Possible functions of P21 CDKN1A within mouse spermatogonia ISSN 2753-8176 (online) (DOI: 10.13140/RG.2.2.27322.06086)

 Possible functions of P21 CDKN1A within mouse spermatogonia 

Ana Pedro¹

¹Gwyntwr1386 Pharmacy, Regus Chester Business Park, Heronsway, Chester, CH49QR, UK

DOI: 10.13140/RG.2.2.27322.06086

Abstract

The cyclin dependent kinase inhibitor P21 CDKN1A is upregulated during differentiation and upon DNA damage in somatic cells. We analysed the expression of P21 CDKN1A , γ-H2AX, PCNA and BrDu in differentiating mouse spermatogonia in mouse testis. P21 CDKN1A was expressed in in a variable manner in different spermatogonia types such as spermatogonia A and B. In addition, in agreement with γ-H2AX, PCNA and BrDu expression, P21 might have roles in cell cycle checkpoints, chromosome instability, replication, mitosis control and DNA repair but not apoptosis in spermatogonia.

Keywords: P21 CDKN1A, Spermatogonia

1. Corresponding author: anapedrolaboratories@gmail.com

The cyclin dependent kinase inhibitor P21 CDKN1A is upregulated during differentiation and upon DNA damage both in somatic cells (1) and in germ cells (2). The levels of P21 increase following P53 tumor suppressor activation. Inhibition of CDK by P21 triggers the arrest of cell cycle in the G1 and G2 phases. In the absence of exogenous insults which cause replication stress, only vestigial P21 levels are prevalent. Nevertheless, these vestigial P21 levels control DNA replication speed and origin firing during S phase in order to preserve genomic stability. This P21 S-phase function depends on its ability to displace other partners from chromatin-bound proliferating cell nuclear antigen (PCNA). In addition, PCNA also regulates P21 because it prevents its upregulation during S phase (3). P21 is also involved in differentiation, cell migration, cytoskeletal dynamics, apoptosis, transcription, DNA repair, reprogramming of induced pluripotent stem cells, autophagy and the onset of senescence, besides, its roles in cell cycle regulation including mitosis. P21 can act either as a tumor suppressor or as an oncogene what depends on the cellular context, its subcellular localization and post-translational modifications (1). It was previously found that P21 is a factor which might play an important role in spermatocytes and repair mechanisms in these cells, but does not play any function in spermatogonia (4). However, we found P21 expression in spermatogonia of the mouse seminiferous epithelium and therefore analysed its expression in spermatogonia of the mouse testis together with the expression of PCNA, BrDU and γ-H2AX.

First we analysed the expression of P21, PCNA and γ-H2AX in A spermatogonia which are in the initial stage of differentiation (fig.1) . P21 is expressed in a variable manner in A spermatogonia from different stages from homogeneous nuclear and cytoplasmic staining to small foci in the nuclei. In relation to P21/PCNA expression in A spermatogonia, PCNA and P21 might appear both dispersed or PCNA might be associated to late replication sites (5, 6), while P21 appears dispersed.

Further, we compared the expressions of P21 CDKN1A with those of γ-H2AX, BrDu in B spermatogonia, which represent the last stage of spermatogonial differentiation before dividing to give rise to preleptotene spermatocytes(fig.2). P21 is expressed in a variable mode in B spermatogonia, from dispersed foci in the nuclei and cytoplasm staining to staining mitotic B spermatogonia (suggesting a role for P21 in mitosis, 1) and its presence in heterochromatic regions, suggesting a role in DNA replication control (1,3). In B spermatogonia, P21 might appeared dispersed while BrdU appears dispersed too, or BrdU can appear alone associated to heterochromatin without the presence of P21. Finally, when γ-H2AX appears associated with heterochromatin, P21 appears dispersed.

Conclusions

Our results suggest that P21 might be involved in cell cycle checkpoints, chromosome instability, replication, mitosis control and DNA repair in spermatogonia. We don’t think P21 is involved in spermatogonia apoptosis as per our previous results (7) and in concordance with other studies where it was found that, the P53 apoptosis pathway in testicular cells is trigerred by DNA damage independently of P21 expression (8)

Materials and methods

This work was performed in the scope of Ana Pedro’s PhD Thesis approved on the 07/07/2009 by the Dean of University of Valladolid, Spain.

Mice

Control mice (9) with the same background (129sv/C57BL6; 50:50) were used in this study, which were provenient from Manuel Serrano’s lab, CNIO, Madrid. Mice testes were included in paraffin blocks.

We used for brightfield P21 study the following antibodies: P21 CDKN1A Neomarkers, ab-9, #RB032-P0, 1:150), Santa Cruz Biotechnology, Inc; Rabbit ABC Staining System; #sc-2018.

P21 CDKN1A, PCNA and γ-H2AX staining . For double immunofluorescence we used mouse anti-γ-H2AX, 1:2000 (Upstate, #05-636), P21 CDKN1A Neomarkers, ab-9, #RB-032-P0, 1:150), Santa Cruz Biotechnology, Inc., mouse anti-PCNA 1:1500 (Santa Cruz Biotechnology, Inc; # sc-56), rabbit anti-PCNA 1:200 (Santa Cruz Biotechnology, Inc; #sc-7907), anti-rabbit Alexa Fluor ®488 , 1:500 (Molecular Probes, # A-11034), anti-mouse Alexa Fluor ® 488, 1:1500 (Molecular Probes, # A-21202), anti-rabbit Alexa Fluor ® 594, 1:1000 (Molecular Probes # A-21207), antimouse Alexa Fluor ® 594 (Molecular Probes, # A-21203)

 BrdU labelling

BrdU labelling technique was performed as previously described (10)

References

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