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The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse.

Zheng K, Wu X, Kaestner KH, Wang PJ - BMC Dev. Biol. (2009)

Bottom Line: The percentage of Ngn3-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA. kezheng@vet.upenn.edu

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.

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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.
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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.

Mentions: Spermatogonial stem cells (SSCs) are believed to be a subset of As cells [3]. Currently, there are no cytological markers that could distinguish SSCs from "non-stem" As cells. To examine whether LIN28 is expressed in SSCs, we performed double immunostaining of cultured spermatogonia highly enriched for SSCs with anti-LIN28 and anti-PLZF or anti-GFRA1 antibodies. PLZF is required for maintenance of SSCs [18,19]. We found that LIN28 was expressed in cultured SSCs, but the abundance of LIN28 in SSCs was not uniform, suggesting the heterogeneity of in vitro cultured SSCs (Fig. 4A and Additional file 2).


The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse.

Zheng K, Wu X, Kaestner KH, Wang PJ - BMC Dev. Biol. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2719617&req=5

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.
Mentions: Spermatogonial stem cells (SSCs) are believed to be a subset of As cells [3]. Currently, there are no cytological markers that could distinguish SSCs from "non-stem" As cells. To examine whether LIN28 is expressed in SSCs, we performed double immunostaining of cultured spermatogonia highly enriched for SSCs with anti-LIN28 and anti-PLZF or anti-GFRA1 antibodies. PLZF is required for maintenance of SSCs [18,19]. We found that LIN28 was expressed in cultured SSCs, but the abundance of LIN28 in SSCs was not uniform, suggesting the heterogeneity of in vitro cultured SSCs (Fig. 4A and Additional file 2).

Bottom Line: The percentage of Ngn3-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA. kezheng@vet.upenn.edu

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.

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