Carlos A. Suárez‐Quian

Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex

Previous studies demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) regulates steroid biosynthesis. In this study we investigate further PBR action by examining its subcellular localization in mouse adrenal gland using anti-peptide PBR antiserum and employing biotin-streptavidin peroxidase immunocytochemistry. Results demonstrated PBR immunostaining exclusively in the cortex. Within this region, however, PBR staining was homogeneously distributed in cells of the zona glomerulosa, whereas in cells of the zona fasciculata both cytoplasmic and prominent plasma membrane immunostaining was evident. Next, PBR distribution was examined using confocal microscopy. Confocal optical sections were obtained, 3-D reconstructions of these sections generated, and vertical, z-sections of the 3-D reconstruction recreated. The immunostaining pattern observed was consistent with a cell surface distribution of PBR. The demonstration of a subset of PBR at the plasma membrane may account for actions of PBR ligands not related to mitochondrial function.

Meiotic Arrest and Germ Cell Apoptosis in Androgen-Binding Protein Transgenic Mice

The fundamental role of androgen-binding protein (ABP) in spermatogenesis remains obscure after nearly 25 yr since its first characterization. In the present investigation, we used a transgenic mouse model that overexpresses rat ABP to examine the potential involvement of this protein in the regulation of processes occurring during spermatogenesis. Specifically, homozygous or heterozygous transgenic mice were analyzed in terms of spermatogenic progression, DNA fragmentation pattern, and germinal cell ploidy status. All animals homozygous for transgenic ABP exhibited an increased accumulation of primary spermatocytes and cells at metaphase with abnormal morphology and localization within the seminiferous epithelium. Analysis of DNA fragmentation by in situ techniques and agarose gel electrophoresis provided evidence for an increased occurrence of apoptosis in the transgenic animals, principally involving pachytene spermatocytes and cells at metaphase. Flow cytometric analysis of the DNA content of isolated...

Estrogen Receptor β Expression and Apoptosis of Spermatocytes of Mice Overexpressing a Rat Androgen-Binding Protein Transgene

Abstract Progression of the first meiotic division in male germ cells is regulated by a variety of factors, including androgens and possibly estrogens. When this regulation fails, meiosis is arrested and primary spermatocytes degenerate by apoptosis. Earlier studies showed that overexpression of rat androgen-binding protein (ABP) in the testis of transgenic mice results in a partial meiotic arrest and apoptosis of pachytene spermatocytes. In view of the recent localization of estrogen receptor β (ERβ) in primary spermatocytes and data suggesting the ability of ERβ to repress cellular proliferation, we tested the hypothesis that variations in the testicular steroid microenvironment caused by excess ABP produce changes in ERβ expression in this cellular type that could be associated to the meiotic arrest and, eventually, to the induction of germ cell apoptosis observed in the ABP transgenic mice. Increased levels of ERβ mRNA and protein were demonstrated in the testis of rat ABP transgenic mice compared with nontransgenic littermates by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments, Northern blotting, and Western Blotting. The major differences were found when isolated germ cells of transgenic and nontransgenic littermates were analyzed by RT-PCR. In keeping with this finding, ERβ was strongly immunolabeled in pachytene spermatocytes of rat ABP transgenic mice and localized in tubular stages in which TUNEL labeling was maximal. Confocal microscopy analysis of a fluorescent TUNEL assay and ERβ immunohistochemistry revealed that degenerating pachytene spermatocytes overexpressed ERβ. The present results are consistent with the interpretation that ERβ is associated with the events that regulate negatively the progression of meiosis or that lead to spermatocyte apoptosis.

Relationship between submandibular gland epidermal growth factor and spermatogenesis in C3H mice

Epidermal growth factor (EGF), a potent mitogen produced primarily in the submandibular gland of adult male mice, has been implicated in modulating processes known to be of vital importance in the regulation of spermatogenesis. In the present investigation we demonstrate that submandibular gland EGF from adult male mice is indeed capable of displacing radiolabeled EGF from testicular membranes. Scatchard analysis of this binding site reveals that it is of high affinity (Kd = 0.77 nM) and low capacity (Bmax = 8.15 fmol/mg protein). Cross-linking of 125I-EGF to the identical membrane preparation resulted in the SDS-PAGE/autoradiography identification of a single band at approximately 170 kDa. Next, we examined the cellular distribution of the EGF receptor in the testis using biotin-streptavidin immunoperoxidase and employing different antisera probes generated to a conserved sequence of the EGF receptor. The Scatchard and cross-linking data described above, along with the immunocytochemistry results, suggest strongly that there is only one functional binding site for EGF in the adult testis and that this receptor is present in Sertoli and Leydig cells.