Analysis of LIN28A in early human ovary development and as a candidate gene for primary ovarian insufficiency

Abstract

Lin28 proteins are emerging as important regulators of microRNAs in endocrine systems. Lin28a regulates primordial germ cell development and puberty timing in mice, whereas the related protein LIN28B is associated with age at menarche in genome-wide association studies in humans. Here, we studied expression of LIN28A and LIN28B in early human gonad development. LIN28A increased in the developing ovary between 6 and 9weeks post conception, but not in the developing testis. Immunohistochemistry demonstrated LIN28A in peripheral germ cells. LIN28B was expressed at lower levels in both tissues and did not increase with time. As disruption of Lin28a affects germ cell development in mice, LIN28A was considered a candidate gene for primary ovarian insufficiency (POI) in humans. However, no significant changes were found in 50 women studied. These findings show LIN28A is strongly expressed in germ cells during early human ovary development, but disruption of LIN28A is not a common cause of POI.

Fig. 1

Expression of LIN28A and LIN28B in the gonad between 6 and 9 weeks post conception (wpc). (A) Ovary and testis samples showed higher expression of LIN28A compared to control tissue (heart; 8wpc). Similar results were seen when other control samples were studied (data not shown). A progressive increase in LIN28A expression was detected in the ovary up until 9 wpc. (B) LIN28B expression was higher in the testis and ovary compared to control, but did not show a marked increase with ovarian age. Data are shown as fold change above control, with LIN28A and LIN28B expression levels normalized to GAPDH and relative quantification of gene expression performed according to the 2^−ΔΔCt method. Bars represent 1SD. Note the different scales in panels A and B.

Fig. 2

Immunohistochemistry showing expression of POU5F1 (OCT4) (red) and LIN28A (green) in the human ovary at 7 wpc. (A) Low power image showing a population of LIN28A expressing cells in the peripheral cortical region of the developing gland. Lower level expression of LIN28A was seen in some somatic cells. Scale bar, 100 μm. (B) LIN28A shows strong staining in this population of POU5F1 (OCT4)-expressing germ cells. Scale bar, 100 μm. (C) High power image showing LIN28A staining mainly in the cytoplasm of germ cells whereas POU5F1 (OCT4) staining was intense in the nuclei. Scale bar, 10 μm. (D) The omission of primary antibody resulted in very minimal signal. Scale bar, 100 μm. Nuclei are counterstained with DAPI (blue).

Fig. 3

Immunohistochemistry showing expression of POU5F1 (OCT4) (red) and LIN28A (green) in the human testis at 9 wpc. (A) Low power image showing generalized low level expression of LIN28A with denser signal in the developing seminiferous cords. Scale bar, 100 μm. (B) High power image showing no clear increased intensity of LIN28A in POU5F1 (OCT4) expressing cells. Scale bar, 25 μm. (C) Control image showing omission of primary antibody resulted in no significant signal. Scale bar, 100 μm. Nuclei are counterstained with DAPI (blue).