Immunohistochemical study of nuclear changes associated with male germ cell death and spermiogenesis

Abstract

In a previous study on the effects of gestational and lactational exposure of para-nonylphenol on male rats, we noted in both induced and uninduced rats, that variations in cleaved caspase-3 immunostaining patterns were associated with distinct nuclear alterations in mainly basally located germ cells (spermatogonia and preleptotene spermatocytes). These were re-analysed and compared with cleaved caspase-3-labeled germ cells in the aging human and the spermatogenically active catfish testis. In the rat testes, cytoplasmic immunostaining was progressively associated with lateral compression of the nucleus, its break up into large pieces which can contain immunostained marginated chromatin masses. The pale remnants of the nucleus continued to shrink in size concomitant with the appearance of blue-purplish stained regions in the cytoplasm similar in color to the condensed chromatin in spermatids, a condition which was TUNEL-negative. These large clumps of chromatin also eventually disappeared, giving rise to cells resembling cytoplasmic ghosts, a condition which was TUNEL-positive. By contrast, the immunolabeled nuclei of human and catfish germ cells condensed into a single mass, after which they lost immunoreactivity. To exclude the possibility that these observations could reflect alterations in Sertoli nuclei, rat testicular sections were probed with a mouse anti-human GATA-4 monoclonal (MHM) antibody. The MHM was, however, the second of two GATA-4 antibodies tested, with a goat anti-mouse polyclonal (GMP) initially used to label the rat Sertoli nuclei. GMP unexpectedly, but distinctly labeled the complete development of the acrosome in the rat testis, a fortuitous finding with utility for staging of the seminiferous epithelium.

Testicular abnormalities in mice with Y chromosome deficiencies†

“…Immunohistochemistry was performed according to a standard protocol as described before [29]. The following additions were made to suit our specific needs: addition of nuclear opening step using a proteinase K digest solution (20 μg/mL Proteinase K in 10 mM Tris pH 7.5 and 5 mM EDTA) after rehydration and before antigen decloaking; blocking for endogenous avidin and biotin using a blocking kit (Life Technologies, Invitrogen, Waltham, MA, #00-4303) immediately before primary antibody incubation; and counterstaining with Mayers Hematoxylin (MHS 16-500 mL, Sigma-Aldrich, St. Louis, MO) instead of 0.5% crystal violet.…”

“…Antibodies (Supplemental Table S1) were diluted in a 10% serum (goat serum for anti-SOX9, and rabbit serum anti-GATA4) solution of phosphate-buffered saline. Sections were incubated with rabbit polyclonal anti-human SOX9 antibody (1:100, AB5535, EMD Millipore Corporation, Millerica, MA) [30] to detect Sertoli cells or goat polyclonal anti-mouse GATA-4 polyclonal antibody (1:200, sc-1237, Santa Cruz Biotechnology, Dallas, TX) [29] to detect spermatids. Although GATA-4 antibody is commonly used to detect Sertoli cells, the antibody that we have chosen has been shown to be specifically reactive to cells with a developing acrosome and has been proposed as an alternative to lectin for spermatid-based staging of the seminiferous epithelium [29].…”

“…Furthermore, males transgenic for Eif2s3x had significantly depleted round spermatid populations compared to males transgenic for Eif2s3y ( Table 2). The counts of testicular cells performed on PAS-H-stained sections [20] were verified using immunohistochemistry with anti-SOX9 antibody for Sertoli cell detection [30] and anti-GATA4 antibody for spermatid detection [29] ( Figure 1). The counts of Sertoli cells in XY control males and the counts of spermatids in males with limited Y gene contribution were similar when defined with the two approaches ( Figure 1 and Supplemental Figure S2).…”

GATA4 regulates Sertoli cell function and fertility in adult male mice☆

“…Asterisks highlight Sertoli cells that are weakly immunoreactive for GATA4, presumably reflecting recombination of a single floxed allele. The punctate perinuclear staining in germ cells reflects direct unspecific binding of the goat anti-mouse secondary antibody to acrosomal components (McClusky et al, 2009). Bar = 50 μm.…”

Sertoli cell anatomy and cytoskeleton

“…Androgen receptor (AR) in adult hamster; Wilm's tumor protein 1 (WT1) in adult mouse; GATA-binding factors: GATA1 in adult hamster; GATA4 in adult rat. WT1, modified from Wang et al [64]; GATA1, modified from Tarulli et al [73]; GATA4, modified from McClusky et al[77]. Used with permission of the publishers.…”