During the late leptotene to the mid-pachytene stages, the number of ALKBH4rich patches decreased and localized in the periphery of the nucleus. Expression of ALKBH4 diminished during the late pachynema and diakinesis. In Alkbh4D/D mice treated with tamoxifen for one and two weeks, an overall decrease of ALKBH4-specific immunofluorescence was observed compared to the Alkbh4L/L samples. We speculated if the ALKBH4 positive structures in the nuclei could be related to nucleolar compartments, as has been reported previously for ectopically expressed ALKBH4 in cell culture studies. Zotarolimus Co-immunostaining with anti-ALKBH4 and antifibrillarin, a nucleolar marker, verified the localization of ALKBH4 in the nucleoli. Although the molecular defects leading to meiotic arrest at pachynema in Alkbh4D/D mice are unknown, the disordered axial elements seen after depletion of ALKBH4 leads us to propose that the loss of cells at the pachytene stage is related to a failure of SC formation, with subsequent activation of pachytene checkpoint control mechanisms and apoptosis. A fraction of ALKBH4depleted spermatocytes did nevertheless complete meiosis, but these surviving cells gave rise to a lower than expected number of haploid spermatids. Although we cannot exclude the possibility that the incomplete inducible knockout after Cre recombinase induction is responsible for this effect, the reduction of spermatids may indicate that lack of ALKBH4 also affects spermatogenesis beyond the pachytene stage. We have previously described ALKBH4 as a modulator of specific Udenafil actin-myosin dynamics in the cytoplasm, via regulation of the K84me1-level in actin in cell culture. Interestingly, in this study we found ALKBH4 to localize to distinct euchromatic aggregates/patches in the nucleus of spermatogenic cells and in the nucleolus of Sertoli cells, which may support the involvement of ALKBH4 in regulation of specific actin dynamics in the nucleus. These dynamics may be required for normal development of pre- and post-meiotic germ cells, via regulation of the K84me1 modification.