culture.     

Transplacental diffusion in a bicornuate uterus: comparison of uterine blood flow and oxygen uptake between horns

The purpose of this study was to determine whether samples from the veins of the pregnant and the nonpregnant horn of the uterus lead to similar estimates of uterine blood flow and oxygen consumption. To accomplish this, a comparison of uterine blood flow, arteriovenous differences of oxygen content, and oxygen consumption measured by sampling the venous drainages of the two uterine horns was performed on eight pregnant sheep during the last 20 days of pregnancy. Each sheep carried a single fetus. Umbilical and uterine blood flows were measured with the test substances ethanol and antipyrine by application of the steady-state diffusion method. Twenty-three measurements of uterine blood flow comparing the two horns were not significantly different (P greater than 0.1), and were highly correlated (r = 0.98). The ratio of the oxygen content arteriovenous difference in the pregnant to that in the nonpregnant horn and the ratio of the uterine blood flow in the nonpregnant to that in the pregnant horn were significantly correlated (r = 0.7). As a consequence, paired calculations of oxygen consumption for the whole pregnant uterus had a small coefficient of variation (+/- 3.7%). These results demonstrate that the use of highly diffusible test substances for the measurement of uterine blood flow in pregnant sheep can provide accurate data for the calculation of uterine oxygen uptake, in part because the oxygen and test substance molecules are similarly affected by local variations in placental perfusion.