There are numerous, well-characterized histopathological endpoints for assessing testicular toxicity, including germ cell death, sloughing, multi-nucleated germ cells, Sertoli cell vacuoles, altered seminiferous tubule diameter, and atrophy (Moffit et al. 2007
). Even testis weight is a sensitive indicator of toxicity (Creasy 2002
). Toxicants with different cellular targets and mechanisms of action exhibit different endpoint responses (Moffit et al. 2007
). In adult rats, testis weight and sloughing were the most sensitive endpoints 24h after carbendazim (CBZ) exposure, germ cell death was the most sensitive endpoint 12h after mono-2-ethylhexyl phthalate (MEHP) exposure, and the presence of elongated spermatid heads in the basal half of the seminiferous tubule epithelium at stages IX-XI was the most sensitive endpoint of toxicity after exposure to 2,5-hexanedione (HD) in drinking water for 18d. This study characterizes the dose-dependence, onset, and clearance of retained spermatid heads (RSH) in the basal compartment of the seminiferous tubule after exposure to the Sertoli cell toxicant, HD.
It is important to be familiar with the spermatogenic cycle as certain histopathological events occur at specific time points in spermatogenesis. The spermatogenic cycle is a coordinated development of spermatogonia, spermatocytes and spermatids. A comprehensive review is available in Russell et al. 1990
, and will be described in brief here. This cycle has been divided into 14 stages in the rat based on histological characteristics of the germ cell components. The entire cycle takes approximately 14 days, and each stage lasts a fixed amount of time. At the beginning of the spermatogenic cycle (Stage I), elongated spermatids are near the lumen, and then move basally through stage V. These cohorts of spermatids are in crypts surrounded by ectoplasmic specializations, Sertoli cell structures consisting of adhesion proteins with interacting microtubules (MT) that assist in moving the spermatids luminally at stage VI (Russell 1993
). Spermatids reach the lumen at stage VII and the ectoplasmic specializations dissociate while the spermatids remain adherent to the Sertoli cell by the tubulobulbar complex, which is primarily made up of actin filaments. At the end of stage VIII, spermiation occurs as the tubulobulbar complex dissociates in a testosterone-dependent manner, releasing the step 19 spermatids into the lumen (Saito et al. 2000
). It is generally accepted that step 19 spermatids that fail to undergo spermiation are engulfed by Sertoli cells and transported to the basal compartment for degradation (Russell 1991
) where they are observed as RSH from stages IX-XII. The basal movement of unreleased spermatids immediately after spermiation will be referred to as spermatid head retention. At stage IX, the oldest population of spermatids commences morphological changes into elongated spermatids that will be released in the next cycle of spermatogenesis.
Retained spermatid heads have been observed after suppression of FSH or testosterone and after exposure to ethane dimethanesulfonate (EDS), boric acid, dibromoacetic acid (DBA) and sodium dichloroacetic acid (NaDCA) (Bartlett et al. 1986
; Toth et al. 1992
; Saito et al. 2000
; Linder et al. 1997
; Linder et al. 1994
; Treinen and Chapin 1991
). Selective in vivo
suppression of FSH, testosterone, or both for 1 week caused 11, 14, and 50% failure in spermiation, respectively (Saito et al. 2000
). Toxicants such as EDS destroy Leydig cells, the testosterone producing cells in the testis, thereby lowering testicular testosterone (Bartlett et al. 1986
). Three days after an injection of EDS, testosterone levels in an adult rat were undetectable and RSH were seen at stages X and XI 4d later. Similarly, exposure to boric acid decreased levels of basal testosterone after 4d of dosing and inhibited spermiation after 7d of dosing (Treinen and Chapin 1991
). Exposure to the drinking water disinfection by-product, DBA, but not NaDCA, decreased serum testosterone in rats (Linder et al. 1994
, Toth et al. 1992
). The presence of RSH was noted after exposure to either toxicant.
2,5-Hexanedione (HD), a metabolite of n-hexane, is a testicular toxicant that targets the Sertoli cell, the support cell within the seminiferous tubule. Manifestations of in vivo
HD toxicity take some time to appear as HD must form adducts with proteins, including tubulin, and alter MT dynamics, an important structural element in Sertoli cells. Tubulin purified from rats exposed to 1% HD for 2 wk exhibited decreased nucleation time and more rapid assembly into microtubules (Boekelheide 1988b
). Extensive and irreversible atrophy of the seminiferous tubule occurred in adult rats after exposure to 1% HD in drinking water for 5 weeks (Hall et al. 1991
). However, 18d of exposure to 1% HD in drinking water resulted in few histopathological changes, with the exception of an accumulation of step 19 spermatid heads in the basal compartment of stage IX-XI tubules (Moffit et al. 2007
). Testosterone levels do not change after exposure to 1% HD (unpublished data), so the retention of spermatid heads is likely caused by another mechanism, potentially the disruption of microtubules.
None of the studies that reported the observation of RSH in the basal compartment of the seminiferous tubule in adult rats investigated the dynamics of this phenomenon, nor characterized its utility and sensitivity as an endpoint for assessing toxicity. In this report, we investigate the dynamics of the retention and degradation of spermatid heads and characterize spermatid head retention as a sensitive marker for subacute HD toxicity.