Virgin Animals Research Articles

Life cycle studies of the earthworm Lumbricus friendi (Cognetti, 1904)

The objective of this work was to obtain data on aspects of the lifecycle of Lumbricusfriendi. Field-collected specimens from NW Spain were grown to maturity and maintained under controlled laboratory conditions. Thereafter experiments were undertaken to determine mode of reproduction, reproductive output, cocoon viability and development, and hatchling growth rate. Effects of density and temperature on growth were also examined. Plastic culture vessels containing a substrate of Kettering loam were incubated in temperature-controlled chambers, with dried horse manure as food. Paired, mature animals were sampled monthly over a period of 9 months for cocoon production. Cocoons were incubated on moist filter papers in Petri dishes. Results showed that at 15 °C, individuals matured at a live mass of 3.5 g. Zero cocoons were produced by virgin animals maintained in isolation, but recently matured, paired L. friendi produced 2.16 cocoons individual−1 wk−1. Cocoons were lemon-shaped with a smooth pale yellow-coloured outer surface and had a mean mass of 28 mg. At 15 °C, cocoons required 90 days to hatch and hatchability from the first 5 months of cocoon production was 85% successful. The majority of cocoons produced a single hatchling (twins recorded < 1%). Growth of hatchlings to maturity was achieved within a period of 5 months, with tubercula pubertatis developed after 3 months. Increased temperatures in the range of 5–15 °C led to significantly increased growth rates, whereas increased earthworm density led to a significant decrease in rate of growth. These findings permit comparisons with more closely studied anecic species such as Lumbricus terrestris.

Open field behavior and intra-nucleus accumbens dopamine release in vivo in virgin and lactating rats.

behavioral, endocrine, and neurochemical implications. This experience causes behavioral and neurochemical changes that involve several brain areas important for the expression of maternal behavior. The present study showed that lactating rats exhibited reduced general locomotor activity in the open field test compared with virgin animals. Our hypothesis was that nucleus accumbens dopamine, which regulates maternal behavior in lactating rats, is also involved in the low expression of maternal locomotion in the open field test observed during the early stages of lactation and reflects decreased motivation. Initially we compared open field behavior in virgin and lactating rats to confirm our previous data. Thus, the in vivo release of dopamine in the nucleus accumbens in virgin and lactating female rats was measured. Perfusate concentrations of extracellular dopamine and its metabolites showed no differences between virgin and lactating rats. Thus, the reduced general activity observed in lactating rats might not be related to intra-nucleus accumbens dopamine control. Keywords: maternal behavior, virgin rats, lactating rats, nucleus accumbens, dopamine.

Parity is associated with an expanded macrophage population in the mammary gland

Pregnancy is a well established protective factor against breast cancer. One explanation for protection is the increased differentiation status of the parous epithelium. However, this does not explain the association of parity with increased aggressiveness of breast cancers, particularly cancers that occur soon after pregnancy. Because tumor aggressiveness can be influenced by the cell population that surrounds the mammary epithelium, we examined the potential role of the immune system in establishing a long-term difference between the mammary glands of primiparous and virgin animals. Specific mRNA levels, enzyme activities and antigen expressing cells were quantified in primiparous and virgin mammary glands from Sprague-Dawley rats in diestrous. Our results show that macrophages, but not neutrophils or B-cells, are specifically increased in fully involuted glands compared with age-matched virgin mammary glands. Macrophages play a dual role in tumor progression, both opposing and supporting the process. Our finding of an increased macrophage population in the primiparous mammary gland could explain the dichotomy of the reported association of parity with decreased breast cancer incidence and increased breast cancer aggressiveness.

Impact of Pregnancy and Vaginal Delivery on the Passive and Active Mechanics of the Rat Vagina

Remodeling of vaginal extracellular matrix and smooth muscle likely plays a critical role in reducing the risk of maternal injury during vaginal delivery by altering the mechanical properties to increase distension and reduce stress. Long-Evans rats were divided into five groups to examine the passive mechanical and active contractile properties throughout pregnancy and postpartum: virgin (n=17), mid-pregnant (Day 14-16, n=12), late-pregnant (Day 20-22, n=14), immediate postpartum (0-2 h after delivery, n=14), and 4 week postpartum (n=15). Longitudinal sections of vaginal tissue were loaded to failure uniaxially for passive mechanical or active contractile properties were examined. For passive mechanics, the tangent modulus decreased 45% by mid-pregnancy and immediately postpartum (p<0.001). The ultimate strain continuously increased up to 43% higher than virgin animals (p=0.007) in the immediate postpartum group. For active mechanics, the maximal contractile force was 36-56% lower through immediate postpartum animals, and was significantly more sensitive to K+ throughout pregnancy and postpartum (p=0.003). The changes observed in the passive and active properties of the rat vagina are consistent with what would be expected from a tissue that is remodeling to maximize its ability to distend at the time of vaginal delivery to facilitate passage of the fetus with minimal injury.

BRCA1 Basal-like Breast Cancers Originate from Luminal Epithelial Progenitors and Not from Basal Stem Cells

Breast cancers in BRCA1 mutation carriers frequently have a distinctive basal-like phenotype. It has been suggested that this results from an origin in basal breast epithelial stem cells. Here, we demonstrate that deleting Brca1 in mouse mammary epithelial luminal progenitors produces tumors that phenocopy human BRCA1 breast cancers. They also resemble the majority of sporadic basal-like breast tumors. However, directing Brca1 deficiency to basal cells generates tumors that express molecular markers of basal breast cancers but do not histologically resemble either human BRCA1 or the majority of sporadic basal-like breast tumors. These findings support a derivation of the majority of human BRCA1-associated and sporadic basal-like tumors from luminal progenitors rather than from basal stem cells. They also demonstrate that when target cells for transformation have the potential for phenotypic plasticity, tumor phenotypes may not directly reflect histogenesis. This has important implications for cancer prevention strategies.

Redefining the Expression and Function of the Inhibitor of Differentiation 1 in Mammary Gland Development

The accumulation of poorly differentiated cells is a hallmark of breast neoplasia and progression. Thus an understanding of the factors controlling mammary differentiation is critical to a proper understanding of breast tumourigenesis. The Inhibitor of Differentiation 1 (Id1) protein has well documented roles in the control of mammary epithelial differentiation and proliferation in vitro and breast cancer progression in vivo. However, it has not been determined whether Id1 expression is sufficient for the inhibition of mammary epithelial differentiation or the promotion of neoplastic transformation in vivo. We now show that Id1 is not commonly expressed by the luminal mammary epithelia, as previously reported. Generation and analysis of a transgenic mouse model of Id1 overexpression in the mammary gland reveals that Id1 is insufficient for neoplastic progression in virgin animals or to prevent terminal differentiation of the luminal epithelia during pregnancy and lactation. Together, these data demonstrate that there is no luminal cell-autonomous role for Id1 in mammary epithelial cell fate determination, ductal morphogenesis and terminal differentiation.

Collagen scaffold: a treatment for simulated maternal birth injury in the rat model

We sought to determine the impact of a collagen scaffold on the healing response after simulated birth injury in a rodent model. A total of 52 virgin animals were divided into the following groups: control (n = 18), injured untreated (n = 18), and injured treated with porcine small intestinal submucosa (SIS) (n = 16). Histopathology, immunofluorescence of collagens, and vaginal mechanical properties were used to assess the impact of injury and the subsequent healing response. Collagen I/V decreased by 44% after birth injury relative to the controls (P = .001). Birth injury resulted in inferior mechanical properties of the vagina with a decrease of 38% in the tangent modulus and 44% in the tensile strength. SIS improved the collagen I/V and I/III ratios by 28% and 46%, respectively, paralleling the trend in the mechanical properties. Simulated birth injury negatively affected vaginal biochemical and biomechanical properties long term. SIS treatment mitigated the impact of birth injury by enhancing tissue quality.

It's not my nerves! Augmented pressor response to stress in multiparous rats is not associated with increased sympathetic outflow.

There is epidemiological evidence that multiparity (multiple pregnancies) increases the long‐term risk for cardiovascular disease. We have shown that multiparous rats have an augmented pressor response to stress. This is due, at least in part, to an oxidative stress induced increase in vascular reactivity. We proposed that the augmented pressor response in the multiparous rats was also the result of a greater increase in sympathetic outflow. Age‐matched multiparous and virgin rats were implanted with telemetric devices to monitor renal sympathetic nerve activity (Telemetry Research) and blood pressure (DSI). One week later, they were exposed to 10 sec air jet. The stress‐induced increase in integrated mean amplitude of nerve activity was significantly higher in the virgin animals (Virgin: 3.2±1.6 uV to 36.5±12.2 uV; Multiparous: 0.8±0.6 uV to 8.9±6.2 uV; P&lt;0.05). There was no difference between the groups with respect to the increase in spike frequency (Virgin: 28.2±7.8 to 36.6±10.0 spikes/sec; Multiparous: 16.6±2.8 to 28.6±1.4 spikes/sec; P&gt;0.05). We conclude that, contrary to our hypothesis, the augmented pressor response of multiparous rats is due solely to increased vascular reactivity, not to a greater increase in sympathetic outflow. (HSFC)

Use of a prolactin-Cre/ROSA-YFP transgenic mouse provides no evidence for lactotroph transdifferentiation after weaning, or increase in lactotroph/somatotroph proportion in lactation.

In rats, a shift from somatotroph dominance to lactotroph dominance during pregnancy and lactation is well reported. Somatotroph to lactotroph transdifferentiation and increased lactotroph mitotic activity are believed to account for this and associated pituitary hypertrophy. A combination of cell death and transdifferentiation away from the lactotroph phenotype has been reported to restore non-pregnant pituitary proportions after weaning. To attempt to confirm that a similar process occurs in mice, we generated and used a transgenic reporter mouse model (prolactin (PRL)-Cre/ROSA26-expression of yellow fluorescent protein (EYFP)) in which PRL promoter activity at any time resulted in permanent, stable, and highly specific EYFP. Triple immunochemistry for GH, PRL, and EYFP was used to quantify EYFP+ve, PRL−ve, and GH+ve cell populations during pregnancy and lactation, and for up to 3 weeks after weaning, and concurrent changes in cell size were estimated. At all stages, the EYFP reporter was expressed in 80% of the lactotrophs, but in fewer than 1% of other pituitary cell types, indicating that transdifferentiation from those lactotrophs where reporter expression was activated is extremely rare. Contrary to expectations, no increase in the lactotroph/somatotroph ratio was seen during pregnancy and lactation, whether assessed by immunochemistry for the reporter or PRL: findings confirmed by PRL immunochemistry in non-transgenic mice. Mammosomatotrophs were rarely encountered at the age group studied. Individual EYFP+ve cell volumes increased significantly by mid-lactation compared with virgin animals. This, in combination with a modest and non-cell type-specific estrogen-induced increase in mitotic activity, could account for pregnancy-induced changes in overall pituitary size.

Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells

IntroductionIn humans, an early full-term pregnancy reduces lifetime breast cancer risk by up to 50% whereas a later pregnancy (>35 years old) can increase lifetime risk. Several mechanisms have been suggested, including changes in levels of circulating hormones, changes in the way the breast responds to these hormones, changes in gene expression programmes which may alter susceptibility to transformation and changes to mammary stem cell numbers or behaviour. Previous studies have shown that the mammary tissue isolated from both virgin and parous mice has the ability to repopulate a cleared mammary fat pad in transplant experiments. Limited dilution transplant assays have demonstrated that early pregnancy (at 5 weeks of age) reduces stem/progenitor cell numbers in the mouse mammary epithelium by twofold. However, the effects on stem/progenitor cell numbers in the mammary epithelium of a pregnancy in older animals have not yet been tested.MethodsMice were put through a full-term pregnancy at 9 weeks of age, when the mammary epithelium is mature. The total mammary epithelium was purified from parous 7-week post-lactation and age-matched virgin mice and analysed by flow cytometry and limiting dilution cleared fat pad transplants.ResultsThere were no significant differences in the proportions of different mammary epithelial cell populations or numbers of CD24+/Low Sca-1- CD49fHigh cells (stem cell enriched basal mammary epithelial compartment). There was no significant difference in stem/progenitor cell frequency based on limiting dilution transplants between the parous and age-matched virgin epithelium.ConclusionsAlthough differences between parous and virgin mammary epithelium at later time points post lactation or following multiple pregnancies cannot be ruled out, there are no differences in stem/progenitor cell numbers between mammary epithelium isolated from parous animals which were mated at 9 weeks old and virgin animals. However, a recent report has suggested that animals that were mated at 5 weeks old have a twofold reduction in stem/progenitor cell numbers. This is of interest given the association between early, but not late, pregnancy and breast cancer risk reduction in humans. However, a mechanistic connection between stem cell numbers and breast cancer risk remains to be established.

Administration of D-alanine-[Ang-(1-7)] (A-779) prior to pregnancy in Sprague Dawley rats produces antidiuresis in late gestation

We previously demonstrated that angiotensin-(1-7) [Ang-(1-7)], which is increased in the kidney and urine during pregnancy, influences normal fluid expansion of pregnancy. These previous studies were completed by chronic administration of the Ang-(1-7) receptor antagonist D-Alanine-[Ang-(1-7)] (A-779) at a dose of 48 μg/kg/hour after the start of pregnancy (gestational days 11 to 19). To further explore the role of Ang-(1-7) on kidney function during early, middle, and late pregnancy, Sprague Dawley rats were chronically pretreated 8 days prior to pregnancy and throughout pregnancy (gestational days 0 to 19) with vehicle or A-779 at a dose of 24 μg/kg/hour. Metabolic studies were completed in virgin animals and throughout pregnancy (gestational days 4 to 5, 14 to 15, and 18 to 19). Chow consumption and water intake increased throughout pregnancy while the difference between intake and output (balance) was increased only at late (day 19) pregnancy with both vehicle and A-779 administration. Urine volume and urinary osmolality were significantly increased and decreased, respectively, throughout pregnancy in vehicle-treated rats only. In late (19 days) pregnancy, A-779 administration significantly decreased chow consumption and water intake. In virgin animals, A-779 administration significantly increased urine volume, while during late pregnancy (19 days), urine volume was significantly decreased with A-779 administration. These studies using pretreatment with a lower dose of A-779 prior to pregnancy confirm results of higher dose A-779 administration after the start of pregnancy. These studies show that Ang-(1-7) produces antidiuresis in virgin rats and diuresis in late gestation. Ang-(1-7) also contributes to the enhanced water intake during pregnancy allowing maintenance of the normal volume expanded state despite diuresis.

A profound decrease in maternal arginine uptake provokes endothelial nitration in the pregnant rat

While a specific role for nitric oxide (NO) in inducing the hemodynamic alterations of pregnancy is somewhat controversial, it is widely accepted that excess NO is generated during pregnancy. L-Arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate L-arginine uptake, cationic amino acid transporter-1 (CAT-1) acts as the specific arginine transporter for endothelial NO synthase. The present study was designed to test the hypothesis that, during pregnancy, when arginine consumption by the fetus is significantly increased, compensatory changes in maternal arginine uptake affect the endothelium. Uptake of radiolabeled arginine (L-[3H]arginine) by freshly harvested maternal aortic rings from pregnant rats decreased by 65 and 30% in mid- and late pregnancy, respectively, compared with those obtained from virgin animals. This decrease was associated with a significant increase in endothelial protein nitration (the footprint of peroxynitrite generation), as shown by both Western blotting and immunohistochemistry utilizing anti-nitrotyrosine antibodies, reflecting endothelial damage. Northern blot analysis revealed that steady-state aortic CAT-1 mRNA levels did not change throughout pregnancy, whereas CAT-1 protein abundance was significantly increased, peaking at mid-pregnancy. Protein content of protein kinase C (PKC)-alpha, which was previously shown to decrease CAT-1 activity, increased significantly in the pregnant animals and was associated with a significant increase in CAT-1 phosphorylation. Intraperitoneal injection of alpha-tocopherol, a PKC-alpha inhibitor, prevented the decrease in arginine transport and attenuated protein nitration. In conclusion, aortic arginine uptake is reduced during pregnancy, through posttranslational modulation of CAT-1 protein, presumably via upregulation of PKC-alpha. The aforementioned findings are associated with an increase in protein nitration and, therefore, in selected individuals, may lead to the development of certain forms of endothelial dysfunction, like preeclampsia.

Estrogen and Progesterone are Critical Regulators of Stat5a Expression in the Mouse Mammary Gland

Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the prepubertal gland and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution, and then increased again after weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double-labeling experiments in animals treated with E plus P for 3 d demonstrated that Stat5a was localized exclusively to cells containing both E and P receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland and suggest that these hormones act at the cellular level through their cognate receptors.

Suppression of endotoxin-induced fever in near-term pregnant rats is mediated by brain nitric oxide

Over the last three decades, experiments in several mammalian species have shown that the febrile response to bacterial endotoxin is attenuated late in pregnancy. More recent evidence has established that the expression of nitric oxide synthase (NOS) enzymes is increased in the brain late in pregnancy. The current study investigated the possible role of brain nitric oxide in mediating the phenomenon of fever suppression. Core body temperature (Tb) of near-term pregnant rats (day 19 and 20) was measured following inhibition of brain NOS and intraperitoneal injection of LPS (50 microg/kg); they were compared with both day 15 pregnant and virgin animals. Intracerebroventricular injection with an inhibitor of NOS, NG-monomethyl-L-arginine citrate (L-NMMA; 280 microg), in near-term pregnant rats restored the febrile response to LPS. As expected, near-term dams that received intracerebroventricular vehicle + IP LPS did not increase Tb, in contrast to the 1.0 +/- 0.2 degrees C rise in Tb in dams treated with ICV L-NMMA + IP LPS (P < 0.01). In virgin females and day 15 pregnant controls receiving this treatment, the increases in Tb were 1.5 +/- 0.3 degrees C and 1.6 +/- 0.4 degrees C, respectively. Thus, blockade of brain NOS restored the febrile response to LPS in near-term dams; at 5 h postinjection, Tb was 60-70% of that observed in virgins and day 15 pregnant animals. Intracerebroventricular L-NMMA alone did not induce a significant change in Tb in any group. These results suggest that the mechanism underlying the suppression of the febrile response in near-term pregnancy is mediated by nitric oxide signaling in the brain.

Charles River Sprague Dawley Rats Lack Early Age-Dependent Susceptibility to DMBA-Induced Mammary Carcinogenesis

Developmental stages of mammary glands influence their susceptibility to initiating events related to carcinogenesis. The "window of susceptibility" to mammary carcinogenesis is classically defined as the time in early puberty when the mammary gland morphology is most sensitive to initiation events. Administration of the polyaromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene (DMBA), in a single oral dose yields maximal mammary tumor formation when administered in this "window". We examined the DMBA treated mammary glands, precursor lesions, and morphology of the uninvolved mammary epithelium for the first 100 days of life for Charles River Sprague Dawley CD(R) IGS. Our goal was to determine the DMBA dose at which 50% of the rats (IC50) developed carcinoma in situ (CIS) within three months of dosing. Here we demonstrate, rather than the classical U-shaped dose curve in which there is maximum sensitivity for DMBA at 50 days, there is an increasing degree of sensitivity with age in the CD(R) IGS rat. Additionally, we report that vehicle-treated animals developed mammary CIS without any known initiator, and 100 day virgin animals demonstrated lactational changes, independent of DMBA exposure or dose. Lastly, we demonstrate this strain of virgin female rats has elevated pituitary prolactin immunoreactivity independent of the level of mammary differentiation. We conclude this strain of Charles River Sprague Dawley rats has prolactin-induced pituitary stimulation, and therefore, the window of susceptibility for mammary tumorigenesis is absent.

Haploinsufficiency of the corepressor of estrogen receptor activity (REA) enhances estrogen receptor function in the mammary gland

Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphogenesis, function, and carcinogenesis. The repressor of ER activity (REA) is an ER-interactive protein that counterbalances estrogen-induced ER transcriptional activity. Our previous study showed that genetic deletion of both REA alleles resulted in embryonic lethality. This study demonstrates that REA and ERalpha are coexpressed in mammary epithelial cells. REA heterozygous (REA(+/-)) mutant mice exhibit faster mammary ductal elongation in virgin animals, increased lobuloalveolar development during pregnancy, and delayed mammary gland involution after weaning. These morphological phenotypes of REA(+/-) mice are associated with significantly increased cell proliferation and ER transcriptional activities, as indicated by the estrogen response element (ERE)-luciferase reporter in the WT/ERE-Luc and REA(+/-)/ERE-Luc bigenic mice and by the higher expression levels of estrogen-responsive genes such as progesterone receptor and cyclin D1 in the mammary gland. Our analysis also revealed that REA is an important repressor of ER transcriptional activity in the mammary gland under natural, as well as ovariectomized and estrogen-replaced, hormonal conditions. Our results indicate that REA is a physiological modulator of ER function in the mammary gland and that its correct gene dosage is required for maintenance of normal ER activity and normal mammary gland development. Consequently, a reduction or loss of REA function may cause overactivation of ER and increase breast cancer risk in humans.

Local over-expression of prolactin in differentiating mouse mammary gland induces functional defects and benign lesions, but no carcinoma

Experimental, clinical, and epidemiological data support the growth-promoting role of endocrine prolactin (PRL) in mammary tumors. PRL is also produced by the breast, where it is now recognized to act as a growth/survival factor via autocrine/paracrine mechanisms. Recent transgenic (Tg) mouse models have revealed the pro-oncogenic effect of PRL over-expression in virgin mammary glands. To address the question whether PRL tumorigenicity was maintained on differentiated mammary glands, we generated mammary-specific Tg mice expressing human (h)PRL under the control of the milk whey acidic protein promoter, which directs autocrine hPRL over-expression in late gestation throughout lactation. Minimal levels of transgene expression were detected in the mammary glands of virgin animals, which at best induced partial ductal branching and lobulo-alveolar structures in older nulliparous females. As expected, expression of mammary hPRL dramatically increased at the end of first pregnancy, and from this point it never returned to baseline, although it peaked at each gestation/lactation cycle. Over-expression of hPRL that starts when the gland is already well into the differentiation process led to various morphological mammary alterations, including abnormally differentiated epithelium, atropy of the myoepithelial layer, dilated ducts, cysts, and lymphocytic infiltrates. These phenotypes tended to worsen with successive pregnancies, also reflecting cumulative damage of failure of involution. Although some older, multiparous females developed benign tumors (papillomas and metaplasias), none of the animals studied developed mammary carcinomas. In addition, we noticed that half of the Tg females exhibited lactation defects, leading to significantly increased pup mortality. This phenotype was due neither to failure of milk production nor to modification of its protein content, but rather it was correlated to lipid enrichment of the milk, which, in combination with profoundly altered morphology of the gland, led to impaired milk extrusion through the nipple. In summary, these data show that over-expression of autocrine hPRL in a differentiating mammary gland induces dramatic functional and morphological defects, but not carcinoma. This deserves further investigations on the emerging concept that autocrine PRL may have different effects on pathological development of the mammary gland depending on the differentiation state of the latter.

Tales of two snails: sexual selection and sexual conflict in Lymnaea stagnalis and Helix aspersa

Sexual selection and sexual conflict have been shown to play key roles in the evolution of species with separate sexes. Experimental evidence is accumulating that this is also true for simultaneous hermaphrodites. For example, many species of land snails forcefully stab their mating partners with love darts. In the brown garden snail (Helix aspersa, now called Cantareus asperses), this dart increases sperm storage and paternity, probably via the transfer of an allohormone that inhibits sperm digestion. A recent interspecies comparison of dart-possessing land snails revealed coevolution between darts and spermatophore-receiving organs that is consistent with counteradaptation against an allohormonal manipulation. The great pond snail (Lymnaea stagnalis) seems to use a seminal product to manipulate its partner and mates in the male role when enough seminal fluid is available in the prostate gland. Receipt of semen not only initiates egg laying in virgin animals, but also feminizes the mating partner later in life. These increases in the female function have been shown to go at the expense of growth and seminal fluid production of the sperm recipient. Although in Helix, and probably also Lymnaea, the sperm donor benefits from the induced changes through increased fertilization success, the sperm recipient may experience injury, imposed reallocation of resources, and altered sperm storage. These findings support the existence of sexual conflict in simultaneously hermaphroditic snails, and its importance for the evolution of mating behaviors and reproductive morphologies is discussed.

Renal PDE‐5 and the blunted natriuretic response to ANP in the gravid rat

Normal pregnancy is characterized by a large plasma volume expansion accompanied by sodium retention, which has been shown to be as a result of a blunted response to natriuretic stimuli, such as pressure natriuresis and atrial natriuretic peptide (ANP). PDE-5 protein expression has been shown to be increased in renal medullary cells during pregnancy. PDE-5 degrades cGMP to a non-active form, therefore we propose that increased PDE-5 availability causes in a fall in renal cGMP levels and blunts the natriuretic effect of ANP. We aimed to investigate the effect of intrarenal infusion of a selective PDE-5 inhibitor, sildenafil at 0.2μg/min, on the response to ANP in pregnancy. We performed acute experiments in anesthetized 16-day pregnant and virgin rats. ANP infusion caused a fall in BP in all groups and sildenafil induced a further reduction. Sodium excretion for the kidney receiving the intrarenal infusion is shown in Table 1. The sildenafil treatment resulted in restoring the increase in sodium excretion in the pregnant animals to levels equal to virgin animals. This data indicates that PDE-5 expression in the gravid rat kidney plays a role in the blunted natriuretic response to ANP during pregnancy and selective PDE-5 inhibition restores the natriuretic effect of ANP in the pregnant rat. Financial support for this work was provided by NIH, NICHD grant # R01 HD041571.

A quantitative study of rat uterine sympathetic innervation during pregnancy and post partum

In mammals, pregnancy induces a transient and extensive degeneration of uterine sympathetic innervation. We used the models of unilateral oviduct ligation and in oculo myometrium transplant in pregnant rats to address the role of stretching forces and/or hormone milieu in the loss of sympathetic innervation. The sympathetic fibres of the uterine horn and in oculo myometrial transplants were quantified on tissue sections processed by the glyoxylic acid technique. In normal pregnant rats, the density of uterine horn innervation was significantly reduced at late pregnancy and recovery took place during post partum. The empty horn of pregnant rats showed no significant changes in density of myometrial innervation during pregnancy or post partum. In oculo myometrial transplants were organotypically reinnervated in virgin animals. When the transplants were exposed to gestational hormonal milieu, few or no fibres were observed to the end of pregnancy; however, a significant increase at post partum was observed. Results showed that both the effects of stretching and the hormone milieu derived from the fetus-placenta complex play a role as inductors of changes on sympathetic myometrial innervation during pregnancy and support the idea that immature muscular uterine fibres are more susceptible to the effects of pregnancy than those originating from adult animals.