Changes in hypothalamic expression of the Lin28/let-7 system and related microRNAs during postnatal maturation and after experimental manipulations of puberty

Abstract

Lin28 and Lin28b are related RNA-binding proteins that inhibit the maturation of miRNAs of the let-7 family and participate in the control of cellular stemness and early embryonic development. Considerable interest has arisen recently concerning other physiological roles of the Lin28/let-7 axis, including its potential involvement in the control of puberty, as suggested by genome-wide association studies and functional genomics. We report herein the expression profiles of Lin28 and let-7 members in the rat hypothalamus during postnatal maturation and in selected models of altered puberty. The expression patterns of c-Myc (upstream positive regulator of Lin28), mir-145 (negative regulator of c-Myc), and mir-132 and mir-9 (putative miRNA repressors of Lin28, predicted by bioinformatic algorithms) were also explored. In male and female rats, Lin28, Lin28b, and c-Myc mRNAs displayed very high hypothalamic expression during the neonatal period, markedly decreased during the infantile-to-juvenile transition and reached minimal levels before/around puberty. A similar puberty-related decline was observed for Lin28b in monkey hypothalamus but not in the rat cortex, suggesting species conservation and tissue specificity. Conversely, let-7a, let-7b, mir-132, and mir-145, but not mir-9, showed opposite expression profiles. Perturbation of brain sex differentiation and puberty, by neonatal treatment with estrogen or androgen, altered the expression ratios of Lin28/let-7 at the time of puberty. Changes in the c-Myc/Lin28b/let-7 pathway were also detected in models of delayed puberty linked to early photoperiod manipulation and, to a lesser extent, postnatal underfeeding or chronic subnutrition. Altogether, our data are the first to document dramatic changes in the expression of the Lin28/let-7 axis in the rat hypothalamus during the postnatal maturation and after different manipulations that disturb puberty, thus suggesting the potential involvement of developmental changes in hypothalamic Lin28/let-7 expression in the mechanisms permitting/leading to puberty onset.

Figure 1.

Expression profiles of Lin28 and Lin28b genes in different tissues in the adult rat. A panel of tissues from adult male and female (ovary) rats were screened for expression of Lin28 and Lin28b mRNAs, using conventional RT-PCR, as described in Materials and Methods.

Figure 2.

Expression profiles of the components of the Lin28/let-7 axis and related factors in the hypothalamus of male and female rats during postnatal maturation. Expression analyses of Lin28, Lin28b, and c-Myc mRNAs, as well as let-7a, let-7b, mir-9, mir-132, and mir-145 miRNAs were conducted in whole hypothalamic fragments from male rats at different stages of postnatal development. Different letters above bars indicate statistical differences (ANOVA with post hoc Tukey test).

Figure 3.

Expression profiles of the components of the Lin28/let-7 axis in preoptic area (POA) and medial basal hypothalamus (MBH) regions from male and female rats and female rhesus monkeys at different stages of postnatal/pubertal maturation (A, B). *P ≤ .05 vs neonatal in each region. (a) P ≤ .05 vs MBH for each age. (b) P ≤ .05 vs infantile in each region. (c) P ≤ .05 vs juvenile in each region (two-way ANOVA with post hoc Tukey test). In addition, expression levels of LIN28b measured by qPCR in the MBH of female rhesus monkeys during postnatal development are shown in panel C. *P ≤ .05 vs juvenile group (ANOVA with post hoc Tukey test). Abbreviations: NEO, neonatal; INF, infantile; JUV or J, juvenile; PUB, pubertal; EP, early pubertal; LP, late pubertal.

Figure 4.

Expression profiles of the components of the Lin28/let-7 axis and related factors in the hypothalamus of pubertal male and female rats following neonatal estrogenization or androgenization. In males (upper panels), expression analyses were conducted in whole hypothalamic fragments from rats subjected to a standard protocol of neonatal estrogenization (estradiol benzoate, EB); samples were obtained from peripubertal (postnatal day, PND-45) animals. In females (lower panels), expression analyses were conducted in whole hypothalamic fragments from rats subjected to a standard protocol of neonatal estrogenization (EB) or androgenization (testosterone propionate, TP); samples were obtained from peripubertal (PND-35) animals. Expression analyses included Lin28 (males only), Lin28b, and c-Myc mRNAs, as well as let-7a, let-7b, mir-9, mir-132, and mir-145 miRNAs. *P ≤ .05; vs control (vehicle) (t Student test).

Figure 5.

Expression profiles of the components of the Lin28/let-7 axis and related factors in the hypothalamus of male and female rats following photoperiod manipulation (CD, constant darkness from postnatal day [PND]10–15). In males (upper panels), studies in hypothalamic samples obtained at PND-15 are shown. In females (lower panels), studies were conducted at PND-15 and PND-35. For male studies, *P ≤ .05 vs control (normal photoperiod) group (t Student test). For female studies, *P ≤ .05 vs. PND-15 group for each photoperiodic regimen. (a) P ≤ .05 vs control (normal photoperiod) group for each age. (Two-way ANOVA with post hoc Tukey test.)

Figure 6.

Expression profiles of the components of the Lin28/let-7 axis and related factors in the hypothalamus of male and female rats following postnatal undernutrition. In males (upper panels), analyses were conducted at postnatal day (PND)–5, -15, and -45. *P ≤ .05 vs PND-5 group for each pups litter group. (a) P ≤ .05 vs. 12 pups/litter for each age. (b) P ≤ .05 vs corresponding PND-15 in each pups litter (two-way ANOVA with post hoc Tukey test). In females (lower panels), analyses were conducted at PND-5, -15, and -35. *P ≤ .05 vs PND-5 in each pups litter group. (a) P ≤ .05 vs. 12 pups/litter in each age. (b) P ≤ .05 vs PND-15 in each pups litter group (two-way ANOVA with post hoc Tukey test). P ≤ .05 was considered significant.