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.