Fetal nutrition and timing of puberty.

Over the last decade growing evidence has been documented on the relationship between intrauterine growth retardation (IUGR) and pubertal development indicating changes in timing and progression of puberty. These changes in pubertal development are part of a growing list of IUGR-related diseases, which includes type 2 diabetes mellitus, cardiovascular disease, short stature and polycystic ovary syndrome. The influence of IUGR on the mechanisms behind the onset of puberty is still elusive. In the absence of prospective studies on gonadotropin-releasing hormone pulse patterns in IUGR children, other markers of pubertal development such as age at menarche in girls and progression of puberty have been employed. We investigated pubertal development and DHEAS levels in children born small for gestational age (SGA) after third trimester growth retardation and children born appropriate for gestational age (AGA). A faster progression of puberty was found in girls but not in boys. DHEAS levels tended to be higher in SGA children than in AGA children. In animal studies using two rat models, growth and onset of puberty based on perinatal undernutrition were also investigated. In one model intrauterine growth retardation was induced by ligation of the uterine arteries (IUGR) at day 17 of gestation and in the other model postnatal food restriction (FR) was induced by increasing litter size after birth until weaning. In both models, the rats showed a persistent growth failure. Onset of puberty was defined by vaginal opening (VO) in female rats and by balanopreputial separation (BPS) in male rats. At onset of puberty IUGR and FR rats had a lower body weight compared to controls, indicating that no threshold for body weight is needed for the onset of puberty. In the IUGR female rats, the onset of puberty was delayed and in the FR female rats the onset of puberty was in time. In both IUGR and FR female rats VO and first cycle were uncoupled. In IUGR female rats, at VO, at first cycle and at the age of 6 months the ovaries showed a decline in number of follicles indicating that intrauterine malnutrition in the female rat has a permanent influence on the growth and development of follicles. In the FR female rats, at VO, the ovaries showed a normal number of follicles but an abnormal maturation pattern. At the time of first cycle and at the age of 6 months normalization in follicle growth pattern was observed. These findings suggest that postnatal undernutrition has a transient influence on follicle growth and development. In male rats, both models showed delayed onset of puberty and impaired testicular function, as shown by decreased testosterone levels. These data indicate that early malnutrition during different critical developmental time windows may result in different long-lasting effects on pubertal development in both humans and rats.

Endocrine regulation and extended follow up of longitudinal growth in intrauterine growth-retarded rats.

Bilateral uterine artery ligation in late gestation was performed in pregnant dams in order to determine the effects of intrauterine growth retardation (IUGR) on long-term postnatal somatic growth and the GH neuroendocrine axis in the adult female and male rat. Body weight (BW), nose-anus length (NAL) and tail length (TL) were recorded at regular intervals in both the IUGR and control (CON) offspring until the age of 93 days. Spontaneous 6-h GH secretory profiles and serum IGF-I were determined around the age of 100 days in both the IUGR and the CON group. No catch-up growth in BW, NAL or TL was observed in young adult male IUGR rats. Female IUGR rats did catch up in NAL beyond the age of 57 days and in TL before weaning, but did not catch up at any time in BW. Spontaneous 6-h GH secretory profiles in female and male IUGR rats at a mean age of 100+/-4 days were similar to their controls at a mean age of 101+/-4 days. Overall median 6-h rat GH plasma concentrations, rat GH peak amplitude, number of rat GH peaks and sum of peak area were not significantly different. Median serum IGF-I levels in young adult female and male IUGR rats showed no difference when compared with their respective controls. These results demonstrate that IUGR, after bilateral uterine artery ligation in late gestation, leads to incomplete BW catch-up growth in young adult rats of both sexes with physiological GH/IGF-I secretion, suggesting intrauterine modulation of tissue responsiveness to GH and IGF-I. Female IUGR rats do catch up in NAL and TL, developing disturbed body proportions.

Delayed first cycle in intrauterine growth-retarded and postnatally undernourished female rats: follicular growth and ovulation after stimulation with pregnant mare serum gonadotropin at first cycle.

In the present study we examined the consequences of intrauterine growth retardation and postnatal food restriction on the maturational process of sexual development by studying onset of first cycle. In addition, we investigated the effect of pregnant mare serum gonadotropin (PMSG) on ovarian growth and ovulation in intrauterine growth-retarded (IUGR) and postnatally food-restricted (PFR) rats. Intrauterine growth retardation was induced by uterine artery ligation on day 17 of gestation and food restriction was achieved by enlarging the litter to 20 pups per mother from day 2 after birth until weaning (day 24). In control rats, vaginal opening and the first cycle took place on the same day. In IUGR rats, uncoupling occurred between vaginal opening and the first cycle. Vaginal opening was delayed (P<0.05) and the first cycle was even further delayed (P<0.01) compared with controls. Body weight in IUGR rats was lower (P<0.05) at vaginal opening, but at first cycle and after stimulation with 50 IU PMSG in the first cycle it was similar to that in controls. In the ovaries of IUGR rats, the numbers of primordial (P<0.05), growing (P<0.01) and antral follicles (P<0.01), and the total number of follicles (P<0.01) were lower than in controls after stimulation with 50 IU PMSG at first cycle. The number of corpora lutea in the ovaries of the IUGR rats and the controls was similar and reflected superovulation. In the PFR rats, vaginal opening occurred at the same time as in control rats, but at a lower body weight (P<0.01). First cycle was much delayed (P<0.01), by which time body weight was greater (P<0.01) than that of controls at first cycle. On the basis of the differences in weight and age between PFR rats and controls at first cycle, we performed two studies. In study A, ovaries were analysed histologically 42 h after stimulation with PMSG at first cycle of control rats and age-matched PFR rats. In study B, the ovaries of PFR rats at first cycle and age-matched control rats were examined 42 h after PMSG stimulation. In the ovaries of the PFR rats in study A, a greater total number of follicles (P<0.05) was observed, represented by a greater number of primordial follicles (P<0.01) and a lower number of antral follicles (P<0.05), including corpora lutea. The number of corpora lutea in the ovaries of the PFR rats was significantly lower than that in controls (P<0.01). The total number of follicles in the ovaries of the PFR rats of study B did not differ from the age-matched controls after PMSG stimulation at first cycle, and neither did the number of the follicles in the different classes. We conclude that, in IUGR rats at first cycle, PMSG can induce multiple follicular growth and development followed by superovulation comparable to that in controls, despite a decreased total number of follicles in the ovaries. However, in PFR rats of the same age, the ovary is not capable of responding adequately to PMSG, despite a greater total number of follicles. Stimulation with PMSG at first cycle resulted in follicular growth and superovulation comparable to those in age-matched controls. Undernutrition in different critical time periods around birth in the rat leads to ovarian development in such a way that, in both groups, an increased risk of reduced reproductive capacity can be expected.

Growth hormone binds to a single high affinity receptor site on mouse osteoblasts: modulation by retinoic acid and cell differentiation.

Growth hormone (GH) exerts direct differentiative and proliferative effects on osteoblasts. We studied 125I-labeled human (h) GH binding to primary mouse osteoblasts derived from collagenase-treated 18-day fetal mouse calvaria. Scatchard analysis of the data revealed a single class of high affinity GH receptors (apparent Ka = 5.74 x 10(9) M-1) with 2200 sites per cell. Affinity crosslinking and SDS-PAGE electrophoresis showed two bands with apparent molecular masses of 120 and 70 kDa. Mouse osteoblasts express GH receptor mRNA with gene transcripts of 4.2 and 1.2 kb, at levels which reach approximately 1/6 of those in mouse liver and 1/3 of those in mouse muscle. Two populations of undifferentiated and diffentiated osteoblasts, obtained by sequential collagenase digestion of mouse calvaria, were used to study the relationship between osteoblastic phenotype and GH receptor expression. Although the affinity of the receptors in undifferentiated and differentiated cells was the same, the capacity was significantly higher (1.45 +/- 1.0% vs 2.39 +/- 0.9%, P = 0.03) in differentiated cells. This stresses the specific importance of the osteoblast as a target cell for GH. The differentiating potential of the vitamin A derivative retinoic acid was subsequently used experimentally to induce differentiation in the cells. Retinoic acid increased 125I-hGH binding to preosteoblasts (153%, P = 0.02). Together, these data demonstrate the presence of a high affinity GH receptor in mouse osteoblasts which is related to differentiation.

The effect of exercise on systemic and bone concentrations of growth factors in rats.

Exercise can prevent bone loss and increase bone density. Growth factors such as insulin-like growth factor-I (IGF-I) and transforming growth factor beta (TGFbeta) are thought to be involved in the local response to mechanical loading, resulting in bone remodelling. We tested the effect of additional weight bearing during exercise on the systemic response of IGF-I and local bone response of IGF-I and TGFbeta. Thirty-four female Wistar rats (aged 3 months, weight 226.9 +/- 20.2 g) were randomly divided in four groups: group 1 baseline controls; group 2 sedentary controls; group 3 ran 15 min a day on a motor-driven exercise belt; group 4 ran 15 min a day with a backpack containing 40 g. The animals ran 5 days a week, for 6 weeks, with an average velocity of 16.6 +/- 4.4 m/min, and a slope of 5 degrees uphill. The serum growth hormone (GH) concentration was significantly higher in the running rats (group 3, P = 0.009) than in the sedentary controls (group 2). The IGF-I and IGF binding protein 3 (IGFBP3) levels in serum and the IGF-I levels in liver were similar in all groups. In the tibia no significant differences were observed in IGF-I, IGFBP3 and TGFbeta concentration. In the humerus, the IGF-I concentration was lower in the running rats (group 3) than in the sedentary controls (P = 0.04), but it was higher in the rats that ran with additional weight than in those without (P = 0.009). The TGFbeta concentration in the humerus was lower in both group 3 (P = 0.001) and 4 (P = 0.03) than in the sedentary controls. The effects in bone caused by mechanical stimulation cannot be explained by changes in serum IGF-I and IGF-I produced in the liver. The concentrations of IGF-I and TGFbeta in bone appeared to be modulated by running exercise.

Body mass index, body composition, and leptin at onset of puberty in male and female rats after intrauterine growth retardation and after early postnatal food restriction.

In this study we examined the body composition at onset of puberty in intrauterine growth retarded (IUGR), postnatal food restricted (FR), and control male and female rats. IUGR was induced by ligation of the uterine artery on d 17 of gestation and FR by litter enlargement to 20 pups per mother from d 2 after birth until weaning (d 24). We defined onset of puberty as balanopreputial separation in male rats and vaginal opening in female rats. We calculated body mass index, measured body composition with dual-energy x-ray absorptiometry, and measured leptin concentrations in serum. It was reported previously that early malnutrition, either during late gestation or immediately postnatally, results in a delayed onset of puberty in IUGR and FR male rats and in IUGR female rats, but not in FR female rats. In IUGR male rats at balanopreputial separation and in IUGR female rats at vaginal opening no differences were found in body mass index, body composition, and leptin levels compared with controls. FR male rats had a significantly lower percentage of fat and serum leptin concentrations at balanopreputial separation. FR female rats had a significantly lower body mass index, percentage of fat, and serum leptin concentrations at vaginal opening. We conclude that the onset of puberty in the rat is not dependent on a certain percentage of body fat or a certain threshold of circulating levels of leptin and that food deprivation during different "critical" time periods around birth results in different effects in later life.

The effects of intra-uterine growth retardation and postnatal undernutrition on onset of puberty in male and female rats.

The nutritional status, prenatally and early postnatally, plays a critical role in postnatal growth and development. Early malnutrition may change the original programming of organs, especially those in developmental phases, which can result in long-term changes in metabolism. The association between a low birth weight and the increased risk on type 2 diabetes, hypertension and cardiovascular disease is well known. In the present study we investigated whether intrauterine malnutrition or direct postnatal food restriction affects the onset of puberty in male and female rats. Intrauterine growth retardation (IUGR) was induced by uterine artery ligation on day 17 of gestation and postnatal food restriction (FR) by litter-enlargement to 20 pups per mother from day 2 after birth until weaning (24 d). Both models of malnutrition resulted in a persistent growth failure postnatally. The parameter of the onset of puberty was balano-preputial-separation (BPS) in the male rat and vaginal opening (VO) in the female rat. In both male IUGR (n = 26) and FR (n = 20) rats, the age at BPS was significantly delayed, with 48.1 +/- 1.9 d (p < 0.0001) and 50.4 +/- 2.9 d (p < 0. 0001), respectively, compared with controls (n = 30) with 45.8 +/- 1.4 d. In female IUGR rats (n = 37) the age at VO was significantly delayed, with 37.4 +/- 2.7 d (p < 0.04) compared with 36.1 +/- 1.5 d in controls (n = 23), but not in female FR rats (n = 18) with 36.5 +/- 2.2 d. Weight at onset of puberty did not differ between male IUGR and control rats, 194.5 +/- 20.0 g and 201.7 +/- 16.8 g, respectively, but was significantly lower in male FR rats with a weight of 175.6 +/- 17.5 g (p < 0.0001). In female IUGR as well as in female FR rats, weight at onset of puberty was significantly lower compared with controls: weight in IUGR 106.1 +/- 13.1 g (p < 0.001), weight in FR 85.3 +/- 7.6 g (p < 0.0001) and weight in controls 116.9 +/- 9.3 g. We conclude that early malnutrition, during late gestation or direct postnatally, results in a delayed onset of puberty in IUGR and FR male rats and in IUGR female rats, but not in FR female rats. The onset of puberty in these growth retarded rats as well as in controls does not depend on the achievement of a certain, crucial weight. The perinatal period appears to be a "critical time period" for the maturational process of pubertal development.

Ligation of the uterine artery and early postnatal food restriction - animal models for growth retardation.

Intrauterine growth restriction (IUGR) is one of the major causes of short stature in child- and adulthood. The cause of IUGR is unknown, however, an impaired uteroplacental function during the second half of human pregnancy might be an important factor, by affecting the programming of somatotropic axis and leading to postnatal growth failure into adulthood. Two rat models with perinatally induced growth retardation were used to examine the long-term effects of perinatal insults on growth. IUGR rats were prepared from pregnant dams, with a bilateral uterine artery ligation at day 17 of their pregnancy. Since the rat is relatively immature at birth, an early postnatal food restriction model was included as another model to broaden the time window of sensitive period of organogenesis. An individual growth curve was calculated of each animal (n = 813). From these individual growth curves the predicted growth curve for each experimental group was calculated by multilevel analysis. The proposed mathematical model allows us to estimate the growth potentials of these rat models with precision and could provide basic information to investigate the relationships among a number of other variables in future studies. Furthermore, we concluded that both pre- and early postnatal malnutrition leads to irreversible slowing down of postnatal growth.