IMMUNOCYTOCHEMICAL LOCALIZATION OF THE YA, YC, YB-1, AND YB-2, SUBUNITS OF GLUTATHIONE S-TRANSFERASES IN THE TESTIS AND EPIDIDYMIS OF ADULT-RATS

Glutathione S-transferases (GSTs) are dimeric proteins that come from a multigene family. They can be grouped into five classes (alpha, mu, pi, sigma, theta) based on the degree of amino acid homology of their subunits. These GST isozymes are able to catalyze the conjugation of g lutathione with a wide variety of electrophiles, thereby protecting im portant cellular constituents from electrophilic attach. In the presen t study, the distribution of the Ya and Ye subunits from the alpha fam ily, as well as the Yb-1 and Yb-2 subunits from the mu gene family was examined using immunocytochemistry in the adult rat testis and epidid ymis. The results of these four GST subunits were also compared with t wo other subunits, the Yf and Yo proteins, which have already been inv estigated in our laboratory [Veri et al. (1993), J. Androl., 14:23-44; Veri et al. (in press), J. Androl.]. In the testis, Leydig cells were intensely stained for all six subunits. Within the seminiferous epith elium, Sertoli cells were reactive only for antibodies raised against the Ya, Yb-1 and Yf subunits. Among germ cells, all spermatogonia, spe rmatocytes and step 1-15 spermatids were virtually unreactive for each of the six GSTs. However, moderate to intense staining was seen over steps 16-19 spermatids with the anti-Yo and anti-Ya antibodies, and in tense staining over step 19 spermatids with the anti-Yb, and anti-Yb-2 antibodies. In the rete testis, Yf, Yo, Yb-1, and Yb-2 subunits were intensely reactive over the epithelial cells with weak staining for Yc and no staining for Ya antibodies. Interestingly, in the efferent duc ts the Yc, Yb-1, and Yf proteins were intensely reactive over ciliated cells, whereas only the Ye protein was intensely reactive over noncil iated cells. In the epididymis, immunoreactivity varied among the prin cipal and basal cells of a given epididymal region for each GST antibo dy. In the case of principal cells, several of the GSTs showed a simil ar immunostaining pattern along the tubule. Although not identical in intensity of reaction, the Yc, Yb-1, Ya and Yo proteins showed an incr ease in staining intensity from the proximal to distal segments of the epididymis. In contrast, the Yb-2 protein was intensely expressed onl y in the distal caput with weak levels throughout the rest of the epid idymis. The Yf reactivity was strongest from the distal initial segmen t to the distal caput and unreactive in the corpus and proximal cauda epididymidis. In the case of the Yb-2 subunit, reactivity was more int ense over the nucleus of principal cells than the cytoplasm in the pro ximal cauda epididymidis. Similar results were seen for Yb-1 protein o ver principal cells from the middle region of the initial segment to t he proximal caput. In the distal caput, principal cells displayed a ch eckerboard-like staining pattern with the Yb-1 and Yf proteins. A popu lation of apically located cells of the proximal initial segment was i ntensely reactive with the Yb-1 and Yo proteins and of the middle init ial segment with the Yf protein; Intense staining was also noted for s everal GSTs in basal cells. The Yf and Yc proteins were stained in bas al cells along the entire length of the epididymis. The Yb-1, Ya and Y b-2 proteins were intensely reactive over basal cells of the corpus ep ididymidis with weak to no staining in other regions. The Yo protein w as negative in basal cells. Clear cells were always unreactive. These results indicate that expression of GST proteins varies considerably a long the length of the epididymis. Changes in levels of GST expression in principal and basal cells along the length of the epididymis may r esult from the need to protect spermatozoa from a changing environment of harmful electrophiles. The results also suggest that expression of the various GSTs is complex and under the control of different regula tory factors. (C) 1995 Wiley-Liss, Inc.