The complete mitochondrial genome sequence of the Canada goose (Branta canadensis).

The Canada goose (Branta canadensis) entire mitochondrial genome of a bird from Western Pennsylvania has 16,760 bp (GenBank accession number NC 007011) and has been analyzed for gene locations, length, start codon and stop codons. This genome from a bird harvested during the non-migratory season is the REFSEQ and the haplotype is designated GCC-A. There are two rRNAs, 22 tRNAs, 13 protein-coding regions, and 1 displacement loop region. The base composition of mtDNA was A (30.2%), G (15.1%), C (32.1%), and T (22.6%), so the percentage of A and T (52.8%) was slightly higher than G and C. All genes except ND6 and eight tRNA genes (Gln, Ala, Asn, Cys, Tyr, Ser, Pro and Glu) are encoded on the heavy strand. The gene arrangement is the same as most birds and differs from mammals by an inversion of the mtDNA at the connection between the D-loop and the ND5 junctions.

Decreased recognition of SUMO-sensitive target genes following modification of SF-1 (NR5A1).

SUMO modification of nuclear receptors, including the constitutively active receptor steroidogenic factor 1 (SF-1; NR5A1), is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines (Lys119 and Lys194) that reside adjacent to the DNA-binding domain (DBD) and ligand-binding domain (LBD), respectively. Surprisingly, while previous loss-of-function studies predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein was either unchanged or modestly impaired. Sumoylation at Lys194 also modestly reduced Ser203 phosphorylation. In contrast to these findings, sumoylation of the DBD at Lys119 resulted in a marked and selective loss of DNA binding to noncanonical SF-1 targets, such as inhibinalpha; this binding deficit was extended to all sites when the sumoylated human mutant (R92Q) protein, which exhibits lower activity, was used. Consistent with this result, the K119R mutant, compared to wild-type SF-1, was selectively recruited to a "SUMO-sensitive" site in the endogenous inhibinalpha promoter, leading to increased transcription. DNA binding and sumoylation of Lys119 appeared to be mutually exclusive, suggesting that once SF-1 is bound to DNA, sumoylation may be less important in regulating SF-1 activity. We propose that sumoylation of nuclear receptors imposes an active posttranslational mark that dampens recognition of SUMO-sensitive target genes to restrain their expression.

Phosphorylation of mitogen-activated protein kinase 8 (MAPK8) is associated with germ cell apoptosis and redistribution of the Bcl2-modifying factor (BMF).

Successful spermatogenesis requires that germ cells remain in physical contact with Sertoli cells until spermiation. Previous studies have shown that the Bcl2-modifying factor (BMF) is a proapoptotic protein found in many epithelial cells which, when phosphorylated by the active form of mitogen-activated protein kinase 8 (p-MAPK8), initiates apoptosis in response to loss of adhesion of the cells to their basal lamina. Based on this, we hypothesized that p-MAPK8 and BMF may play important roles in the apoptotic death of testicular germ cells in response to their detachment from Sertoli cells. Immunohistochemical analysis of the normal rat testis revealed p-MAPK8 expression in spermatocytes and elongated spermatids but not in round spermatids. This localization was opposite to that of BMF, which is expressed in round spermatids but not in spermatocytes or elongated spermatids. When freshly isolated germ cells were cultured in the absence of Sertoli cells, a condition in which there was widespread germ cell apoptosis, an increase in p-MAPK8 relative to overall MAPK8 protein, was seen by Western blot analysis. Additionally, immunocytochemical analysis showed an increase in immunoreactive p-MAPK8 in round spermatids and spermatocytes in association with BMF expression. From these correlative data, we propose that the activation of MAPK8 and redistribution of BMF may be integrally involved in the mechanism by which specific germ cells undergo programmed cell death in response to their detachment from Sertoli cells.

Aging and the brown Norway rat leydig cell antioxidant defense system.

Previous studies have shown that testosterone production by the Leydig cells of aged Brown Norway rats is reduced from the relatively high levels produced by Leydig cells of young rats and that this reduction is not secondary to decreased serum luteinizing hormone concentration. The free radical theory of aging proposes that imbalance between pro-oxidants and the antioxidant defense system ultimately results in oxidative damage to cellular processes. With this in mind, we hypothesized herein that age-related reductions in steroidogenesis by Brown Norway rat Leydig cells may be associated with the impairment of the antioxidant defense system of these cells. To begin to test this hypothesis, we compared the activities and steady-state mRNA and protein levels of the antioxidant enzymes copper zinc (CuZn) superoxide dismutase (CuZnSOD, SOD1), manganese (Mn) superoxide dismutase (MnSOD, SOD2), and glutathione peroxidase (GPx) and the levels of reduced and oxidized glutathione in Leydig cells isolated from the testes of young (4-month-old) and aged (20-month-old) Brown Norway rats. For some studies, Leydig cells were isolated separately from aged testes that either had regressed because of age-related losses of germ cells or that were nonregressed. SOD (total) and GPx activities were found to decrease significantly with age whether or not the testes were regressed. CuZnSOD and MnSOD mRNA levels decreased with aging, though the magnitude of the decreases were considerably lower than the respective decreases in enzyme activities. GPx mRNA levels also decreased, which is consistent with the decreases seen in enzyme activity. MnSOD protein expression declined with age, and to a lesser extent, CuZnSOD did as well. Reduced and oxidized glutathione also exhibited age-related reductions in cells from both normal and regressed aged testes. The age-related decreases in Leydig cell antioxidant enzyme activities, gene expression, and protein levels and in glutathione were consistent with the hypothesis that the loss of steroidogenic function that accompanies Leydig cell aging may result in part from a decrease in the fidelity of the cellular antioxidant defense system.

Protein C inhibitor expression by adult rat Sertoli cells: effects of testosterone withdrawal and replacement.

Protein C inhibitor (PCI), a member of the plasma serine protease inhibitor family, has been reported to be abundantly expressed in the seminal vesicles and testes. In this study, we examine the localization and regulation of the PCI gene and protein expression in testes and freshly isolated Sertoli cells from control rats, rats treated with luteinizing hormone-suppressive testosterone/estradiol (TE)-containing Silastic capsules for 7, 14, 28, and 56 days, and rats treated with TE for 56 days, followed by high levels of testosterone for 7 or 14 days. The administration of the TE capsules for 56 days resulted in reduced testicular testosterone, from approximately 100 ng/mL in the controls to approximately 10 ng/mL, accompanied by a 73% reduction in testicular weight. PCI mRNA levels in freshly isolated Sertoli cells were reduced by 30% and 54% following TE treatment for 28 and 56 days, respectively. When rats that had received TE capsules for 56 days were provided replacement testosterone, there was a 40% increase in PCI mRNA levels within 7 days in the absence of any change in testicular weight, and PCI mRNA levels returned to control values by 14 days. The decrease in PCI mRNA levels in TE-treated rats was paralleled by a decrease in PCI protein levels in whole testis lysates and in seminiferous tubule fluid (STF). Protease activity was significantly increased in STF following 56 days of TE treatment. Taken together, these results indicate that 1) PCI in the testis is expressed by Sertoli cells; 2) the testicular expression of PCI is responsive to intratesticular testosterone levels; and 3) protease activity within the seminiferous epithelium is elevated when intratesticular concentration is decreased, perhaps as a consequence of decreased PCI.

An ex vivo analysis of Sertoli cell actin dynamics following gonadotropic hormone withdrawal.

The receptors for the steroid hormone testosterone and the peptide hormone follicle-stimulating hormone are localized to the somatic Sertoli cell in the seminiferous epithelium. In the rat, prolonged gonadotrophic hormone withdrawal has been shown to result in substantial germ cell apoptosis. Previous studies have shown that, coincident with the loss of germ cells following hypophysectomy, the actin cytoskeleton of the Sertoli cell becomes disorganized and diffuse throughout the cell's cytoplasm. The molecular mechanisms that govern Sertoli cell actin filament dynamics in response to the loss of gonadotrophic hormones remain undefined. It was therefore hypothesized that hypophysectomy brings about a decrease in the amount of polymerized actin (F-actin) within the Sertoli cell and that this decrease is associated with changes in the expression of genes known to govern Sertoli actin dynamics. To this end, Sertoli cells were isolated from adult control and hypophysectomized rats. Sertoli cells from hypophysectomized rats were found to contain significantly less (72%) F-actin relative to untreated controls, although overall, beta-actin protein and mRNA expression remained constant. The expression levels of genes known to directly influence the amount of F-actin in cells were then examined by Northern blot analysis. Cofilin and profilin I gene expression was unaffected by hypophysectomy, whereas the expression of profilin II and espin both decreased significantly (47% and 42%, respectively). Taken together, these results suggest that, following hypophysectomy, the actin cytoskeleton of the Sertoli cell shifts to a predominantly depolymerized state, perhaps in part because of decreases in profilin II and espin gene products.

Testicular expression and distribution of the rat bcl2 modifying factor in response to reduced intratesticular testosterone.

The Bcl2 modifying factor (Bmf) is a pro-apoptotic member of the Bcl2 family of apoptosis-related proteins that has been shown to initiate apoptosis in response to the loss of attachment of cells from their basal lamina (anoikis). Experimental reduction in intratesticular testosterone concentration brings about the death of spermatids as a consequence of their sloughing from Sertoli cells. Given the role of Bmf in anoikis in other systems, we hypothesized that Bmf would be expressed in germ cells and that its expression and normal distribution might be altered under conditions that induce widespread germ cell loss. To test these hypotheses, we demonstrated that Bmf indeed is expressed in the testis and cloned the full-length rat Bmf cDNA. Immunohistochemistry revealed that Bmf is present in the subacrosomal space of postmeiotic spermatids from step 4 to 16 of spermiogenesis. To test the hypothesis that Bmf expression and distribution are altered by conditions that elicit anoikis, intratesticular testosterone was reduced by implanting Silastic capsules containing testosterone and estradiol into adult rats for 8 weeks. As hypothesized, this resulted in a significant change in Bmf distribution relative to untreated animals. In particular, Bmf exhibited a loss of its normal subacrosomal distribution, becoming redistributed throughout the cytoplasm and nucleus, and appeared in cells in which it is not normally expressed (e.g., pachytene spermatocytes). Additionally, Bmf mRNA expression increased in response to lowered testosterone. These results suggest that Bmf may well be involved in germ cell apoptosis and/or anoikis in response to decreased intratesticular testosterone concentration.

Reduced intratesticular testosterone concentration alters the polymerization state of the Sertoli cell intermediate filament cytoskeleton by degradation of vimentin.

The Sertoli cell intermediate filament cytoskeleton is composed of the type III family member vimentin. The distribution of Sertoli cell vimentin varies with the stage of spermatogenesis, with shortening of the filaments at stages VII-VIII, the stages of spermiation. Experimental reduction in intratesticular testosterone (T) concentration also results in the sloughing of advanced spermatids from the Sertoli cells, as well as in the apoptotic death of spermatocytes. We hypothesized that alteration of the distribution of Sertoli cell vimentin might play a role in the loss of germ cells that occurs in response to reduced intratesticular T. To test this hypothesis, intratesticular T was reduced by implanting LH-suppressive SILASTIC brand capsules containing T and estradiol into adult rats for 8 wk. Immunohistochemical analyses revealed that, in response to the implants, the vimentin cytoskeleton collapsed around the Sertoli cell nuclei at all stages of the cycle, losing the extensive branching and structure normally seen at most stages of the cycle. Western blots of isolated Sertoli cells revealed that protein levels did not differ significantly between control and T- and estradiol-treated rats. However, Sertoli cell fractions containing the vimentin monomer revealed that vimentin was cleaved into four to five fragments in Sertoli cells in response to the implants, suggestive of proteolysis. These results indicate that, in response to reduced intratesticular T, the vimentin cytoskeleton of the Sertoli cell collapses to a perinuclear localization, and suggest that this collapse is associated with, and perhaps caused by, the degradation of the vimentin monomer rather than by loss of its expression.