Half Of Rat Pregnancy Research Articles

Maternal undernutrition induces premature reproductive senescence in adult female rat offspring

To determine the effects of maternal undernutrition (MUN) on the reproductive axis of aging offspring. Animal (rat) study. Research laboratory. Female Sprague-Dawley rats. Food restriction during the second half of pregnancy in rats. Circulating gonadotropins, antimüllerian hormone (AMH), ovarian morphology, estrous cyclicity, and gene expression studies in the hypothalamus and ovary in 1-day-old (P1) and aging adult offspring. Offspring of MUN dams had low birth weight (LBW) and by adult age developed obesity. In addition, 80% of adult LBW offspring had disruption of estrous cycle by 8 months of age, with the majority of animals in persistent estrous. Ovarian morphology was consistent with acyclicity, with ovaries exhibiting large cystic structures and reduced corpora lutea. There was an elevation in circulating T, increased ovarian expression of enzymes involved in androgen synthesis, an increase in plasma LH/FSH levels, a reduction in E2 levels, and no changes in AMH in adult LBW offspring compared with in control offspring. Hypothalamic expression of leptin receptor (ObRb), estrogen receptor-α (ER-α), and GnRH protein was altered in an age-dependent manner with increased ObRb and ER-α expression in P1 LBW hypothalami and a reversal of this expression pattern in adult LBW hypothalami. Our data indicate that the maternal nutritional environment programs the reproductive potential of the offspring through alteration of the hypothalamic-pituitary-gonadal axis. The premature reproductive senescence in LBW offspring could be secondary to the development of obesity and hyperleptinemia in these animals in adult life.

Effect of Food Restriction of Plasma Leptin Concentrations in Pregnant and Nonpregnant Female Mice.

Our long term goal is to test whether leptin depletion is responsible for some of the effects of maternal nutrient restriction during early pregnancy on the placenta and embryo. To test this hypothesis, it is first necessary to establish whether plasma leptin concentrations decrease in response to food restriction in pregnant female mice. Leptin, a peptide hormone produced by adipose tissue, has been found to correlate to body mass index or other measures of adiposity in multiple species. Leptin release is also known to be regulated by circadian rhythm, food intake, insulin, and corticosteroids. Accordingly, leptin levels fall even more rapidly following food restriction than would be explained by fat loss alone. However, there is reason to doubt that this is the case in female mice, particularly during pregnancy. During later pregnancy, leptin concentrations increase independently of adiposity, a process that begins around day 11 in mice. A previous study found that leptin actually increases in response to food restriction during the latter half of pregnancy in rats. Another found that restriction to 60% of ad libitum consumption led to significantly decreased plasma leptin concentrations in male, but not female mice. The goal of this study was to determine whether 50% food restriction affects plasma leptin concentrations in pregnant and non-pregnant female mice. Thirty-six female mice were fed ad libitum (ad lib) or fed 50% of the food consumed by ad lib-fed mice (restricted) at 1600h each day for 10 days. On the tenth day, terminal blood samples were taken from three animals on each diet every four hours. Leptin concentrations differed most between diet groups in the afternoon and evening, but were not different in the morning. We then compared plasma leptin concentrations in samples taken at 1600h after 10 days in each treatment group: non-pregnant, ad lib fed (n=8), non-pregnant, restricted (n=8), pregnant (d11.5) ad lib (n=7) and pregnant restricted (n=4). Two additional plug-positive mice on the restricted diet did not maintain pregnancy, and were excluded from analysis. Initial mouse weights were the same across treatment groups. Final weights were significantly higher in pregnant vs non-pregnant animals fed ad lib, but similar in pregnant and non-pregnant animals on the restricted diet. Mouse weights on the restricted diet were significantly lower than those of ad lib fed mice. Leptin concentrations were similar in pregnant and non-pregnant mice fed ad lib. Leptin was significantly lower in both pregnant and non-pregnant mice following food restriction (p<0.0001), but fell more dramatically in the pregnant animals. Preliminary results indicated that when both groups of pregnant mice were fed in the morning, and blood samples were collected in the morning, leptin concentrations were higher in the restricted group, perhaps due to observed differences in the timing of food consumption. We conclude that plasma leptin concentration does fall in response to food restriction in pregnant and non-pregnant female mice, but the timing of sampling and feeding are critical. This work is supported by NIH HD055231 (poster)

Relative Roles of the Epithelial and Stromal Tissue Compartment(s) in Mediating the Actions of Relaxin and Estrogen on Cell Proliferation and Apoptosis in the Mouse Lower Reproductive Tract

Relaxin and estrogen are secreted by the ovary during the second half of pregnancy in rats and mice. Relaxin promotes marked growth of the lower reproductive tract in both species. Relaxin promotes accumulation of epithelial and stromal cells in the cervix and vagina by both stimulating cell proliferation and inhibiting apoptosis. Estrogen acting through estrogen receptor alpha (ERalpha) plays an essential permissive role in relaxin's actions. A fundamental step toward understanding the actions of relaxin and estrogen is to identify the tissue compartments that initiate their effects. Limited studies using either antibodies to human relaxin receptor (LGR7, RXFP1) or an IRES-LacZ reporter cassette in the LGR7 gene revealed relaxin receptors in subepithelial stroma cells and smooth muscle cells but not in epithelial cells in rodent vaginal and/or cervical tissues. ERalpha has been reported in both stromal and epithelial compartments in the rodent reproductive tract. This chapter describes ongoing studies that use relaxin bioactivity as a means of identifying the tissue compartment(s) that initiates the actions of relaxin and estrogen on the lower reproductive tract. Specifically, a tissue separation-recombination methodology in combination with LGR7 knockout mice was initially used to obtain functional evidence that stromal LGR7 is both necessary and sufficient to promote proliferation and inhibit apoptosis in both stromal and epithelial cells in mouse cervix and vagina. The tissue separation-recombination method is currently being used in conjunction with ERalpha knockout mice to determine if the obligatory permissive effect of estrogen on relaxin-induced cell proliferation occurs through stromal and/or epithelial ERalpha.

Prenatal choline deficiency increases choline transporter expression in the septum and hippocampus during postnatal development and in adulthood in rats

Supplementation of maternal diet with the essential nutrient, choline, during the second half of pregnancy in rats causes long-lasting improvements in spatial memory in the offspring and protects them from the memory decline characteristic of old age. In contrast, prenatal choline deficiency is associated with poor performance in certain cognitive tasks. The mechanism by which choline influences learning and memory remains unclear; however, it may involve changes to the hippocampal cholinergic system. Previously, we showed that the hippocampi of prenatally [embryonic days (E) 11–17] choline-deficient animals have increased synthesis of acetylcholine (ACh) from choline transported by the high-affinity choline transporter (CHT) and reduced ACh content relative to the control and to the E11–17 choline-supplemented rats. In the current study, we found that, during postnatal period [postnatal days (P) 18–480], prenatal choline deficiency increased the expression of CHT mRNA in the septum and CHT mRNA and protein levels in the hippocampus and altered the pattern of CHT immunoreactivity in the dentate gyrus. CHT immunoreactivity was more prominent in the inner molecular layer in prenatally choline-deficient rats compared to controls and prenatally choline-supplemented animals. In addition, in all groups, we observed a population of hilar interneurons that were CHT-immunoreactive. These neurons are the likely source of the hippocampal CHT mRNA as their number correlated with the levels of this mRNA. The abundance of hippocampal CHT mRNA rose between P1 and P24 and then declined reaching 60% of the P1 value by P90. These data show that prenatal availability of choline alters its own metabolism (i.e., CHT expression). While the upregulated CHT expression during the period of prenatal choline deficiency may be considered as a compensatory mechanism that could enhance ACh synthesis when choline supply is low, the persistent upregulation of CHT expression subsequent to the brief period of prenatal deprivation of choline in utero might be beneficial during choline deficiency in adulthood.

Pregnancy in rats is modulated by ganglionic cholinergic action

The control of ovarian steroidogenesis during pregnancy is mainly of endocrine origin. At present, there is little information about the influence of neural factors on the gestation physiology. The purpose of this work was to study the action of cholinergic agents in celiac ganglion upon the liberation of progesterone and ovarian androstenedione in the second half of pregnancy in rats. We used the ex vivo celiac ganglion-superior ovarian nerve-ovary integrated system (celiac ganglion-SON-ovary) that was incubated in buffer solution for 180 min, with the celiac ganglion and the ovary located in different compartments and linked by the SON. The results obtained indicate that the control values of ovarian androstenedione vary according to the pregnancy day analyzed. The addition of acetylcholine in ganglion decreased the liberation of both steroids on Day 15 whereas at the end of pregnancy it decreased the liberation of androstenedione without modifying progesterone. Due to the effect observed with atropine and hexametonium, acetylcholine action might occur through unspecific ganglionic pathways (Days 15 and 21) or through muscarinic ganglionic receptors (Days 19 and 20). Thus, we conclude that the cholinergic sympathetic system from the celiac ganglion might be a fine modulator of the pregnancy physiology.

The Effects of Blocking the Actions of Estrogen and Progesterone on the Rates of Proliferation and Apoptosis of Cervical Epithelial and Stromal Cells During the Second Half of Pregnancy in Rats1

Serum levels of the ovarian hormones relaxin, estrogen, and progesterone are elevated during the second half of 23-day rat pregnancy when dramatic growth of the cervix occurs. Recently, we demonstrated that relaxin contributes to cervical growth by both promoting cell proliferation and inhibiting apoptosis of cervical cells during late pregnancy. The objective of this study was to determine the influence of estrogen and progesterone on the rates of proliferation and apoptosis of cervical cells at 3-day intervals during the second half of rat pregnancy. The actions of estrogen and progesterone were blocked with s.c. injections of estrogen antagonist ICI 182,780 and progesterone antagonist RU486, respectively. To evaluate cell proliferation, 5'-bromo-2'-deoxyuridine was injected s.c. 8 h before cervixes were collected. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling was used to detect apoptotic cells. Proliferating and apoptotic cells were identified by immunohistochemistry, and the rates at which these processes occurred were determined by morphometric analysis. Blocking the actions of estrogen and progesterone decreased the rates of proliferation and increased the rates of apoptosis of both cervical epithelial and stromal cells during late pregnancy. However, blocking the actions of progesterone had the opposite effects on apoptosis of both cervical epithelial and stromal cells during the middle of pregnancy. In conclusion, this study provides evidence that estrogen and progesterone, like relaxin, contribute to the increase in the cervical cell content during late pregnancy by promoting proliferation and inhibiting apoptosis of cervical cells.

Vascular expression of adrenomedullin is increased in Wistar rats during early pregnancy

Objective Circulating levels of adrenomedullin (ADM)—a vasodilator peptide with long-lasting effects—increase in the course of pregnancy. Neither the site nor the concomitant rate of ADM synthesis in pregnancy is known. The aim of this study was to test the hypothesis that the rise in plasma levels of ADM during pregnancy is paralleled by increased gene expression and protein levels in the vascular bed. Study design We determined in cardiovascular and reproductive tissues of non-pregnant ( n = 10) and 10-days pregnant ( n = 10) Wistar rats ADM gene expression by semi-quantitative RT-PCR (normalized to GAPDH). As a support for the mRNA data, protein concentrations were measured by both ELISA and Western blot analysis. Finally, ADM in these tissues was localized by immunohistochemical staining. Statistical analysis was carried out by applying Mann–Whitney U-test. Results ADM mRNA levels in the abdominal aorta, renal artery and the kidney were increased during pregnancy. In addition, immunohistochemical staining in the kidney, uterus, abdominal aorta, renal, uterine and superior mesenteric artery was more intense as compared to non-pregnant rats. However, we observed lower concentrations of tissue ADM protein in pregnant rats, indicating an increased release of the hormone by the producing cells. Conclusion Vascular ADM gene expression is increased in the first half of rat pregnancy. This coincides and may be functionally related to the institution of a high flow/low resistance circulation in pregnancy.

The Extent to which Relaxin Promotes Proliferation and Inhibits Apoptosis of Cervical Epithelial and Stromal Cells Is Greatest during Late Pregnancy in Rats

Relaxin promotes marked growth of the cervix during the second half of rat pregnancy, and this growth is accompanied by an increase in both epithelial and stromal cells. The objective of this study was to test the hypothesis that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical cells is greatest during late pregnancy in rats. The influence of neutralization of circulating relaxin by iv injection of 5 mg monoclonal antibody against rat relaxin (MCA1) was examined at 3-d intervals throughout the second half of pregnancy. Controls were injected with either 5 mg monoclonal antibody against fluorescein or 0.5 ml PBS vehicle. To evaluate cell proliferation, 5'-bromo-2-deoxyuridine was injected sc 8 h before cervixes were collected. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling and electron microscopy were used to detect apoptotic cells. Neutralization of relaxin with MCA1 decreased the rate of proliferation and increased the rate of apoptosis of cervical cells by d 13. However, the extent to which relaxin influenced these processes was greatest and dramatic by late pregnancy. In MCA1-treated rats on d 22 of pregnancy, the rates of proliferation of both epithelial and stromal cells were less than 20% those in controls, and the rates of apoptosis in epithelial cells and stromal cells were more than 10- and 3-fold, respectively, greater than those in controls. In conclusion, this study provides evidence that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy.

Ação da Betametasona em Ratas Prenhes: Impacto sobre os Níveis de Corticosterona e Glândulas Adrenais Maternas e Fetais

Purpose: the repetitive use of antenatal corticosteroid therapy for acceleration of fetal lung maturation has been common in cases at risk of preterm delivery. We studied the corticosterone levels at term and the morphologic aspects in the maternal and fetal adrenal glands submitted to the effect of betamethasone in the second half of rat pregnancy in order to verify its consequences. Methods: thirty female pregnant rats were divided into three groups of ten animals each. Group I received betamethasone on the 11th, 12th, 18th and 19th day of pregnancy. Group II received distilled water on the same days (control group). Group III did not receive any drug (stress control group). All rats were sacrificed on the 20th day of pregnancy when plasma corticosterone levels of dams were assessed and the maternal and fetal adrenal glands were studied by light microscopy. Results: plasma corticosterone level of dams was lower in the group treated with betamethasone (4.8 mg/dL) when compared with the control groups (17.7 and 26.8 mg/dL). The light microscopy study revealed cytoplasmatic vacuolation in the fasciculate zone in the maternal and fetal adrenal glands, which indicates adrenal suppression. Conclusions: the antenatal repetitive and prolonged use of corticosteroids in pregnant rats for acceleration of lung fetal maturation causes maternal and fetal adrenal suppression.

Evidence that relaxin inhibits apoptosis in the cervix and the vagina during the second half of pregnancy in the rat.

The growth of the cervix and vagina that occurs during the second half of rat pregnancy is accompanied by an increase in both epithelial and stromal cells. Neither the mechanism(s) that regulates this accumulation of cells nor its hormonal control is known. To test the hypothesis that the rate of apoptosis declines during the second half of pregnancy, cervices and vaginas were collected on days 5, 10, 15, 18, and 21 of pregnancy. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling was used to detect apoptotic cells. The rate of apoptosis declined (P < 0.05) in epithelial and stromal cells in both the cervix and vagina during the second half of pregnancy, when blood levels of relaxin are increasing. To test the hypothesis that relaxin inhibits apoptosis, cervices and vaginas were collected 6, 12, 24, 48, and 72 h after the neutralization of endogenous relaxin, on days 19-21 of pregnancy, with a monoclonal antibody for rat relaxin. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling method and electron microscopy were used to detect apoptotic cells. Withdrawal of relaxin caused an increase in the rate of apoptosis in both the cervix and the vagina (P < 0.05). It is concluded that the rate of apoptosis declines in the cervix and the vagina during the second half of rat pregnancy, and that relaxin likely contributes to this process.

Induction of relaxin messenger RNA expression in response to prolactin receptor activation requires protein kinase C delta signaling.

The ability of PRL or rat placental lactogen (rPL)-1 to induce relaxin mRNA expression was analyzed in a luteinized rat granulosa cell culture model. PRL receptor activation induced relaxin mRNA expression in a concentration- and time-dependent manner. High concentrations of PRL receptor agonist, equivalent to those of the second half of pregnancy in rats, were required to elicit relaxin mRNA expression. A 40-fold induction of relaxin mRNA was observed in cells treated 24 h with 1 microg/ml of rPL-1. Estrogen enhanced relaxin expression induced by PRL but did not affect relaxin expression on its own. PRL/rPL-1 induction of relaxin expression was independent of the extracellular regulated kinase (ERK) members of the mitogen-activated protein kinase (MAPK) pathway, based on the inability of the ERK kinase inhibitor PD98059 to block induction of relaxin expression. PRL/rPL-1 induction of relaxin expression required protein kinase C (PKC) delta, based on the ability of the preferential PKC delta inhibitor rottlerin to abolish induction of relaxin expression. Direct activation of PKC by phorbol myristate acetate, however, was not sufficient to promote induction of relaxin mRNA expression. Stats (signal transducers and activators of transcription) 3 and 5 DNA binding activities were induced by PRL/rPL-1 treatment of luteinized granulosa cells but only Stat 3 DNA binding was reduced by rottlerin. PRL/rPL-1 treatment of luteinized granulosa cells resulted in increased phosphorylation on tyrosine-705 and serine-727 of Stat 3, and these responses were reduced and blocked, respectively, by rottlerin. Tyrosine and serine phosphorylations of Stat 3 in the corpus luteum were also increased in the second half of pregnancy when PL levels are highest. Stat 3, but not Stat 1 or 5, coimmunoprecipitated with luteal PKC delta during pregnancy; Stat 3 transiently coimmunoprecipitated with PKC delta from luteinized granulosa cells in response to PRL receptor activation; and Stat 3/PKC delta complex formation required PKC delta kinase activity. Taken together, these results show that PKC delta is obligatory for PRL/rPL-1-dependent relaxin expression, that PKC delta complexes with Stat 3 in response to PRL receptor activation, and that PKC delta is involved in the regulation of Stat 3 phosphorylation downstream of the PRL receptor. These results demonstrate that PRL/rPL-1 promotes relaxin expression in luteal cells and that this event is mediated, at least in part, via PKC delta.

Effects of naloxone infusion on nocturnal prolactin secretion and Fos/FRA expression in pregnant rats.

Dopamine (DA) secreted by tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus is the major inhibitory factor controlling prolactin (PRL) secretion from the anterior pituitary. Endogenous opioid peptides (EOPs), mainly the neuropeptide beta-endorphin, have a stimulatory influence on PRL secretion. During the first half of pregnancy in rats, PRL secretion is characterized by two daily surges, the nocturnal surge and the diurnal surge. We tested the hypothesis that EOPs are critical stimulatory factors in regulating the nocturnal PRL surge, possibly via inhibition of TIDA neuronal activity. Naloxone (NAL), an opioid antagonist, was continuously infused (0.2 mg/10 microL/min i.v.) during the expected time of the nocturnal PRL surge in day 8 pregnant rats. Radioimmunoassay (RIA) was used to measure plasma PRL levels, and the immunocytochemical (ICC) staining of Fos/FRA was performed to detect changes in transcriptional activity of neurons in the hypothalamus. ICC of tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis, was performed to visualize TIDA neurons in the arcuate nucleus. The results showed that the nocturnal surge of PRL was markedly delayed and dampened in NAL-treated rats (p < 0.01). The numbers of both Fos/FRA and (Fos/FRA)/TH dual-staining neurons increased in the arcuate nucleus following NAL treatment (p < 0.05 for both comparisons). These data indicate that EOPs are critical stimulatory factors for the nocturnal PRL surge and that the actions of EOPs are partially mediated via decreasing TIDA neuronal activity.

Maternal Factors Modulate the Increase in Vasoactive Substances During Rat Pregnancy

Objective: To explore if the changes in vasoactive substances observed during early pregnancy in the rat are modulated by maternal or fetoplacental factors.Methods: Urinary excretion of cGMP, 6-keto-prostaglandin-Fla (6-keto-PGFla), thromboxane B2 and kallikrein activity was measured in pregnant (P, n= 11), pseudopregnant (PSP, n= 12), and virgin (n = 13) rats and in ovariec-tomized virgin rats supplemented with slow-release pellets containing either progesterone (50 mg/pellet) or estradiol (0.5 mg/pellet) or a combination of both hormones, for 21 days.Results: The cGMP excretion was higher in PSP rats than in virgin rats at day 5 (virgin = 82 ± 7, P = 93 ± 5, PSP = 110 ± 8 nmol/24 h,p < 0.05); at day 10, values were significantly increased in P and PSP rats. 6-keto-PGFla excretion was similarly elevated in P and PSP rats at day 5 (virgin = 120 ± 10, P = 160 ± 10, and PSP = 174 ± 14 ng/24 h,p < 0.01). This trend was still present at day 10. Thromboxane B2 excretion showed a nonsignificant increase in P and PSP rats in day 5; at day 10, values were significantly elevated in both experimental groups (virgin = 23 ± 2, P = 32 ± 4, and PSP = 32 ± 2 ng/24 h, p < 0.05). Kallikrein excretion was significantly increased in PSP and P rats at days 5 and 10. Estradiol or progesterone administration caused a significant decrease in serum aldosterone and an increase in urinary kallikrein activity.Conclusions: These results indicate that during the first half of rat pregnancy, the increment in vasoactive substances is modulated by maternal and not by fetoplacental factors.

Monoclonal antibodies specific for rat relaxin. X. Endogenous relaxin induces changes in the histological characteristics of the rat vagina during the second half of pregnancy.

This study employed morphometric analysis to evaluate changes in the histological characteristics that accompany relaxin-induced growth and softening of the vagina during the second half of rat pregnancy. There were three treatment groups (N = 4/group). Five milligrams of a monoclonal antibody for rat relaxin, designated MCA1, were injected i.v. daily on days 12-21 of gestation to treatment group MCA1. Control groups received either 5 mg of monoclonal antibody for fluorescein (MCAF; monoclonal antibody control) or 0.5 ml PBS (vehicle control). Vaginas were removed on day 22 of pregnancy, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Tissue sections (5 microm) were stained with Gomori's trichrome to visualize collagen, or orcein to visualize elastin. Measurements were performed with a light microscope equipped with a video camera connected to a computer. Within the vaginal stroma, the density of collagen fiber bundles was lower, the length of elastin fibers was shorter, and the cross-sectional area and wall thickness of arteries were greater in relaxin-replete control rats than in relaxin-deficient MCA1-treated rats. These relaxin-induced changes in the stroma appear to account, at least in part, for the hormone's softening effect on the vagina. Within the epithelium, there were approximately 2-fold more basal and mucus-secreting cells in relaxin-replete control rats than in MCA1-treated rats. The relaxin-induced accumulation of epithelial cells appears to contribute to vaginal growth. We conclude that relaxin plays a role in preparing the vagina as well as the cervix for rapid and safe delivery in pregnant rats.

Relaxin increases the accumulation of new epithelial and stromal cells in the rat cervix during the second half of pregnancy.

Both cervical and vaginal growth are relaxin dependent during rat pregnancy. We recently reported a relaxin-dependent 1.5-fold increase in cervical and vaginal DNA content from midpregnancy until term. This finding indicated that relaxin probably promotes cervical and vaginal growth at least in part by promoting cellular proliferation. The objective of this study was to identify and quantify cells in the cervix and vagina that proliferate during the second half of rat pregnancy in response to relaxin. Primiparous pregnant rats were ovariectomized or sham ovariectomized (group C; n = 8) on day 9 of pregnancy (D9). Ovariectomized rats were then treated with physiological doses of progesterone plus estrogen (n = 7) or progesterone, estrogen, and porcine relaxin (n = 7). Cellular proliferation was determined by continuously administering a low dose of 5-bromo-2'-deoxyuridine (BrdU) via miniature osmotic pump from D9-D22. On D22, cervices and vaginas were collected, fixed in formalin, paraffin embedded, and serially sectioned (4 microm). Adjacent serial sections were either immunostained for BrdU to assess cell proliferation or stained with hematoxylin to determine total cell number. Cell proliferation was evaluated by counting BrdU-positive nuclei and total nuclei in the same area on adjacent sections. Cell counts were determined using computerized digital morphometric analysis at x575. In control rats, nearly 75% of the epithelial cells and 55% of the stromal cells within the cervix at term had proliferated during the second half of pregnancy. The accumulation of approximately half of the new cells was relaxin dependent. Within the cervical stroma, relaxin increased the accumulation of cells associated with blood vessels and also the number of isolated cells (probably fibroblasts). Relaxin did not appear to affect smooth muscle cell proliferation in the cervix. In contrast to the cervix, a minority of vaginal epithelial cells (45%) and stromal cells (20%) proliferated during the second half of pregnancy. Although relaxin appeared to have a tendency to increase the accumulation of new vaginal epithelial and stromal cells, morphometric analysis did not provide support for such an effect. In conclusion, this study demonstrates that relaxin promotes a marked increase in the accumulation of new epithelial cells and stromal cells within the cervix. The relaxin-induced increase in new epithelial and stromal cells probably contributes to relaxin's effects on growth and remodeling of the cervix that are required for rapid and safe delivery.

Evidence that systemic relaxin promotes moderate water consumption during late pregnancy in rats.

The protein hormone relaxin is secreted by the ovaries throughtout the second half of the 23 day pregnancy in the rat. We recently reported that neutralization of endogenous relaxin with monoclonal antibodies for rat relaxin decreases water consumption during the daily light period during the second half of pregnancy in rats. The apparent effects of relaxin on water consumption, however, were extremely modest. One explanation for the failure to observe a greater relaxin-dependent effect on water consumption is failure of the monoclonal antibody for rat relaxin to neutralize all circulating relaxin. A second explanation is that circulating relaxin has only slight effects on water consumption. This investigation was conducted with an experimental model in which circulating relaxin was removed in order to re-examine the effects of relaxin on water consumption during the daily light period in late pregnancy in rats. On day 9 (D9) of pregnancy, before the presence of relaxin (R) in the circulation, primiparious pregnant rats were ovariectomized (O) or sham ovariectomized (C). Throughout the remainder of pregnancy, rats were treated with combinations of either progesterone (P) and estrogen (E, group OPE) or progesterone, estrogen and porcine relaxin (group OPER) in doses that restore physiological parameters to values similar to those that occur during the second half of pregnancy in intact rats. Progesterone and estrogen were administered by Silastic tubing implants and porcine relaxin was administered via miniature osmotic pump. Sham-ovariectomized animals received either the hormone vehicles (group SC) or no implants (group IC). Water consumption was measured daily from D4 to D20 at both 0700 and 2100 h which was when the lights went on and off respectively. Water consumption increased as pregnancy continued from D10 to D20 during the daily 10 h dark periods (P < 0.01), but not during the 14 h light periods for all four groups. Daily water consumed by rats in group OPE was significantly lower (P < 0.05) than that consumed by shamovariectomized rats from D17 to D20 and lower than that consumed by rats in group OPER on D20. During the dark period there was no difference in water consumption among groups. During the light period, however, group OPE consumed significantly less water (P < 0.05) than group C from D18 to D22. Moreover, there was a consistent tendency (P < 0.13) for the water consumption to be greater in rats in group OPER than in those in the relaxin-deficient group OPE during the daily light period from D11 to D20 of pregnancy. We conclude that the increase in water consumption that occurs during the daily dark periods during the second half of pregnancy is not attributable to circulating relaxin. Circulating relaxin promotes only modest increases in water consumption during the daily light periods during late pregnancy in the rat.

Evidence that endogenous relaxin promotes growth of the vagina and uterus during pregnancy in gilts.

Recently, it was demonstrated that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats and that administration of porcine relaxin promotes growth of the uterus in nonpregnant or early pregnant gilts. This study examined the effects of circulating relaxin on growth of both the vagina and uterus during the last two thirds of the 114-day gestation period in gilts. Furthermore, this study employed an in vitro immunohistochemical localization technique to determine whether the vagina and uterus in pigs have specific relaxin-binding sites. Three groups of pregnant gilts were used: sham-ovariectomized controls (group C; n = 8), ovariectomized progesterone-treated (group OP; n = 6), and ovariectomized progesterone- plus relaxin-treated (group OPR; n = 7). Gilts were either sham ovariectomized or ovariectomized on day 40 of gestation. Hormone replacement therapy with progesterone (group OP), progesterone plus relaxin (group OPR), or hormone vehicles (group C) began on day 38 (progesterone) or day 40 (relaxin) and continued until day 110. On day 110, the vagina and uterus were collected, and wet weight, dry weight, and percent hydration were determined. Small pieces (2-3 cm3) of the vagina and uterus from groups C and OP were frozen and cryosectioned for the immunohistochemical localization of relaxin-binding sites. Relaxin promoted growth of both the vagina and uterus. The wet weights of both the vagina and uterus in relaxin-deficient gilts (group OP) were lower (P < 0.05) than those in controls (group C), and relaxin replacement therapy (group OPR) restored the wet weights of both tissues to values that did not differ from those in controls. The mean dry weights and percent hydrations in the vagina and uterus did not differ among treatments. Immunohistochemical localization studies in the vagina and uterus demonstrated that specific and saturable binding of relaxin was localized in the same cell types of both tissues, namely epithelial cells (luminal in vagina, and both luminal and glandular in uterus), smooth muscle cells (both circular and longitudinal in vagina, and myometrial in uterus), and cells associated with blood vessels. In conclusion, this study provides evidence that circulating relaxin promotes growth of both the vagina and uterus during pregnancy in the pig. Furthermore, this study provides evidence that both the vagina and uterus contain specific and saturable relaxin-binding sites in epithelial cells, smooth muscle cells, and cells associated with blood vessels. We conclude that these cells probably initiate relaxin's effects on the vagina and uterus of the pregnant pig.

Evidence That Endogenous Relaxin Promotes Growth of the Vagina and Uterus during Pregnancy in Gilts

Recently, it was demonstrated that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats and that administration of porcine relaxin promotes growth of the uterus in nonpregnant or early pregnant gilts. This study examined the effects of circulating relaxin on growth of both the vagina and uterus during the last two thirds of the 114-day gestation period in gilts. Furthermore, this study employed an in vitro immunohistochemical localization technique to determine whether the vagina and uterus in pigs have specific relaxin-binding sites. Three groups of pregnant gilts were used: sham-ovariectomized controls (group C; n = 8), ovariectomized progesterone-treated (group OP; n = 6), and ovariectomized progesterone- plus relaxin-treated (group OPR; n = 7). Gilts were either sham ovariectomized or ovariectomized on day 40 of gestation. Hormone replacement therapy with progesterone (group OP), progesterone plus relaxin (group OPR), or hormone vehicles (group C) began on day 38 (progesterone) or day 40 (relaxin) and continued until day 110. On day 110, the vagina and uterus were collected, and wet weight, dry weight, and percent hydration were determined. Small pieces (2–3 cm3) of the vagina and uterus from groups C and OP were frozen and cryosectioned for the immunohistochemical localization of relaxin-binding sites. Relaxin promoted growth of both the vagina and uterus. The wet weights of both the vagina and uterus in relaxin-deficient gilts (group OP) were lower (P < 0.05) than those in controls (group C), and relaxin replacement therapy (group OPR) restored the wet weights of both tissues to values that did not differ from those in controls. The mean dry weights and percent hydrations in the vagina and uterus did not differ among treatments. Immunohistochemical localization studies in the vagina and uterus demonstrated that specific and saturable binding of relaxin was localized in the same cell types of both tissues, namely epithelial cells (luminal in vagina, and both luminal and glandular in uterus), smooth muscle cells (both circular and longitudinal in vagina, and myometrial in uterus), and cells associated with blood vessels. In conclusion, this study provides evidence that circulating relaxin promotes growth of both the vagina and uterus during pregnancy in the pig. Furthermore, this study provides evidence that both the vagina and uterus contain specific and saturable relaxin-binding sites in epithelial cells, smooth muscle cells, and cells associated with blood vessels. We conclude that these cells probably initiate relaxin’s effects on the vagina and uterus of the pregnant pig.

In vitro studies of the stimulation of insulin secretion and B-cell proliferation by rat placental lactogen-II during pregnancy in rats

The present study investigated the effect of rat placental lactogen-II (rPL-II) on insulin secretion and B-cell proliferation of the maternal islets during the last half of pregnancy in rats using a homologous system. Pancreatic islets were isolated from nonpregnant rats and rats at day 13 of pregnancy and cultured for 8 days in medium containing trophoblast culture medium or purified hormones. The medium was changed daily and insulin concentrations were determined by measuring immunoreactivity. The number of proliferating B cells were determined by double staining for both insulin and 5'-bromo-2'-deoxyuridine (BrdU) incorporated into replicating DNA during the last 24 h of incubation. rPL-II-enriched medium, in which trophoblasts of placentae from rats at day 13 of pregnancy were incubated for 8 days, was added to the islet culture system. Insulin concentration in the medium of non-pregnant rat islets was significantly increased and doubled on incubation in 100% trophoblast culture medium. Addition of purified rPL-II to the culture medium of pregnant rat islets stimulated insulin secretion at the concentrations of 50-500 ng ml-1 in a dose-dependent manner. The stimulatory effect of rPL-II on insulin secretion was found to be more than double that with rat prolactin (rPRL) and rat growth hormone (rGH) at 100 ng ml-1. For determination of B-cell proliferation, non-pregnant rat islets were incubated with rPL-II, rPRL or rGH at 1000 ng ml-1 for 8 days and then 10 mmol BrdU l-1 was added during the last 24 h of incubation. rPL-II (5.30 +/- 0.36%), rPRL (3.79 +/- 0.34%) and rGH (2.87 +/- 0.29%) significantly increased the rate of incorporation of BrdU into the B cells compared with that of the control (1.34 +/- 0.18%). These results indicate that rPL-II directly regulates insulin secretion and B-cell proliferation of maternal islets during the last half of pregnancy in rats.

Monoclonal antibodies specific for rat relaxin. IX. Evidence that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats.

It is established that endogenous relaxin promotes the growth and development of the cervix, mammary glands, and nipples in pregnant rats. Additionally, the observation that porcine relaxin promotes growth of the vagina in both nonpregnant and pregnant rats provides indirect evidence that endogenous relaxin may effect growth of the vagina during rat pregnancy. The purpose of this study was to determine whether endogenous relaxin promotes growth of the vagina in pregnant rats. To that end, a monoclonal antibody, specific for rat relaxin, designated MCA1, was used to passively neutralize endogenous relaxin throughout the second half of pregnancy in intact rats. Five milligrams of highly purified MCA1 were injected iv to rats daily from days 12-22 of pregnancy. Controls received either a monoclonal antibody for fluorescein or PBS. The vaginal wet weight, dry weight, length, diameter, inner surface area, DNA content, and percent water content were determined. No differences were found between monoclonal antibody for fluorescein and PBS controls for any of the measured vaginal parameters. In contrast, values for all physical parameters, except percent water content, were significantly lower in MCA1-treated rats than in controls. Vaginal DNA content was also lower in MCA1-treated rats than in controls; and this observation supports the view that relaxin induces vaginal growth at least in part by promoting cell proliferation. To examine the mechanism of relaxin's apparent action on the vagina, specific relaxin-binding sites were localized immunohistochemically. Relaxin-binding sites were found in epithelial and smooth muscle cells, and the binding was specific for relaxin. We conclude that endogenous relaxin promotes growth of the vagina in pregnant rats.