The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse

Abstract

Background: Life-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells - undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into As (single), Apr (paired), and Aal (aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.

Results: In the present study, we show that LIN28, a pluripotency factor, is specifically expressed in undifferentiated spermatogonia (As, Apr, and Aal) in mouse. Ngn3 also specifically labels undifferentiated spermatogonia. We used Ngn3-GFP knockin mice, in which GFP expression is under the control of all Ngn3 transcription regulatory elements. Remarkably, Ngn3-GFP is only expressed in approximately 40% of LIN28-positive As (single) cells. The percentage of Ngn3-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia.

Conclusion: Our study demonstrates that LIN28 specifically marks undifferentiated spermatogonia in mice. These data, together with previous studies, suggest that the LIN28-expressing undifferentiated spermatogonia exist as two subpopulations: Ngn3-GFP-negative (high stem cell potential) and Ngn3-GFP-positive (high differentiation commitment). Furthermore, Ngn3-GFP-negative cells are found in chains of Ngn3-GFP-positive spermatogonia, suggesting that cells in the Aal spermatogonia could revert to a more primitive state.

Figure 1

Expression of LIN28 in mouse testis. Western blot analysis was performed on 20 μg of protein extracts for each sample. β-actin served as a control. Molecular weight standards were marked in kDa. (A) Western blot analysis of LIN28 in adult mouse tissues. (B) Absence of LIN28 in germ cell-deficient XX^Y* testes. Testes were collected from adult and post-natal day 10-old mice. V6.5 mouse embryonic stem (ES) cells served as a positive control. LIN28 was absent in fibroblast feeder cells. (C) Developmental expression of LIN28 in postnatal testes. Testes were collected from mice of postnatal day 1 through adulthood.

Figure 2

Seminiferous epithelium stage-dependent distribution of LIN28-positive spermatogonia. (A) Expression of LIN28 in representative tubules. Adult testis sections were immunostained with anti-LIN28 antibody (green) and anti-ACRV1 antibody (red). Chromatin was stained with DAPI (blue) but was presented in black and white in the second row of panels to show nuclear morphology and the amount of heterochromatin. The morphology of spermatid acrosomes and nuclei was used to determine the stages of seminiferous tubules and distinguish among various types of germ cells. The stage of each seminiferous tubule is shown as roman numerals in the center. LIN28-positive spermatogonia are indicated by arrows. Note that strong signal in interstitial cells (Leydig cells) is due to autofluorescence. Pa, pachytene spermatocyte; Lp, leptotene spermatocyte; RS, round spermatid; ES, elongating spermatid. Scale bar, 50 μm. (B) Frequency of LIN28-positive spermatogonia during spermatogenesis. A total of 177 seminiferous tubule cross-sections were examined for LIN28-positive spermatogonia. The count of tubule sections examined is shown above each stage (I-XII). The number of LIN28-positive cells per tubule section (mean ± SE) is plotted.

Figure 3

LIN28 is specifically expressed in undifferentiated spermatogonia. Whole-mount immunofluorescence of seminiferous tubules from adult mice was performed with anti-LIN28 antibody. (A) Whole-mount examination of LIN28 expression in seminiferous tubules. Examples of A_s, A_pr, and A_al (up to 32 interconnected cells) spermatogonia are shown. LIN28 is predominantly cytoplasmic with punctate nuclear staining. Note that the A_pr and A_al spermatogonia are interconnected by intercellular cytoplasmic bridges due to incomplete cytokinesis. The background fluorescence helps orient the tubules. Scale bars, 25 μm. (B) Frequency of spermatogonia clusters comprising different numbers of chained LIN28-positive cells. A total of 205 isolated cells and clusters were counted. Only clearly identified clusters were included. The percentage of clusters with a longer chain of cells might be underestimated, since such large clusters extended around the tubule edge as shown in Fig. 3A and thus were excluded. Clusters with a non-2ⁿ number of cells or too many chained cells were grouped as "other".

Figure 4

Expression and siRNA knockdown of LIN28 in cultured spermatogonia highly enriched for spermatogonial stem cells (SSCs). (A) Immunostaining of SSCs with anti-LIN28 and anti-PLZF or anti-GFRA1 antibodies. Scale bar, 50 μm. (B) Quantitative PCR measurement of Lin28 mRNA levels (n = 3, mean ± SE) in SSCs after siRNA treatment for 30 hours. (C) Decreased LIN28 protein abundance (43% compared to the control) in SSCs after 30 hours of siRNA treatment. The control SSCs were not treated with Lin28 siRNA. Feeder cells served as a negative control. β-actin served as a loading control. (D) The number of SSCs (n = 3, mean ± SE) with and without Lin28 siRNA treatment. (E) Quantitative measurement of mature let-7g miRNA levels (n = 3, mean ± SE) in SSCs after siRNA treatment for 30 hours.

Figure 5

Ngn3-GFP labels a more committed subpopulation of LIN28-positive spermatogonia. Seminiferous tubules from adult Ngn3-GFP mice were immunostained with anti-LIN28 and anti-GFP antibodies [26]. We used antibodies to visualize GFP, since the GFP fluorescence was weak. (A) LIN28-positive undifferentiated spermatogonia are divided into Ngn3-GFP-positive and Ngn3-GFP-negative subpopulations. A_s, Apr and the number of A_al spermatogonia were indicated. Ngn3-GFP-negative spermatogonia were circled. Scale bar, 25 μm. (B) Frequency of spermatogonia clusters (2ⁿ cells: 1, 2, 4, 8, 16) with cells that are either all Ngn3-GFP-positive or all Ngn3-GFP-negative. The total number of 2ⁿ-cell clusters examined was shown above each column.

Figure 6

Heterogeneity of Ngn3-GFP-expression in A_pr and A_al spermatogonia. Seminiferous tubules from adult Ngn3-GFP mice were immunostained with anti-LIN28 and anti-GFP antibodies. (A) Presence of Ngn3-GFP-negative cell(s) in A_pr and A_al (4-cell) spermatogonia. Note the unusual 4-cell chain (encircled) that is branched. (B) One cell (arrow) at the end of the 8-cell chain was Ngn3-GFP-negative. (C) Two spermatogonia (arrows) in the middle of 16-cell chain were Ngn3-GFP-negative. Scale bar, 25 μm.