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JM Wallace, DA Bourke, P Da Silva and RP Aitken

Overnourishing adolescent ewes throughout pregnancy promotes maternal tissue synthesis at the expense of placental growth, which in turn leads to a major decrease in lamb birth weight. As maternal dietary intakes are inversely related to peripheral progesterone concentrations in these adolescent dams, it was hypothesized that sup-optimal progesterone concentrations in overnourished dams may compromise the growth of the differentiating conceptus resulting in fewer uterine caruncles being occupied and, hence, fewer placentomes formed. This hypothesis was tested by supplementing overnourished adolescent dams with exogenous progesterone during early pregnancy and determining the impact on pregnancy outcome at term. Embryos recovered from superovulated adult ewes inseminated by a single sire were transferred in singleton to the uterus of peripubertal adolescent recipients. After transfer of embryos, ewes were offered a moderate or high amount of a complete diet (n=11 per group). A further high intake group received a progesterone supplement each day from day 5 to day 55 of gestation (term=145 days) to restore circulating progesterone concentrations to moderate values throughout the first third of pregnancy (n=11). For ewes establishing pregnancies (n=7 per group), live weight gain during the first 100 days of gestation was 66+/-4, 323+/-17 and 300+/-7 g per day, body condition score at term was 2.1+/-0.05, 3.0+/-0.08 and 3.1+/-0.07 units and the duration of gestation after spontaneous delivery was 148+/-1.7, 144+/-0.8 and 143+/-0.8 days for the moderate intake, high intake and high intake plus progesterone groups, respectively. At delivery, fetal cotyledon mass (136+/-12.1 versus 57+/-8.2g, P<0.001) and lamb birth weight (5164+/-151 versus 2893+/-381 g, P<0.001) were higher in moderate intake than in high intake dams. Progesterone supplementation restored circulating concentrations to moderate values during the first third of gestation. Lamb birth weight in the high intake plus progesterone group (4150+/-389 g) was intermediate between the high intake (P<0.02) and moderate intake (P<0.05) groups, but this change in birth weight was not associated with corresponding changes in fetal cotyledon mass (76+/-10.3 g). Moreover, the number of fetal cotyledons was similar in all three groups. Thus, progesterone did not directly affect the growth of the fetal cotyledon but may have influenced placental vascularity, blood flow or nutrient transfer capacity or alternatively the development of the embryonic inner cell mass.

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J Wallace, D Bourke, P Da Silva and R Aitken

Human adolescent mothers have an increased risk of delivering low birth weight and premature infants with high mortality rates within the first year of life. Studies using a highly controlled adolescent sheep paradigm demonstrate that, in young growing females, the hierarchy of nutrient partitioning during pregnancy is altered to promote growth of the maternal body at the expense of the gradually evolving nutrient requirements of the gravid uterus and mammary gland. Thus, overnourishing adolescent dams throughout pregnancy results in a major restriction in placental mass, and leads to a significant decrease in birth weight relative to adolescent dams receiving a moderate nutrient intake. High maternal intakes are also associated with increased rates of spontaneous abortion in late gestation and, for ewes delivering live young, with a reduction in the duration of gestation and in the quality and quantity of colostrum accumulated prenatally. As the adolescent dams are of equivalent age at the time of conception, these studies indicate that nutritional status during pregnancy rather than biological immaturity predisposes the rapidly growing adolescents to adverse pregnancy outcome. Nutrient partitioning between the maternal body and gravid uterus is putatively orchestrated by a number of endocrine hormones and, in this review, the roles of both maternal and placental hormones in the regulation of placental and fetal growth in this intriguing adolescent paradigm are discussed. Impaired placental growth, particularly of the fetal component of the placenta, is the primary constraint to fetal growth during late gestation in the overnourished dams and nutritional switch-over studies indicate that high nutrient intakes during the second two-thirds of pregnancy are most detrimental to pregnancy outcome. In addition, it may be possible to alter the nutrient transport function of the growth-restricted placenta in that the imposition of a catabolic phase during the final third of pregnancy in previously rapidly growing dams results in a modest increase in lamb birth weight.

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P Da Silva, RP Aitken, SM Rhind, PA Racey and JM Wallace

The influence of maternal nutrition during pregnancy on anterior pituitary gonadotrophin gene expression and ovarian development in sheep fetuses during late gestation was investigated. Embryos recovered from superovulated adult ewes that had been inseminated by a single sire were transferred, singly, into the uteri of adolescent recipients. After embryo transfer, adolescent ewes were offered a high or moderate amount of a complete diet. Pregnancies were terminated at day 131 +/- 0.6 of gestation and the fetal brain, anterior pituitary gland and gonads were collected. Gonadotrophin gene expression (LHbeta and FSHbeta subunits) in the fetal pituitary gland was examined using in situ hybridization. Ovarian follicular development was quantified in haematoxylin- and eosin-stained ovarian sections embedded in paraffin wax. Six dams that were offered a high nutrient intake carried normal-sized fetuses (weight within +/- 2 SD of mean weight for control fetuses from dams fed a moderate level of complete diet) and 13 dams carried growth-restricted fetuses (weight +/- 2 SD of mean weight for control fetuses from dams fed a moderate level of complete diet). Mean placental masses in these groups were 354 +/- 24.5 and 230 +/- 21.1 g, respectively, compared with 442 +/- 54.3 g in the dams that were offered a moderate nutrient intake (n = 6). Growth-restricted fetuses from dams offered a high nutrient intake showed higher pituitary LHbeta mRNA expression (P < 0.05) than normal-sized fetuses from dams offered a moderate nutrient intake (252 +/- 21.6 and 172 +/- 23.6 nCi g(-1), respectively). FSHbeta mRNA expression was not influenced by growth status. Fewer follicles (primarily in the resting pool) were observed in the ovaries of both growth-restricted (P < 0.002) and normal-sized fetuses from dams offered a high nutrient intake (P < 0.01) compared with normal-sized fetuses from dams offered a moderate nutrient intake. Irrespective of nutritional treatment, the total number of follicles was positively associated with placental mass (P < 0.01). Thus, a high maternal nutrient intake during adolescent pregnancy had a negative influence on ovarian follicular development in fetuses as determined during late gestation.

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P Da Silva-Buttkus, R van den Hurk, ER te Velde and MA Taverne

Epidemiological studies in humans linking adult disease to growth in utero indicate that prenatal life is a critical period for the appropriate development of the reproductive axis. The aim of this study was to compare ovarian development in intrauterine growth-retarded and normally grown piglets originating from the same litter. Intrauterine growth-retarded piglets (runts) were identified on the basis of statistical analysis of the birth weight distribution within each litter. At birth, ovaries were collected from runt piglets (n=14) and their respective mean weight (normal, n=14) littermates. Ovaries were weighed and fixed, and development of ovarian germ cells was quantified in haematoxylin-eosin-stained paraffin wax sections using an image analysis system. Germ cell loss, using an in situ TdT-mediated dUTP nick-end labelling (TUNEL) assay for DNA fragmentation, and follicle cell activity, using immunohistochemistry to demonstrate vimentin, were studied in ovarian sections. At birth, body weight and absolute ovarian mass were significantly lower in runt piglets compared with their respective normally grown littermates (body weight: 733+/-38.5 versus 1530+/-39.7 g; ovarian mass: 51+/-3.0 versus 108+/-9.6 mg; P<0.001 for both). In the ovary, the proportion of nests of oogonia, the number of oocytes and TUNEL-positive cells, and the localization and intensity of vimentin immunoreactivity were not different between runt and normal littermates. However, runt piglets had more primordial follicles (268+/-18.6 versus 235+/-20.1 per mm(2) of cortex; P<0.05), fewer primary follicles (11+/-2.0 versus 20+/-3.0 per mm(2) of cortex; P<0.001) and no secondary follicles compared with normal piglets. These findings indicate that intrauterine growth retardation delayed follicular development in pig ovaries at birth.

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P Da Silva, RP Aitken, SM Rhind, PA Racey and JM Wallace

The onset of puberty in prenatally growth-restricted versus normally grown lambs of both sexes, born in April and housed under natural photoperiod, was examined. Singleton pregnancies were established and adolescent ewes were offered a high or moderate nutrient intake throughout gestation. Placental mass was reduced (P < 0.001) in high compared with moderate intake dams and resulted in the birth of growth-restricted and normal birth weight offspring, respectively. At birth, female lambs weighed 3.43 kg versus 5.03 kg (P < 0.001; n = 14 per group) and male lambs weighed 2.75 kg versus 5.18 kg (P < 0.001; n = 7 per group) in growth-restricted and normal birth weight groups, respectively. Lambs suckled for 12 weeks and thereafter were fed ad libitum until week 43 of age. Growth-restricted lambs had lower preweaning live weight gains and this difference was more pronounced in male (P < 0.05) than in female lambs (P = 0.07). Thereafter, live weight remained lower (P < 0.05) in growth-restricted than in normally grown lambs of both sexes until week 25 of age. In females, the time of onset of puberty was similar in the two groups. All females ovulated and there were no differences in the number of ovarian cycles recorded or in the incidence of aberrant ovarian function. In males, testosterone concentrations and testicular volume were lower in growth-restricted compared with normally developed lambs from birth until weeks 28 and 35 of age, respectively (P < 0.05). The seasonal increase in plasma testosterone concentrations occurred later in growth-restricted than in normally developed lambs (P < 0.01) but the timing of maximum peak concentrations was similar. Peak testosterone concentrations were lower (P < 0.05) in growth-restricted than in normal male lambs.

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S J Martins da Silva, J O Gardner, J E Taylor, A Springbett, P A De Sousa and R A Anderson

The ability of an oocyte to support early embryonic development requires both nuclear and cytoplasmic maturation. We have investigated the effects of brain-derived neurotrophic factor (BDNF) on maturation of the bovine oocyte and embryo development after parthenogenetic activation. By RT-PCR and immunohistochemistry, cumulus and oocytes were shown to express mRNA and protein for BDNF and the p75 common neurotrophin receptor. However, mRNA for the BDNF-specific full length and truncated isoforms of the TrkB receptor are only detected in cumulus, suggesting that oocytes and cumulus differ in their capacity to respond to neurotrophin signalling. In in vitro maturation experiments, the proportion of cumulus oocyte complexes maturing to metaphase II was not altered by BDNF in groups lacking fetal calf serum (FCS), but was significantly lower than the positive control containing 10% FCS (P < 0.01). However, after maturation, the proportion of parthenogenetically activated oocytes forming blastocysts was highest for 10 ng/ml BDNF (24%, n = 95) followed by 100 ng/ml BDNF (18%, n = 91) and 10% FCS (15%, n = 103), which in turn were greater than no serum (10%, n = 83; P < 0.01). Maturation in the presence of a BDNF blocking antibody resulted in a blastocyst yield that was comparable to the absence of serum, and lower than in the presence of BDNF (P < 0.01). Similar effects on progression to metaphase II and blastocyst formation were observed using oocytes matured without cumulus. Together, these results provide the first evidence for a role for neurotrophins in promoting oocyte cytoplasmic competence to support embryonic development, despite being insufficient in the absence of serum to enhance nuclear maturation.

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M Plaza Davila, P Martin Muñoz, J M Gallardo Bolaños, T A E Stout, B M Gadella, J A Tapia, C Balao da Silva, C Ortega Ferrusola and F J Peña

To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na+/K+ gradient, which is dependent on an Na+-K+ antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility.

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A J Borg, H E J Yong, M Lappas, S A Degrelle, R J Keogh, F Da Silva-Costa, T Fournier, M Abumaree, J A Keelan, B Kalionis and P Murthi

Abnormal trophoblast function is associated with fetal growth restriction (FGR). The JAK–STAT pathway is one of the principal signalling mechanisms by which cytokines and growth factors modulate cell proliferation, differentiation, cell migration and apoptosis. The expression of placental JAK–STAT genes in human idiopathic FGR is unknown. In this study, we propose the hypothesis that JAK–STAT pathway genes are differentially expressed in idiopathic FGR-affected pregnancies and contribute to abnormal feto-placental growth by modulating the expression of the amino acid transporter SNAT2, differentiation marker CGB/human chorionic gonadotrophin beta-subunit (β-hCG) and apoptosis markers caspases 3 and 8, and TP53. Expression profiling of FGR-affected placentae revealed that mRNA levels of STAT3, STAT2 and STAT5B decreased by 69, 52 and 50%, respectively, compared with gestational-age-matched controls. Further validation by real-time PCR and immunoblotting confirmed significantly lower STAT3 mRNA and STAT3 protein (total and phosphorylated) levels in FGR placentae. STAT3 protein was localised to the syncytiotrophoblast (ST) in both FGR and control placentae. ST differentiation was modelled by in vitro differentiation of primary villous trophoblast cells from first-trimester and term placentae, and by treating choriocarcinoma-derived BeWo cells with forskolin in cell culture. Differentiation in these models was associated with increased STAT3 mRNA and protein levels. In BeWo cells treated with siRNA targeting STAT3, the mRNA and protein levels of CGB/β-hCG, caspases 3 and 8, and TP53 were significantly increased, while that of SNAT2 was significantly decreased compared with the negative control siRNA. In conclusion, we report that decreased STAT3 expression in placentae may contribute to abnormal trophoblast function in idiopathic FGR-affected pregnancies.