Interaction between neonatal maternal deprivation and serum leptin levels on metabolism, pubertal development, and sexual behavior in male and female rats

Abstract

Background: Maternal deprivation (MD) during neonatal life can have long-term effects on metabolism and behavior, with males and females responding differently. We previously reported that MD during 24 h at postnatal day (PND) 9 blocks the physiological neonatal leptin surge in both sexes. It is known that modifications in neonatal leptin levels can affect metabolism in adulthood. Thus, we hypothesized that at least some of the long-term metabolic changes that occur in response to MD are due to the decline in serum leptin during this critical period of development. Hence, we predicted that treatment with leptin during MD would normalize these metabolic changes, with this response also differing between the sexes.

Methods: MD was carried-out in Wistar rats for 24 h on PND9. Control and MD rats of both sexes were treated from PND 9 to 13 with leptin (3 mg/kg/day sc) or vehicle. Weight gain, food intake, glucose tolerance, and pubertal onset were monitored. Sexual behavior was analyzed in males. Rats were killed at PND90, and serum hormones and hypothalamic neuropeptides involved in metabolic control and reproduction were measured. Results were analyzed by three-way analysis of covariance using sex, MD, and leptin treatment as factors and litter as the covariate and employing repeated measures where appropriate.

Results: In males, MD advanced the external signs of puberty and increased serum insulin and triglyceride levels and hypothalamic proopiomelanocortin mRNA levels at PND90. Neonatal leptin treatment normalized these effects. In contrast, MD decreased circulating triglycerides, as well as estradiol levels, in females at PND90 and these changes were also normalized by neonatal leptin treatment. Neonatal leptin treatment also had long-term effects in control rats as it advanced the external signs of puberty in control males, but delayed them in females. Neonatal leptin treatment increased serum insulin and hypothalamic mRNA levels of the leptin receptor and cocaine- and amphetamine-regulated transcript in control males and increased orexin mRNA levels in controls of both sexes. Although pubertal onset in males was advanced by either MD or neonatal leptin treatment in males and delayed by leptin treatment in females, the mRNA levels of hypothalamic neuropeptides and receptors related to reproduction were not affected by MD or neonatal leptin treatment in either sex at PND90.

Conclusions: These findings indicate that some of the long-term changes in metabolic and reproductive parameters induced by MD, such as advanced pubertal onset and increased hypothalamic proopiomelanocortin (POMC) expression, hyperinsulinemia, and hypertriglyceridemia in adult males and decreased serum triglyceride and estradiol levels in females, are most likely due to the decrease in leptin levels during the period of MD.

Keywords: Hypothalamus; Neonatal leptin surge; Neuropeptides; Puberty; Reproduction; Sexual dimorphism; Weight gain.

Fig. 1

Experimental design diagram. This diagram indicates the day or days when maternal deprivation, leptin treatment, weaning, control of pubertal onset, blood sampling, glucose tolerance testing, and behavioral testing were performed. PND postnatal day

Fig. 2

Changes in body mass and food intake throughout the study. a, b Mean body mass (BM), c accumulated BM gain, and d accumulated food intake per rat from the time of weaning until 5 days before being killed. e Glucose tolerance test (area under the curve), in maternal deprivation (MD) or control (Co) male and female rats treated with leptin (Lept) or vehicle (Vh) from postnatal day (PND) 9 until PND13 (n = 10–12). Body mass was reduced by MD in both sexes until approximately PND57, while leptin treatment of MD rats returned body mass to control levels. Males weighed more and ate more than females throughout the study and were less glucose tolerant. a, b Data were analyzed by a repeated measure three-way ANCOVA: (a) main effect of sex; Tukey’s post hoc test: *** CoLeptin vs. MDLeptin (p < 0.005), @@ MDLeptin vs. CoVh (p < 0.01) and ### MDVh vs. MDLeptin (p < 0.005). c–e Data were analyzed by three-way ANCOVA: (a) main effect of sex; (b) main effect of MD. Tukey’s post hoc: **p < 0.01; ***p < 0.005

Fig. 3

Serum levels of leptin (a) and corticosterone at postnatal day (=ND)36 (b) and of corticosterone (c), insulin (d), 17β-estradiol (e), and testosterone (f), at PND90 in maternal deprivation (MD) or control (Co) male and female rats treated with leptin (Lept) or vehicle (Vh) from PND9 until PND13 (n = 7–8). In males, MD decreased leptin and increased insulin and triglyceride levels, with leptin treatment returning insulin and triglycerides to control levels. In contrast in females, MD decreased triglyceride and estradiol levels, which were also normalized by leptin treatment. a Main effect of sex (three-way ANCOVA). b Main effect of MD (two-way ANCOVA). Tukey’s post hoc test:*p < 0.05; **p < 0.01; ***p < 0.005

Fig. 4

Analysis of hypothalamic neuropeptide mRNA levels. Expression levels in the medial basal hypothalamus of the mRNAs encoding NPY (a), AgRP (b), POMC (c), CART (d), orexin (e), and leptin receptor (LepR) (f) in maternal deprivation (MD) or control (Co) male and female rats treated with leptin (Lept) or vehicle (Vh) from postnatal day (PND) 9 until PND13 (n = 5–6). In males, MD increased POMC mRNA levels, but leptin treatment did not normalize this effect. In contrast, leptin treatment of control males increased CART, orexin, and LepR mRNA levels. In females, there was no effect of MD on the expression of these neuropeptides and leptin treatment of control females only increased orexin expression. Three-way ANCOVA: (a) main effect of sex. Tukey’s post hoc test: *p < 0.05; **p < 0.01; ***p < 0.005

Fig. 5

Effect of neonatal maternal deprivation and leptin treatment on pubertal onset. Age of pubertal onset assessed by vaginal opening (VO) in females (a) and balano-prepurcial separation (BPS) in males (b) in maternal deprivation (MD) or control (Co) rats treated with leptin (Lept) or vehicle (Vh) from postnatal day (PND) 9 until PND13 (n = 10–12). The animals were monitored between days 30 and 38 (females) or days 35 and 47 (males) for VO or BPS, respectively. In females, neonatal leptin treatment delayed the mean age of VO in both control and MD rats, with no effect of MD alone. In males, external pubertal signs were advanced with both MD and leptin treatment, with the combination of these factors normalizing pubertal onset. For the presentation of data, cumulative percentage data of VO/BPS are shown beside the mean age of VO/BPS. Two-way ANCOVA: (b) main effect of leptin treatment. Tukey’s post hoc: ***p < 0.005; *p < 0.05

Fig. 6

Sexual behavior in male rats. a Mount latency, b intromission latency, c ejaculation latency, d mount frequency, e intromission frequency, f ejaculation frequency, g mount/intromission frequency, h pseudo-ICI (pseudo intercopulatory interval for non-ejaculating rats), i ICI (inter-copulatory interval: ejaculation latency/intromission frequency in each copulatory series), j copulatory rate (the sum of mounts and intromissions divided by the elapsed time (s) from the first mount until an observed ejaculation), k percentage of rats displaying copulatory activity, and l percentage of rats achieving ejaculation, of maternal deprivation (MD) or control (Co) male and female rats treated with leptin (Lept) or vehicle (Vh) from postnatal day (PND) 9 until PND13 (n = 10–12). Ejaculation frequency was increased by leptin in control rats, but decreased by leptin in MD rats, with the inverse effect seen on ejaculation latency. Two-way ANCOVA: (b) main effect of MD. Tukey’s post hoc: *p < 0.05