Possible functions of P21 CDKN1A within the germ cells of the mouse seminiferous epithelium

The cyclin dependent kinase inhibitor P21 CDKN1A has been shown to be upregulated during differentiation and after DNA damage in somatic cells. We examined the expression of P21 CDKN1A and of γ-H2AX during the differentiation of germ cells in normal mouse testis. P21 CDKN1A was normally expressed in in a variable mode in different cell types (spermatogonia, spermatocytes, spermatids, Sertoli cell) located since the basal till the lumen of the seminiferous tubules. Moreover, in agreement with γ-H2AX expression, P21 maybe involved in different cell cycle checkpoints in spermatocytes such as a pre-replication, XY body inactivation, a midpachytene and a metaphase I checkpoints. Finally, in comparison to control, the p21 deficient mouse testis show elevated numbers of apoptosis of elongated spermatids within stages VIII-IX but any difference in the number of spermatogonia mitosis. These results suggest that P21 also may take part in the regulation of the differentiation of the male germ cells and may have a role in spermatogonia not related with a mitotic checkpoint.

The cyclin dependent kinase inhibitor P21 CDKN1A has been shown to be upregulated during differentiation and after DNA damage in somatic cells (1). Also, it was found that P21 is a factor which maybe important during the meiotic prophase in spermatocytes and repair mechanisms in these cells, but not in the spermatogonial cell cycle (2). Here we further study the role of P21 in the mouse seminiferous epithelium by analysing its expression in mouse testis and by performing apoptosis and proliferation studies in the mouse deficient for p21. We started by analysing the expression of P21 CDKN1A in mouse testes paraffin cuts. P21 is expressed in a variable mode in different cell types (spermatogonia, spermatocytes, spermatids, Sertoli cell) located since the basal till the lumen of the seminiferous tubules ( fig.1). Further, we counted the number of spermatogonia mitotic figures in control and in p21 deficient testis and found there is no significant difference between the number of spermatogonia mitotic figures in control and in p21 deficient testis (9) (not shown). Next, we analysed de relationships of the expression of P21 CDKN1A with those of γ-H2AX in the mouse meiotic germ cells ( fig.2). During preleptotene, when pre-meiotic replication takes place expression of P21 and γ-H2AX are similar, meaning P21 maybe halting cell cycle progression while γ-H2AX identifies sites of stalled replication forks (3). During leptotene, when meiotic doublestrand breaks (DSBs ) occur, γ-H2AX expression is maximal (4) while P21 is much lower, thus allowing cell cycle progression while meiosis is initiated. During zygotene and the zygotenepachytene transition (Z-P), γ-H2AX starts decreasing while P21 raises to a peak of expression at Z-P, when γ-H2AX only appears covering the forming X-Y body (5), suggesting that P21 plays a role in XY body inactivation. Moreover, during pachytene as γ-H2AX continues to fade, P21 shows another peak of expression where it is supposed it works a mid-pachytene checkpoint monitoring those pachytene spermatocytes with meiotic recombination defects (6,7). γ-H2AX totally disappears during diplotene while P21 peaks again at metaphase I (MI), suggesting a role for P21 during a MI checkpoint (8). Finally, we quantified apoptosis in the mouse seminiferous epithelium ( fig.3). We got to the conclusion that in the p21 deficient mouse there are more apoptosis of elongated spermatids at stages VIII and IX.

Conclusions
These results suggest that P21 may take part in the regulation of the differentiation of the male germ cells and may have a role in spermatogonia not related with a mitotic checkpoint. Also, P21 maybe involved in different cell cycle checkpoints in spermatocytes such as a pre-replication, XY body inactivation, a mid-pachytene and a metaphase I checkpoints.

Materials and methods
This study was performed within the scope of Ana Pedro's PhD Thesis approved on the 07/07/2009 by the Dean of University of Valladolid, Spain.

Mice
We used control and p21 deficient (9) mice with the same background (129sv/C57BL6; 50:50) kindly donated by Manuel Serrano, CNIO, Madrid. The mice testes were dessicated and included in paraffin blocks. Apoptosis labelling For apoptosis labelling, we used the Roche, In situ Cell Death Detection Kit, POD, # 1 684 817).

Statistics
We used Prism program. For apoptosis quantification we analysed 10 paraffin cuts from p21 deficient mouse and 10 paraffin cuts from control mouse, counter-stained with PAS and count the number of apoptosis in 30 tubules of each stage in agreement with Oakberg (1956). For mitosis, we counted the number of mitotic figures of spermatogonia in 5 non-consecutive tubules of the stages I, IV, VI, IX, X y XII both in control and p21 deficient mouse. We calculated the percentageof mitosis in relation to the average number of Sertoli cell in control and in p21 deficient mouse.