Sperm release at spermiation is regulated by changes in the organization of actin- and microtubule-based cytoskeletons at the apical ectoplasmic specialization—A study using the adjudin model
The mechanism that regulates sperm release at spermiation is unknown. Herein, we utilized an animal model wherein rats were treated with adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, via oral gavage to induce premature release of elongating/elongated spermatid, followed by round spermatids and spermatocytes. Spermatid release mimicking spermiation occurred within 6-12 h following adjudin treatment and by 96 h, virtually all tubules were devoid of spermatids. Using this model, we tracked the organization of F-actin and microtubules (MTs) by immunofluorescence microscopy, and the association of actin or MT regulatory proteins that either promote or demolish cytoskeletal integrity through changes in the organization of actin-microfilaments or MTs by co-immunoprecipitation. Adjudin treatment induced an increase in the association of: (i) Eps8 (an actin barbed-end capping and bundling protein) or formin 1 (an actin nucleator) with actin, and (ii) EB1 (a MT stabilizing protein) with MT shortly after adjudin exposure (at 6 h), in an attempt to maintain spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES). However, this is followed by a considerable decline of their steady-state protein levels, replacing with an increase in association of (i) Arp3 (a branched actin nucleator that converts actin filaments into a branched/unbundled network) with actin, and (ii) MARK4 (a MT destabilizing protein kinase) with MTs by 12 h after adjudin treatment. These latter changes thus promote actin and MT dis-organization, leading to apical ES disruption and the release of sperm, mimicking spermiation. In summary, spermiation is a cytoskeletal-dependent event, involving regulatory proteins that modify cytoskeletal organization.