Original research article Lonidamine transiently affects spermatogenesis in pubertal CD1 mice Maria Elsa Traina a, T , Maria Guarino a , Elisabetta Urbani a , Luciano Saso b , Patrizia Eleuteri c , Eugenia Cordelli c , Michele Rescia c , Giorgio Leter c , Marcello Spano ` c a Department of Drug Research and Evaluation, Istituto Superiore di Sanita ` , 00161 Rome, Italy b Department of Human Physiology and Pharmacology bVittorio Erspamer,Q University of Rome bLa Sapienza,Q 00161 Rome, Italy c Section of Toxicology and Biomedical Sciences, BIOTEC-MED, ENEA CR, Casaccia, 00060 Rome, Italy Received 1 February 2005; revised 22 April 2005; accepted 22 April 2005 Abstract Lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a well-known antispermatogenic drug, was studied for the first time in pubertal mice to assess its possible effects on spermatogenesis. Male CD1 mice were orally treated on Postnatal Day (PND) 28 with a single dose of LND (100 mg/kg body weight) and sacrificed on PND30, PND42, PND74 and PND123. On PND30 (48 h after dosing), severe testicular effects were evidenced in the treated animals: (a) reduction of the testicular sperm head concentration (approximately 50% of the control value); (b) changes in the spermatogenic cell type distribution (mild decrease of the elongated spermatids and S-phase cells fractions); and (c) morphological alterations of the Sertoli cell cytoplasm and germ cell exfoliation. These changes were recovered in adulthood, on PND74 and PND123. However, no effect on sperm chromatin structure was detected on the epididymal sperm of mature mice by sperm chromatin structure assay, suggesting that LND did not interfere with the process of chromatin reorganization and DNA packaging. D 2005 Elsevier Inc. All rights reserved. Keywords: Lonidamine; Male pubertal mice; Spermatogenesis; Sperm chromatin structure 1. Introduction Lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-inda- zole-3-carboxylic acid] is a well-known antineoplastic [1,2] and antispermatogenic drug [3,4]. Its mechanism of action seems to be different from that of other antispermatogenic agents. In particular, LND is capable of disrupting the inter- Sertoli–germ cell junctions, leading to premature release of germ cells [5] even if other actions could play a significant role such as the inhibition of the energetic metabolism of germ cells [6] but not of Sertoli cells [7] and the inhibition of specific chloride channels in the epididymis [8]. LND is now considered the lead compound of a new class of antispermatogenic agents [4,9,10]. It was stated that the antispermatogenic activity of LND is species specific [11–13] and that rats are more sensitive than mice to its action. So far, only Singh and Dominic [14] have studied the effect of LND in the latter species, giving 28 repeated doses to adult animals. The present study focused on mice in the pubertal age in order to evaluate both the acute and long-lasting testicular effects of a single LND administration. To assess impairment of spermatogenesis, conventional testicular endpoints have been complemented by additional methods, flow cytometric (FCM) DNA content analysis of testis cells and the sperm chromatin structure assay (SCSA) on epididymal sperm. The FCM analysis of testicular cells has become a useful tool for quantitative evaluation of the different germ cell types involved in spermatogenesis and supplies important information for the detection of testicu- lar cytotoxicity [15–20]. SCSA allows the detection of increased sperm DNA susceptibility to in situ denaturation, which indicates derangements of normal chromatin remod- eling and packaging in the nucleus of the male gamete. The induction of an aberrant sperm chromatin structure is a hallmark of germ cell damage [21]. 2. Materials and methods 2.1. Chemicals and reagents 2.1.1. Sperm count and histology Tris(hydroxymethyl) aminomethane and sodium chloride (NaCl) were obtained from Carlo Erba (Italy); Bouin liquid, 0010-7824/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2005.04.008 T Corresponding author. Tel.: +39 06 49902801; fax: +39 06 49387100. E-mail address: traina@iss.it (M.E. Traina). Contraception 72 (2005) 262 – 267