p19 Ink4d and p18 Ink4c cyclin‐dependent kinase inhibitors in the male reproductive axis

The loss of the cyclin‐dependent kinase inhibitors (CKIs) p18 Ink4c and p19 Ink4d leads to male reproductive defects (Franklin et al., 1998 . Genes Dev 12: 2899–2911; Zindy et al., 2000 . Mol Cell Biol 20: 372–378; Zindy et al., 2001 . Mol Cell Biol 21: 3244–3255). In order to assess whether these inhibitors directly or indirectly affect male germ cell differentiation, we examined the expression of p18 Ink4c and p19 Ink4d in spermatogenic and supporting cells in the testis and in pituitary gonadotropes. Both p18 Ink4c and p19 Ink4d are most abundant in the testis after 18 days of age and are expressed in purified populations of spermatogenic and testicular somatic cells. Different p18 Ink4c mRNAs are expressed in isolated spermatogenic and Leydig cells. Spermatogenic cells also express a novel p19 Ink4d transcript that is distinct from the smaller transcript expressed in Sertoli cells, Leydig cells and in other tissues. Immunohistochemistry detected significant levels of p19 Ink4d in preleptotene spermatocytes, pachytene spermatocytes, condensing spermatids, and Sertoli cells. Immunoprecipitation‐Western analysis detected both CKI proteins in isolated pachytene spermatocytes and round spermatids. CDK4/6‐CKI complexes were detected in germ cells by co‐immunoprecipitation, although the composition differed by cell type. p19 Ink4d was also identified in FSH+ gonadotrophs, suggesting that this CKI may be independently required in the pituitary. Possible cell autonomous and paracrine mechanisms for the spermatogenic defects in mice lacking p18 Ink4c or p19 Ink4d are supported by expression of these CKIs in spermatogenic cells and in somatic cells of the testis and pituitary. Mol. Reprod. Dev. 74: 997–1007, 2007. © 2007 Wiley‐Liss, Inc.