LIN28 is selectively expressed by primordial and pre-meiotic germ cells in the human fetal ovary

Abstract

Germ cell development requires timely transition from primordial germ cell (PGC) self-renewal to meiotic differentiation. This is associated with widespread changes in gene expression, including downregulation of stem cell-associated genes, such as OCT4 and KIT, and upregulation of markers of germ cell differentiation and meiosis, such as VASA, STRA8, and SYCP3. The stem cell-expressed RNA-binding protein Lin28 has recently been demonstrated to be essential for PGC specification in mice, and LIN28 is expressed in human germ cell tumors with phenotypic similarities to human fetal germ cells. We have therefore examined the expression of LIN28 during normal germ cell development in the human fetal ovary, from the PGC stage, through meiosis to the initiation of follicle formation. LIN28 transcript levels were highest when the gonad contained only PGCs, and decreased significantly with increasing gestation, coincident with the onset of germ cell differentiation. Immunohistochemistry revealed LIN28 protein expression to be germ cell-specific at all stages examined. All PGCs expressed LIN28, but at later gestations expression was restricted to a subpopulation of germ cells, which we demonstrate to be primordial and premeiotic germ cells based on immunofluorescent colocalization of LIN28 and OCT4, and absence of overlap with the meiosis marker SYCP3. We also demonstrate the expression of the LIN28 target precursor pri-microRNA transcripts pri-LET7a/f/d and pri-LET-7g in the human fetal ovary, and that expression of these is highest at the PGC stage, mirroring that of LIN28. The spatial and temporal restriction of LIN28 expression and coincident peaks of expression of LIN28 and target pri-microRNAs suggest important roles for this protein in the maintenance of the germline stem cell state and the regulation of microRNA activity in the developing human ovary.

FIG. 1.

Expression of LIN28 and LIN28B during human fetal ovarian development. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis reveals expression of LIN28 (A) to be highest at 8–11 wga, and significantly lower at 14–16 wga and 17–20 wga, coincident with the onset of germ cell differentiation [***P<0.001 vs. 8–11 wga (ANOVA with Neumann-Keuls post hoc testing), n=6–8 ovaries per gestational group]. LIN28B expression (B) remained unchanged across gestation. Values denote mean±SEM. ANOVA, analysis of variance; SEM, standard error of the mean.

FIG. 2.

LIN28 expression is germ cell specific and becomes restricted to a subpopulation of germ cells with increasing gestation. At 9 wga (A), all germ cells in the gonad (G) express LIN28 (brown staining) in both the nucleus and cytoplasm. Somatic cells in the gonad (G) and mesonephros (M) display no staining. By 14 wga (B), LIN28-positive cells are concentrated at the periphery of the ovary, although some LIN28-positive germ cells are distributed toward the center of the ovary. At 18 wga (C), LIN28-positive germ cells occupy a thin margin at the edge of the ovary; no staining is detected in more mature germ cells toward the center of the ovary. (D) Magnified image of area identified in (C); meiotic germ cells (identified by their characteristic chromatin arrangement) are LIN28 negative (arrowheads). Somatic cells (SC) in streams and interspersed between LIN28-positive germ cells are also immunonegative. (E) No staining is detected in negative controls. Scale bars in A, B, C, and E: 50 μm; D: 20 μm.

FIG. 3.

LIN28 is expressed in undifferentiated germ cells, but not in those undergoing meiosis. Double immunofluorescence reveals the extensive overlap between the expression domains of LIN28 (green) and OCT4 (red) (A). All OCT4-positive cells express LIN28, although some germ cells are LIN28-positive only (arrows). High-magnification images of the human fetal ovary at 14 wga (B) reveal that LIN28 occupies a distinct subnuclear compartment (arrows) from which OCT4 protein is excluded. No colocalization of LIN28 (green) with the meiosis marker SYCP3 (red) is detected at 15 wga at the periphery (C) or deeper within the cortex (D). No staining is detected on negative controls (insets, A and C). Scale bars in A, C, and D: 50 μm; B: 10 μm, blue staining denotes cell nuclei.

FIG. 4.

Expression of precursor LET-7 pri-microRNA transcripts during human fetal ovarian development. qRT-PCR analysis reveals developmentally regulated expression of (A) pri-LET-7a/f/d and (B) pri-LET-7g during human fetal ovary development. Expression of both transcripts is highest at 8–11 wga, and significantly downregulated at 14–16 and 17–20 wga. ***P<0.001 versus 8–11 wga, **P<0.01 versus 8–11 wga (ANOVA, with Neumann-Keuls post hoc testing n=6–8 ovaries per gestational group). Values denote mean±SEM.