Placental expression of adenosine A(2A) receptor and hypoxia inducible factor-1 alpha in early pregnancy, term and pre-eclamptic pregnancies: interactions with placental renin-angiotensin system.

Hypoxia-inducible factors (HIFs), adenosine and tissue renin-angiotensin-system (RAS) promote angiogenesis and vascularisation. We investigated the temporal expression placental adenosine A2AR receptor and HIF-1α in early pregnancy and at delivery in normotensive (NT) and pre-eclamptic (PE) women. Results were compared to our previously reported angiotensin receptor data. Expression of A2AR and HIF-1α was highest at ≤10 weeks, positively correlated through pregnancy and was higher in PE than NT at delivery. The A2AR associated with the AT4R only in early pregnancy. We suggest adenosine and RAS may interact to promote placentation with a potential adaptation to poor placental perfusion in PE.

Placental expression of eNOS, iNOS and the major protein components of caveolae in women with pre-eclampsia.

Caveolae regulate many cardiovascular functions and thus could be of interest in relation to pre-eclampsia, a pregnancy specific disorder characterised by hypertension and proteinuria. We examined placental mRNA and protein expression/localisation of the caveolae components Caveolin 1-3, Cavin 1-4 as well as eNOS/iNOS in normotensive control (n = 24) and pre-eclamptic pregnancies (n = 19). Placental mRNA expression of caveolin-1, cavin 1-3, was lower and eNOS expression was increased in pre-eclampsia (P < 0.05 for all). Additionally Caveolin-1 protein expression was also reduced in pre-eclampsia (P = 0.007); this could be an adaptive response in pre-eclampsia, possibly to attenuate the oxidative stress/inflammation.

Expression of voltage-dependent potassium channels in first trimester human placentae.

Potassium channel α-subunits encoded by KCNQ1-5 genes form voltage-dependent channels (KV7), modulated by KCNE1-5 encoded accessory proteins. The aim was to determine KCNQ and KCNE mRNA expression and assess protein expression/localisation of the KCNQ3 and KCNE5 isoforms in first trimester placental tissue. Placentae were obtained from women undergoing elective surgical termination of pregnancy (TOP) at ≤ 10 weeks' (early TOP) and >10 weeks' (mid TOP) gestations. KCNQ1-5 expression was unchanged during the first trimester. KCNE5 expression increased in mid TOP vs. early TOP samples (P = 0.022). This novel study reports mRNA and protein expression of KV7 channels in first trimester placentae.

Thyroid hormones and their placental deiodination in normal and pre-eclamptic pregnancy.

Pre-eclampsia is associated with lower serum selenium concentrations and glutathione peroxidase expression/activity; total thyroid hormones are also lower. OBJECTIVES, STUDY DESIGN AND MAIN OUTCOME MEASURES: We hypothesised that the placental selenoprotein deiodinase (D3) will be protected in pre-eclampsia due to the hierarchy of selenoprotein biosynthesis in selenium deficiency. Venous blood and tissue from three standardised placental sites were obtained at delivery from 27 normotensive and 23 pre-eclamptic women. mRNA expression and enzyme activity were assessed for both deiodinases (D2 and D3); protein expression/localisation was also measured for D3. FT4, FT3 and TSH concentrations were measured in maternal and umbilical cord blood. RESULTS: No significant differences in D3 mRNA or protein expression between normotensive and pre-eclamptic pregnancies. There was a significant effect of sampling site on placental D3 activity only in pre-eclamptic women (P = 0.034; highest activity nearest the cord). A strong correlation between D3 mRNA expression and enzyme activity existed only in the pre-eclamptic group; further strengthened when controlling for maternal selenium (P < 0.002). No significant differences were observed between groups for any of the maternal thyroid hormones; umbilical TSH concentrations were significantly higher in the pre-eclamptic samples (P < 0.001). CONCLUSIONS: D3 mRNA and protein expression appear to be independent of selenium status. Nevertheless, the positive correlation between D3 mRNA expression and activity evident only in pre-eclampsia, suggests that in normotensive controls, where selenium is higher, translation is not affected, but in pre-eclampsia, where selenium is low, enzyme regulation may be altered. The raised umbilical TSH concentrations in pre-eclampsia may be an adaptive fetal response to maximise iodide uptake.

The placental renin-angiotensin system and oxidative stress in pre-eclampsia.

There is an inverse correlation between human birthweight and umbilical venous angiotensin II (AngII) concentrations. Oxidative stress and increased pro-renin receptor (PRR) both enhance the cleavage of angiotensin I from angiotensinogen (AGT). Pre-eclampsia, a hypertensive disorder of pregnancy, manifests as high blood pressure and proteinuria, and is a state of increased oxidative stress. HYPOTHESIS: Pre-eclampsia will be associated with increased placental expression of components of the renin-angiotensin system, which could result in reduced infant birthweight. Biopsies were taken 1 cm from the placental edge from 27 normotensive controls and 23 pre-eclamptic White European women. Immunohistochemistry was performed for AGT, PRR, glutathione peroxidase 3 (GPx3) and the AT1R and AT2R AngII receptors. Protein expression was semi-quantitatively assessed (H-score). RESULTS: AT1R expression was significantly increased in pre-eclamptic placentae, and negatively correlated with birthweight (r = -0.529, P = 0.009). AT1R expression was also negatively correlated with GPx3 expression overall (r = -0.647; P = 0.005). AT2R expression positively correlated with AGT (r = 0.615, P = 0.002) in the pre-eclamptic placentae only. CONCLUSIONS: The raised AT1R expression in pre-eclampsia, together with inadequate antioxidant protection, possibly through lower GPx activity, might enhance the vasoconstrictor effect of locally-generated AngII, contributing to the restricted fetal growth characteristic of pre-eclampsia. Conversely, the AT2R:AGT association within the pre-eclamptic placenta may provide a compensatory mechanism.

OS081. Novel KCNQ3/KCNE5 isoform protein and mRNA expression in first trimester human placentae.

INTRODUCTION: Potassium channel α-subunits encoded by KCNQ1-5 genes (Kv7) form voltage-dependent channels that can be modulated by KCNE1-5 encoded accessory proteins. These channels are known to play a role in the reactivity of blood vessels. We have previously shown that both mRNA and protein expression for the novel combination of KCNQ3 and KCNE5 are increased in term and preterm pre-eclampsia (PE) compared to normotensive control placentae [1]. The expression of these isoforms in early placental tissue has not been examined. OBJECTIVES: The aims of this study were to determine whether KCNQ3 and KCNE5 mRNA and proteins are expressed in first trimester placental tissue. METHODS: Placental samples were obtained from women undergoing elective surgical termination of pregnancy between 6 and 12 weeks' gestation (n=7) following informed written consent. KCNQ3 and KCNE5 mRNA expression was measured by qRT-PCR and normalised to stably expressed GAPDH. Immunohistochemistry was used to assess protein expression and localisation of the isoforms. RESULTS: Both mRNA and protein expression of KCNQ3 and KCNE5 were detected in placental tissue at all gestations. KCNE5 mRNA expression remained constant between 6 and 10 weeks with a subsequent rise at 11 and 12 weeks. KCNQ3 mRNA expression was initially lower than KCNE5 but markedly increased at 7 weeks remaining high until 10 weeks and falling below KCNE5 levels by 12 weeks. Protein expression for both KCNQ3 and KCNE5 was localised mainly to the syncytiotrophoblast but was also evident in the mesenchyme; overall KCNQ3 intensity significantly increased with gestational age (p=0.044). CONCLUSION: KCNQ3 and KCNE5 channel isoforms are highly expressed in first trimester placenta. The temporal changes in mRNA expression mirror changes in the placental tissue oxygen tension which increases between 8 and 10 weeks. This would precede the dislocation of the spiral artery plugs enabling maternal blood to flow freely and continuously into the intervillous spaces. We speculate that the increase in mesenchymal protein expression may be related to angiogenesis during this critical window of feto-placental vascular development. Future work will characterise the complete KCNQ/KCNE isoforms in first trimester placental tissue and assess potential functional roles of these channels both in early placentation and in relation to PE. FUNDING: Tommy's Charity (Registered charity 1060508).

OS102. Continuing pathology following a hypertensive pregnancy and the risk of future disease.

INTRODUCTION: New onset hypertension in pregnancy affects up to 6-8% of all pregnancies. For most women, hypertension and proteinuria settle following delivery. The National Institute for Health and Clinical Excellence (NICE) Hypertension in Pregnancy guideline recommends that this group of patients are reviewed by a medical professional postnatally [3]. However, studies have shown that blood pressure and urinalysis are often not checked in the postpartum period [4]. Women with a history of hypertension in pregnancy have a higher risk of future hypertension and cardiovascular disease (CVD) than women who have uncomplicated pregnancies [2]. Risk scores are available for assessing an individual's risk of CVD although they are not validated in women under 30. In UK, the most appropriate is QRISK2 score [1]. OBJECTIVES: To determine the frequency of ongoing problems following a new onset hypertensive pregnancy and assess the risk of future cardiovascular disease. METHODS: 351 women with new onset hypertension in pregnancy were reviewed 6 weeks postnatally. They were assessed for ongoing disease and cardiovascular risk. 10 year QRISK2 scores and heart age (the age at which a matched person has that score) were calculated. RESULTS: 211 women with pre-eclampsia (PE) and 140 with gestational hypertension (GH) were reviewed. 9% and 11% of women with previous PE and GH respectively still required antihypertensive agents at follow-up. Only 1 woman required more than one antihypertensive medication (PE group). 19 women with PE (9%) had ongoing proteinuria (PCR>30). 5% had an estimated GFR <60ml/min. In addition to those with a strong family history of hypertension, 23 patients (6.5%) required investigation for ongoing problems. Risk factors for CVD were common 6 weeks after delivery: Although the overall risk of CVD was low (median 10 year QRISK2 score 0.3, median relative risk 1.0), with 41% of women having the lowest possible heart age, 22% of women had a significantly elevated risk of CVD (QRISK2 heart age ⩾age+10). CONCLUSION: 16% of women had ongoing hypertension or proteinuria, evidence supporting the NICE guidance that all women with hypertension in pregnancy need follow-up after delivery. The overall risk of future CVD in women with previous hypertension in pregnancy is low but about one-fifth of women are at very high risk. A program of risk assessment is required to allow preventative measures to be implemented.

PIERS proteinuria: relationship with adverse maternal and perinatal outcome.

OBJECTIVE: To examine the ability of three different proteinuria assessment methods (urinary dipstick, spot urine protein:creatinine ratio [Pr/Cr], and 24-hour urine collection) to predict adverse pregnancy outcomes. METHODS: We performed a prospective multicentre cohort study, PIERS (Preeclampsia Integrated Estimate of RiSk), in seven academic tertiary maternity centres practising expectant management of preeclampsia remote from term in Canada, New Zealand, and Australia. Eligible women were those admitted with preeclampsia who had at least one antenatal proteinuria assessment by urinary dipstick, spot urine Pr/Cr ratio, and/or 24-hour urine collection. Proteinuria assessment was done either visually at the bedside (by dipstick) or by hospital clinical laboratories for spot urine Pr/Cr and 24-hour urine collection. We calculated receiver operating characteristic area under the curve (95% CI) for each proteinuria method and each of the combined adverse maternal outcomes (within 48 hours) or adverse perinatal outcomes (at any time). Models with AUC ≥ 0.70 were considered of interest. Analyses were run for all women who had each type of proteinuria assessment and for a cohort of women ("ALL measures") who had all three proteinuria assessments. RESULTS: More women were proteinuric by urinary dipstick (≥ 2+, 61.4%) than by spot urine Pr/Cr (≥ 30 g/mol, 50.4%) or 24-hour urine collection (≥ 0.3g/d, 34.7%). Each proteinuria measure evaluated had some discriminative power, and dipstick proteinuria (categorical) performed as well as other methods. No single method was predictive of adverse perinatal outcome. CONCLUSION: The measured amount of proteinuria should not be used in isolation for decision-making in women with preeclampsia. Dipstick proteinuria performs as well as other methods of assessing proteinuria for prediction of adverse events.

Regional variation in angiotensin converting enzyme activity in the human placenta.

Sensitivity of chorionic plate arteries to angiotensin II (AngII) is greatest at the placental periphery. Angiotensin converting enzyme (ACE) is central to the synthesis of AngII and is present in the placental vasculature. We measured vascular ACE activity/mg protein at 8-9 sites between the cord insertion and the periphery in 12 term placentae from normotensive, vaginally-delivered women. ACE increased from insertion to the periphery (P = 0.015); median ACE for each placenta was positively correlated with placental weight (P < 0.05) and placental:birthweight ratio (P < 0.02). We speculate that this may be related to fetal programming since placental:birthweight ratio is related to long-term health.

Expression of AT1R, AT2R and AT4R and their roles in extravillous trophoblast invasion in the human.

The placental renin-angiotensin system (RAS) is active from early pregnancy and may have a role in placentation. Angiotensin II (AngII) acts via binding to receptor types AT1R and AT2R. Recently smaller peptide members of the angiotensin family have been recognised as having biological relevance. Angiotensin (3-8) (AngIV) has a specific receptor (AT4R) and evokes hypertrophy, vasodilatation and vascular inflammatory response. The aim of this study was to characterise placental expression of AT1R, AT2R and AT4R, and to determine whether AngII and AngIV regulate extravillous trophoblast (EVT) invasion, apoptosis and proliferation. Placental samples were obtained from women undergoing elective surgical termination of pregnancy (TOP) at 8-10 weeks gestation (early TOP), 12-14 weeks gestation (mid TOP) or at delivery following normal pregnancy or with pre-eclampsia (PE). Immunohistochemistry and qRT-PCR were performed to determine placental mRNA and protein expression of AT1R, AT2R and AT4R at all gestational ages. EVT invasion following culture with AngII or AngIV was assessed in early placental tissue using Matrigel invasion assays. Invasion was assessed on day 6 of culture and placental explants were harvested for immunohistochemical analysis of apoptosis and proliferation. The results from qRT-PCR and immunohistochemistry showed placental AT1R expression which did not vary with gestation. The highest levels of expression of AT2R were found in early and mid TOP placentae compared to term pregnancy. Expression of AT4R was increased in term placentae, with a significant reduction in PE placentae. Moreover, culture with AngIV or AngII increased EVT invasion from placental explants, which showed increased trophoblast proliferation and reduced apoptosis. This study has characterised expression of AT4R and AT1R and AT2R in human placenta throughout normal pregnancy and in PE. Both AngIV and AngII may play an important role in normal pregnancy.

Differential expression and distribution of placental glutathione peroxidases 1, 3 and 4 in normal and preeclamptic pregnancy.

Preeclampsia is a pregnancy-specific condition affecting 2-7% of women and a leading cause of perinatal and maternal morbidity and mortality; it may also predispose the mother and fetus to increased risks of adult cardiovascular disease. The selenoprotein glutathione peroxidases (GPxs) have critical roles in regulating antioxidant status. OBJECTIVES, STUDY DESIGN AND MAIN OUTCOME MEASURES: Immunohistochemical measurements of GPx1, GPx3 and GPx4 protein expression were performed on samples taken from three standardised sampling sites between the cord insertion and the periphery of the placenta from 12 normotensive, and 12 preeclamptic women to establish if their expression differed between sampling sites. Total GPx activities were also examined from the three sampling sites of these placentae. RESULTS: There were highly significant reductions in overall immunohistochemical staining of all 3 GPxs in the preeclampsia compared to normotensive placentae (GPx1: P=0.016; GPx3: P=0.003; GPx4: P<0.001). Furthermore, graded differences in expression between the standardised placental sampling sites were also found for GPx3 (higher in the inner region, P=0.05) and GPx4 (higher in the periphery, P=0.02) but not GPx1. Placental GPx enzyme activity was also significantly reduced in tissue from preeclamptic women as compared to normotensive women (P=0.007; the difference was more pronounced nearest the cord insertion). CONCLUSIONS: We have shown highly significant reductions in expression of all three major classes of GPx in placentae from women with preeclampsia, and distribution gradients in activity, which may relate to the differential oxygenation of regions of the placenta.

The ontogeny of components of the renin-angiotensin system in the porcine fetal ovary.

There is an autonomous renin-angiotensin system (RAS) in the adult ovary. Renin is present in the primitive kidney, and the fetal ovary develops from the nephrogenic ridge. We hypothesised that components of the ovarian RAS would be present from early gestation, with potential roles in ovarian development. We studied fetal pig ovaries from approximately day 45 (approximately 0.39 gestation) to term and measured mRNA (RT-PCR) for prorenin, angiotensinogen and the angiotensin II (AngII) Type 1 and 2 receptors (AT(1) and AT(2)), and protein expression (Western blot) and localization (immunohistochemistry) of the AT(1) and AT(2) receptors. mRNA for prorenin was present in relatively low abundance from at least day 45 and rose to approximately day 75 of gestation, whilst mRNA for angiotensinogen rose steadily. mRNA for the AT(1) receptor was present from approximately day 45 and did not alter significantly with increasing gestation but AT(2) receptor mRNA was initially high, falling sharply through pregnancy. The AT(1) receptor protein abundance fell steadily to term, whereas the AT(2) receptor protein did not change during gestation. Both receptors were localised in the surface epithelium and egg nests, the granulosa cells of primordial, primary and secondary follicles, and the oocytes of all except the secondary follicles. Collectively, our results support the hypothesis that there is a functional RAS in the fetal ovary from at least approximately day 45 of gestation until term and that it may have a paracrine role in ovarian growth and development.

Smoking in moderate/severe preeclampsia worsens pregnancy outcome, but smoking cessation limits the damage.

We studied phenotypic and clinical outcome data in an observational, multicenter cohort study of 1001 Western European white women and their singleton babies, with stringently defined moderate-to-severe preeclampsia. Ninety women admitted to being current smokers; 71 had stopped smoking before entry to the study. Across the categories of never-smoker, stopped, and current smoker there were significant increases in the proportion of women delivering before 34 weeks' gestation (P=0.011), delivering a baby below the third birth weight centile (P<0.001), or delivering a baby with any adverse outcome (P=0.011). By comparison with never-smokers, smoking during pregnancy was associated with a doubling of risk of being delivered before 34 weeks' (odds ratio: 1.98; 95% CI: 1.24 to 3.16; P=0.004), of delivering babies below the third centile of corrected birth weight (odds ratio: 2.20; 95% CI 1.41 to 3.44; P<0.0001), or for their babies to have any adverse outcome (odds ratio: 1.87; 95% CI: 1.19 to 2.95; P<0.006). Worryingly, the risk of developing eclampsia was increased 5-fold (odds ratio: 4.88; 95% CI: 1.44 to 16.61; P=0.005). The proportion of smokers in these preeclamptic women was lower than in our pregnant population generally. However, preeclampsia still carries significant perinatal morbidity, and cigarette smoking in preeclamptic pregnancies exacerbates this. Stopping smoking decreases the risks. Smoking in young women should be a particular target for advice by general practitioners before pregnancy, with active encouragement after conception to enroll in such trials as the current Smoking, Nicotine and Pregnancy Trial to support cessation.

Polymorphism in the epidermal growth factor gene is associated with birthweight in Sinhalese and white Western Europeans.

Birthweight predicts health later in life and is influenced by inherited factors. We investigated the association of the c.61G > A, and c.2566G > A polymorphisms in the epidermal growth factor (EGF) gene [GenBank NM_001963] with birthweight in three groups of healthy pregnant women, and in women with pregnancies affected by fetal growth restriction (FGR). Subjects comprised 171 Sinhalese women with normal pregnancies (Group A), 64 white Western European women with normal pregnancies (Group B), 101 white Western European women with normal pregnancies and their babies (Group C) and 107 women with pregnancies affected by FGR, their partners and their babies (Group D). Maternal EGF genotypes were associated with birthweight of healthy babies of women in Groups A (P = 0.03), B (P = 0.001) and C (P = 0.01). The association persisted following adjustment for confounding by gestational age, sex, maternal weight, parity and smoking habit. The trend from heaviest to lightest birthweights in all these groups was c.61AA > c.61GA > c.61GG and c.2566GG > c.2566GA > c.2566AA. The EGF haplotype associated with lower birthweight (c.61G, c.2566A) was transmitted at increased frequency from heterozygous parents to babies affected by FGR in Group D (P = 0.02). These findings support the hypothesis that growth factors expressed by the feto-maternal unit affect birthweight, and implicates polymorphism in the EGF gene in the aetiology of birthweight variability.

A prospective, longitudinal study of the renin-angiotensin system, prostacyclin and thromboxane in the first trimester of normal human pregnancy: association with birthweight.

BACKGROUND: Very early human pregnancy is a state of cardiovascular underfilling. The renin-angiotensin system (RAS) is directly concerned with sodium and water homeostasis. Angiotensinogen is known to be the rate-limiting component in the generation of angiotensin I, and hence angiotensin II, in pregnancy. The usual measurement of 'renin activity' does not differentiate between enzyme and substrate. We hypothesized that the RAS is activated from the start of pregnancy; plasma renin concentration (PRC) and angiotensinogen will show differential regulation and might stimulate the rise in prostacyclin. METHODS: A prospective study of 12 nulliparous normal women. PRC and angiotensinogen and excretion of prostacyclin and thromboxane metabolites were measured pre-pregnancy and four to six times after conception to 13 weeks. RESULTS: By 6 weeks gestation, mean PRC was markedly raised and remained stable to 13 weeks. The initial angiotensinogen response varied, but rose consistently after 6-8 weeks. Regression analysis showed angiotensinogen in the first trimester to be strongly associated with corrected birthweight centile (P < 0.001). Excretion of eicosanoid metabolites was very variable, but rose significantly from 6 weeks; the ratio between prostacyclin and thromboxane excretion did not alter over this time. There was no correlation between the various hormones measured. CONCLUSION: Angiotensinogen is known to be rate-limiting in pregnancy. Its association with birthweight may be through effects on early plasma volume expansion and may have implications for intrauterine growth restriction and pre-eclampsia.

Maternal endocrine adaptation throughout pregnancy to nutritional manipulation: consequences for maternal plasma leptin and cortisol and the programming of fetal adipose tissue development.

Maternal nutrient restriction at specific stages of gestation has differential effects on fetal development such that the offspring are programmed to be at increased risk of adult disease. We investigated the effect of gestational age and maternal nutrition on the maternal plasma concentration of leptin and cortisol together with effects on fetal adipose tissue deposition plus leptin, IGF-I, IGF-II ligand, and receptor mRNA abundance near to term. Singleton bearing ewes were either nutrient restricted (NR; consuming 3.2-3.8 MJ/d of metabolizable energy) or fed to appetite (consuming 8.7-9.9 MJ/d) over the period of maximal placental growth, i.e. between 28 and 80 d gestation. After 80 d gestation, ewes were either fed to calculated requirements, consuming 6.7-7.5 MJ/d, or were fed to appetite and consumed 8.0-10.9 MJ/d. Pregnancy resulted in a rise in plasma leptin concentration by 28 d gestation, which continued up to 80 d gestation when fed to appetite but not with nutrient restriction. Plasma cortisol was also lower in NR ewes up to 80 d gestation, a difference no longer apparent when food intake was increased. At term, irrespective of maternal nutrition in late gestation, fetuses sampled from ewes NR in early gestation possessed more adipose tissue, whereas when ewes were fed to appetite throughout gestation, fetal adipose tissue deposition and leptin mRNA abundance were both reduced. These changes may result in the offspring of NR mothers being at increased risk of obesity in later life.

Immunolocalization and protein expression of the alpha subunit of the large-conductance calcium-activated potassium channel in human myometrium.

Large-conductance calcium-activated potassium (BKCa) channels play an important role in the control of myometrial excitability. The aim of the present study was to determine the localization and protein expression of the alpha subunit of BKCa channels in the pregnant and parturient human uterus. An anti-alpha BKCa channel monoclonal antibody (anti-alpha(995-1113)) was used to localize and quantitate immunoreactive BKCa channel protein in myometrium of singleton term pregnant women undergoing either elective (n=26) or emergency Caesarean section following the onset of spontaneous labour (n=25). Data are presented as medians (interquartile range). Differences between groups were analysed using the Mann-Whitney U test. Immunohistochemistry studies localized the alpha subunit of the BKCa channel to the plasma membrane and the cytosol of myometrial cells with similar reaction end product in pregnant women who were or were not undergoing labour. Expression of this subunit, observed as a 125 kDa band in western blots, was significantly higher in pregnant women who were not undergoing labour (30.6% (20.3, 43.9)) than in those who were undergoing labour (15.7% (11.3, 22.4); P<0.01). Reduced BKCa alpha subunit expression in pregnant women during labour may underlie the initiation of uterine contractility during parturition.

In vitro development of pig preantral follicles cultured in a serum-free medium and the effect of angiotensin II.

A novel culture system is reported in which pig preantral follicles (< 300 microm in diameter) with an intact thecal cell layer were isolated and cultured in a serum-free medium for up to 30 days. The medium supported follicle culture after isolation, while maintaining both somatic cell and oocyte viability. Follicles were cultured in groups (n = 3 per group) on collagen-coated wells for 16 days, during which they retained a three-dimensional structure, maintained oocyte viability and increased in diameter and number of somatic cells. Follicle culture for 30 days resulted in a further increase in number of cells, oocyte viability was maintained, and a significant increase in follicle diameter was observed (P < 0.001), with 29% of follicles forming an antrum. Follicles synthesized measurable quantities of progesterone (168 pg per 100 microl per 48 h; no significant increase with time) and increasing quantities of oestradiol (136 pg per 100 microl per 48 h; P < 0.001 with time). Further supplementation of the medium with 100 micromol testosterone l(-1) at day 28 resulted in a significant increase in oestradiol secretion by both antral (P < 0.01) and preantral follicles (P < 0.05). Culture over 30 days in medium with 10(-10) mol angiotensin II l(-1) and further supplementation at day 28 with 100 micromol testosterone l-1 also increased oestradiol synthesis (P < 0.001). These results show that viable preantral follicles may be cultured for extended periods, and indicate that the possible role of angiotensin II in folliculogenesis and steroidogenesis in early development of pig follicles requires further investigation.

The effects of pregnancy and maternal nutrition on the maternal renin-angiotensin system in sheep.

Physiological changes occurring in the mother during pregnancy can determine the outcome of pregnancy in terms of birthweight and neonatal viability. Maternal adaptations include plasma volume expansion linked to enhanced activity of the renin-angiotensin system (RAS). The present study was designed to determine whether these changes occur very early in gestation, and the extent to which maternal nutrient restriction may compromise the maternal RAS. Using sheep, we have investigated the effects of pregnancy per se, maternal nutrient restriction and later restoration of maternal diet on maternal body weight, plasma volume and plasma renin concentration (PRC), and angiotensinogen (Aogen) and arginine vasopressin (AVP) concentration. During the period of placental growth (i.e. 28-80 days gestation) ewes were fed either a nutrient-restricted (NR) diet or were well fed (WF). NR ewes consumed between 3.2 and 3.8 MJ day(-1) of metabolisable energy (ME) which is close to 60 % of requirements taking into account the ME required for both ewe maintenance and growth of the conceptus in order to produce a 4.5 kg lamb at term. WF ewes consumed 150 % of ME requirements. Restoration of maternal diet between 80 and 140 days gestation (i.e. fed to satiety and consuming between 8 and 10.9 MJ day(-1), which is close to 150 % of ME requirements) followed previous nutrient restriction. Between pre-conception and 28 days gestation, plasma volume increased in conjunction with a decline in PRC and Aogen concentration. During the period of nutrient restriction ewe body weight did not increase and plasma volume was lower in NR than WF ewes. During this time there was no effect of maternal nutrition on PRC; however, Aogen concentration was lower in the NR group. From 80 days gestation following the rise in food intake for previously NR ewes, greater increases in ewe body weight, plasma volume and PRC occurred up to term compared with ewes that were well fed throughout gestation. Plasma AVP concentration was not significantly affected by either maternal nutrition or gestational age. In conclusion, the stimulus of moderately severe maternal nutrient restriction evoked smaller rises in maternal weight, plasma volume and Aogen concentration than occurred in ewes that were well fed throughout gestation. Following the restoration of maternal diet after 80 days gestation, PRC gradually rose to peak at term. These adaptations in the maternal RAS during the critical period of placental growth may have long-term effects on fetal development.