Investigation of peripubertal expression of Lin28a and Lin28b in C57BL/6 female mice

Abstract

Genome-wide association studies recently identified 32 loci that associate with the age at menarche (AAM) in humans. Because the locus most robustly associated with AAM is in/near LIN28B, the goal of this study was to investigate how the Lin28 pathway might modulate pubertal timing by examining expression of Lin28b, and its homologue, Lin28a, across the pubertal transition in female mice. Quantitative reverse-transcriptase PCR data indicate that, prior to the onset of puberty, expression of both Lin28b and Lin28a decreases in the ovary, while expression of only Lin28a decreases in the hypothalamus; the expression of Lin28a increases after the onset of puberty in the pituitary. Immunohistochemistry in ovarian tissue verified that Lin28a protein levels decreased in parallel with gene expression. Although these data do not demonstrate cause and effect, they do suggest that decreased expression of Lin28a/Lin28b may facilitate the transition into puberty, consistent with previous data showing that overexpression of Lin28a in transgenic mice leads to delayed puberty. In addition, although Lin28b and/or Lin28a expression significantly decreased prior to puberty, neither Let-7a nor Let-7g miRNA levels changed significantly, raising the possibility that some effects of Lin28b and Lin28a within the hypothalamic-pituitary-gonadal (HPG) axis may be Let-7 miRNA independent. Subsequent studies, such as tissue and age specific modulation of Lin28b and Lin28a expression, could determine whether the expression patterns observed are responsible for modulating the onset of puberty and delineate further the role of this pathway in the HPG axis.

Figure 1. Expression of Lin28a and Lin28b in hypothalamic, pituitary, and ovarian tissue

(A) RT-PCR of Lin28a (190 bp) and Lin28b (123 bp) mRNA in 25-day-old hypothalamic (hypo), pituitary, and ovarian tissue from C57BL/6 female mice. NTC = no template control. (B) Relative expression levels of Lin28a and Lin28b in different tissues at 25 days as determined by qRT-PCR, normalized to β-actin mRNA.

Figure 2. Lin28a and Lin28b mRNA expression levels in hypothalamic, pituitary, and ovarian tissue across the pubertal transition

qRT-PCR for Lin28a and Lin28b mRNA in tissues from prepubertal 15, 20, 25, 30 day old and postpubertal (35–45 days old) C57BL/6 female mice, normalized to β-actin in hypothalamus (A), pituitary (B), and ovary (C). Average age of vaginal opening in the mouse colony is indicated by the arrow (31.5 ± 2.4 days). Values are displayed as means ± SEM (n=5–21 mice for each time point). For Lin28a in hypothalamic tissue, *=p<0.05, and **=p<0.01, when compared to 20-day-old tissue. For Lin28a in pituitary tissue, *=p<0.05, when compared to 20-day-old tissue. For Lin28a in ovarian tissue, †=p<0.001, and ‡=p<0.001, when compared to 15 and 20-day-old tissue, respectively. For Lin28b in ovarian tissue, *=p<0.05, **=p<0.01, and ***=p<0.001, when compared to 20 day old tissue.

Figure 3. Lin28a protein levels decrease prior to the onset of puberty in ovarian tissue

Immunohistochemical staining of Lin28A protein in 20 day (A–C) and 30 day (D–F) ovarian tissue. Lin28a protein expression is identified by diaminobenzidine staining. In 20-day-old tissue, Lin28a is detected in early developing follicles (arrow 1), early secondary follicles (arrows 2, 3, and 4), late secondary follicles and granulosa cells surrounding secondary follicles (arrow 5). In 30-day-old tissue, Lin28a protein is less evident, due to the abundance of atretic follicles (arrows 7, 8, 9 and 10). Lin28a still persists in the granulosa cells and oocytes of secondary follicles (arrow 11), and in early follicles (arrow 12). This pattern of Lin28a protein distribution was consistent in the two biological replicates that were examined

Figure 4. Expression of Let-7a and Let-7g miRNA in hypothalamic, pituitary, and ovarian tissue

qRT-PCR for Let-7a and Let-7g miRNA in 15, 20, 25, and 30-day-old hypothalamic (A) and pituitary (B) tissue, and 20, 25, and 30-day-old ovarian (C) tissue, normalized to snoRNA142. Values are displayed as means ± SEM (n=3–7 for each time point).