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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.

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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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Heterogeneity of Ngn3-GFP-expression in Apr and Aal 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 Apr and Aal (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.
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Figure 6: Heterogeneity of Ngn3-GFP-expression in Apr and Aal 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 Apr and Aal (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.

Mentions: Ngn3 is specifically expressed in undifferentiated spermatogonia (As to Aal) [25]. To determine if Ngn3 and Lin28 mark the same population of undifferentiated spermatogonia, we made use of Ngn3-GFP mice, in which GFP was inserted into the Ngn3 locus by gene replacement [26]. We performed whole-mount immunostaining of Ngn3-GFP seminiferous tubules with anti-LIN28 and anti-GFP antibodies. This analysis revealed that only a subpopulation of LIN28-positive spermatogonia was GFP-positive (Fig. 5A). Overall, ~40% of LIN28-positive As spermatogonia were GFP-positive, supporting that the population of As cells were not homogeneous. The Apr and Aal spermatogonia were either all GFP-positive or all GFP-negative (Fig. 5A) except a few as described later (Fig. 6). Interestingly, the percentage of Ngn3-GFP-positive spermatogonia increased dramatically as spermatogonia develop from As to Aal (16 cells) (Fig. 5B). While ~40% of As cells were GFP-positive, nearly all Aal (16-cell) spermatogonia were GFP-positive. As the number of chained cells increases, spermatogonia become more and more committed to differentiation. Taken together, our data suggested that Ngn3 delineates a more committed subpopulation of undifferentiated spermatogonia, in contrast, the LIN28-positive but Ngn3-GFP-negative spermatogonia are more primitive.


The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse.

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

Heterogeneity of Ngn3-GFP-expression in Apr and Aal 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 Apr and Aal (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.
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Figure 6: Heterogeneity of Ngn3-GFP-expression in Apr and Aal 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 Apr and Aal (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.
Mentions: Ngn3 is specifically expressed in undifferentiated spermatogonia (As to Aal) [25]. To determine if Ngn3 and Lin28 mark the same population of undifferentiated spermatogonia, we made use of Ngn3-GFP mice, in which GFP was inserted into the Ngn3 locus by gene replacement [26]. We performed whole-mount immunostaining of Ngn3-GFP seminiferous tubules with anti-LIN28 and anti-GFP antibodies. This analysis revealed that only a subpopulation of LIN28-positive spermatogonia was GFP-positive (Fig. 5A). Overall, ~40% of LIN28-positive As spermatogonia were GFP-positive, supporting that the population of As cells were not homogeneous. The Apr and Aal spermatogonia were either all GFP-positive or all GFP-negative (Fig. 5A) except a few as described later (Fig. 6). Interestingly, the percentage of Ngn3-GFP-positive spermatogonia increased dramatically as spermatogonia develop from As to Aal (16 cells) (Fig. 5B). While ~40% of As cells were GFP-positive, nearly all Aal (16-cell) spermatogonia were GFP-positive. As the number of chained cells increases, spermatogonia become more and more committed to differentiation. Taken together, our data suggested that Ngn3 delineates a more committed subpopulation of undifferentiated spermatogonia, in contrast, the LIN28-positive but Ngn3-GFP-negative spermatogonia are more primitive.

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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