Right (RVFW) and left (LVFW) ventricular free wall cardiac myocytes were collected from 25 fetal sheep aged 77-146 days gestation (term ϭ 150 days gestation), six salineinfused catheterized fetal sheep (129 GD), and five lambs to measure gestational changes in uni-and binucleated cardiac myocyte numbers and cell volumes by confocal microscopy. At 77 days gestation, 2% of the myocytes were binucleated, which increased to 50% at 135 days gestation and 90% at 4 -6 weeks after birth. RVFW uni-and binucleated myocytes were larger than those in the LVFW, and cell volumes of RVFW uni-and binucleated and LVFW binucleated myocytes (but not LVFW uninucleated myocytes) increased with gestation. Before birth, the approximate number of myocytes was greater in the LVFW than in the RVFW (P Ͻ 0.001). Before 110 GD, cardiac growth appeared to be due to myocyte hyperplasia, as approximate myocyte numbers and VFW weight increased at the same rate. After 110 days gestation, the approximate myocyte number/g VFW weight decreased, which suggests that myocyte hypertrophy, as well as hyperplasia, was occurring in association with the appearance of a greater proportion of binucleated cells after that time. By 4 -6 weeks of age, there was marked hypertrophy of myocytes and an apparent reduction in myocyte number. Anat Rec Part A 274A: 952-961, 2003.

In 17 fetal sheep aged 129 days, the effects of large-dose infusions of cortisol (72.1 mg/day for 2-3 days) on proliferation, binucleation, and hypertrophy of cardiac myocytes, cardiac expression of angiotensinogen, angiotensin receptor subtypes 1 and 2, Glut-1, glucocorticoid and mineralocorticoid receptors, proteins of the MAPK pathways and calcineurin were studied. Cortisol levels were 8.7 +/- 2.3 nM (SE) in 8 control and 1,028 +/- 189 nM in 9 treated fetuses (P < 0.001). Cortisol had no effect on myocyte binucleation. Left ventricular free wall (LVFW) uni- and binucleated myocytes were larger in cortisol-treated fetuses (P < 0.001, P < 0.05). Cortisol-treated fetuses had higher right ventricular free wall (RVFW) and LVFW angiotensinogen (Aogen) mRNA levels (treated: 2.30 +/- 0.37, n = 8 and 2.05 +/- 0.45, n = 7 vs. control: 0.94 +/- 0.12, n = 8 and 0.67 +/- 0.09, n = 7, P < 0.02). Levels of the glucose transporter Glut-1 mRNA were lower in the LVFW of treated fetuses (0.83 +/- 0.23 vs. 1.47 +/- 0.30 in control, P < 0.05, n = 7, 8). The higher the cortisol level, the greater the Aogen mRNA level (RVFW, r = 0.61, P < 0.01, n = 16; LVFW, r = 0.83, P < 0.0003, n = 14). There were no other changes in mRNA levels nor in levels of extracellular kinase, JNK, p38, their phosphorylated forms, and calcineurin. Thus high levels of cortisol such as occur after birth do not affect fetal cardiac myocyte binucleation or number but are associated with higher levels of ventricular Aogen mRNA, lower levels of Glut-1 mRNA, and hypertrophy of LVFW myocytes. These effects could impact on postnatal cardiac development.

In sheep, parturition is initiated by increased fetal hypothalamic-pituitary-adrenal axis (HPAA) activity leading to PGE2 and PGF2␣ production and a rise in the 17␤-estradiol-progesterone (E 2/P4) ratio. Uteroplacental PG production can also increase fetal HPAA activity. Periconceptional maternal undernutrition accelerates fetal HPAA maturation resulting in preterm labor. We determined whether preterm labor was preceded by an increase in PG concentrations and E 2/P4 ratio and whether these increases preceded or followed the corresponding rise in cortisol concentrations. Singleton-bearing ewes were nourished ad libitum (N, n ϭ 9) or undernourished (UN, n ϭ 10) to reduce maternal weight by 15% from Ϫ61 days (d) to ϩ30 d after mating with ad libitum intake thereafter. Paired maternal and fetal blood samples were collected from 126 d until delivery. Half the UN group delivered prematurely (Ͼ2 SD below mean gestation for the flock). PG and cortisol concentrations and E 2/P4 ratio increased before delivery in the same way in both groups. However, the increases occurred 7-10 d earlier in UN than in N animals. In both UN and N fetuses cortisol concentrations rose before fetal and maternal PG concentrations and maternal E 2/P4 ratio. Periconceptional maternal undernutrition induces preterm delivery in sheep by advancing the expected prepartum rise in cortisol and PG concentrations and E 2/P4 ratio. The rise in fetal cortisol concentration precedes the rise in fetal and maternal PG concentrations and maternal E 2/P4 ratio, suggesting that the underlying mechanism is likely to be acceleration of fetal HPAA maturation, resulting in initiation of the normal process of parturition.hypothalamic-pituitary-adrenal axis; prostaglandin; cortisol; estrogenprogesterone ratio

Intrafetal insulin-like growth factor (IGF)-I promotes cardiac hypertrophy in the late-gestation fetal sheep; whether these effects are sustained is unknown. IGF-I was infused for 4 days at 80 microg/h from 121 to 125 days of gestation, and its effects at 128 days, 3 days after the infusion stopped, were determined by comparison with untreated fetal sheep. After IGF-I treatment, fetal weights were similar to those in control fetuses but kidney weights were bigger (P < 0.05), as were spleen weights of male fetuses (P < 0.05). Cardiac myocytes were larger in female than male fetal sheep (P < 0.001). IGF-I increased male (P < 0.001) but not female myocyte volumes. IGF-I did not alter the proportions of uni- or binucleated right or left ventricular myocytes. Female fetal sheep had a greater proportion of binucleated cardiac myocytes than males (P < 0.05). IGF-I-treated fetuses had a slightly greater proportion of right ventricular nuclei in cell cycle phase G(2)/M and a reduced proportion of G(0)/G(1) phase nuclei (P < 0.1). Therefore, evidence for IGF-I-stimulated cardiac cell hyperplasia in fetal sheep in late gestation was limited. In conclusion, the greater sizes and larger proportion of binucleated cardiac myocytes in female fetal sheep suggest that myocyte maturation may occur earlier in females than in males. This may explain in part the male sex-specific responsiveness of cardiac hypertrophy to IGF-I in late gestation. If IGF-I-stimulated cardiomyocyte growth is accompanied by maturation of contractile function, IGF-I may be a potential therapeutic agent for maintaining cardiac output in preterm males.

We and others have shown previously that fetuses at midgestation can survive 30 min of complete umbilical cord occlusion, although hydrops fetalis (or gross fetal edema) results. To investigate whether this hydrops resolves by late gestation and if there are any long-term consequences of the asphyxial insult on the heart and kidneys, eight fetuses were subjected to 30 min of complete umbilical cord occlusion at 0.6 gestation (90 days; term 150 days) and were compared to a sham group (n ¼ 10). During the occlusion period, fetuses became severely hypoxemic, hypercapnemic, and acidotic, with both blood pressure and heart rate decreasing. Most variables had returned to normal by 2-hr recovery. At 129 6 1 days of gestation, approximately 40 days post occlusion, some fetuses were still slightly hydropic as skin fold measurements were increased (P < 0.01), although fetal body weight was not different from the sham group. The two groups had similar heart and kidney weights, ventricular cardiac myocyte nucleation, and glomerular number. By contrast, brain weight was reduced by 37% (P < 0.001) and the cerebral lateral ventricles were grossly dilated. Lungs were 50% smaller than in sham fetuses (P < 0.001). Thus, the hydrops that develops at midgestation as a result of a severe asphyxial episode can, but does not always, fully resolve by late gestation. Also, while fetuses at midgestation can survive this asphyxial episode with no long-term impact in renal or cardiac size, nephron number, or cardiomyocyte nucleation, the brain and lungs are severely affected.