A novel N-terminal domain directs membrane localization of mouse testis-specific calpastatin

Multiple isoforms of calpastatin have been identified with unique N-terminal regions followed by identical calpain inhibitory domains (II-IV). In many instances the isoforms are cell-type specific, although the precise functional differences among these N-terminal regions are largely unknown. Here we report a germ cell-specific isoform of calpastatin (tCAST) that consists of a novel N-terminal peptide of 40 amino acids (domain T) followed by domains II to IV of somatic calpastatin (sCAST). Domain T is responsible for membrane association of tCAST through a protein modification by myristylation. Mutation of the myristylation site eliminates membrane targeting. Unlike most of the isoforms of calpastatin that are generated through alternative RNA splicing or post-translational proteolysis, the testis-specific isoform is transcribed from an intronic promoter in haploid germ cells of the testis. The intronic promoter directs specific expression of a reporter transgene in developing germ cells of the mouse testis.     

Boar proacrosin expressed in spermatids of transgenic mice does not reach the acrosome and disrupts spermatogenesis

Transgenic mice that express boar proacrosin were produced to examine mechanisms for targeting hydrolytic enzymes to the acrosome. A 2.3 kb transgene was constructed by ligating the cDNA for boar preproacrosin with the mouse protamine 2 promoter region. Six founder mice that incorporated the transgene were identified by polymerase chain reaction and Southern blot analysis. Northern blots indicated that the two male founders (Ac.2 and Ac.5) and male progeny from three female founders (Ac.3, Ac.4, Ac.6) expressed the transgene mRNA in testis, but not in somatic tissues. In these transgenic animals boar proacrosin was detected by immunohistochemistry in condensing spermatids, but was not localized in the acrosome. This acrosomal targeting defect of the transgene product may result from its delayed expression during the later steps of haploid differentiation. Furthermore, both male founders and all Ac.4 and Ac.6 males were infertile, as determined by multipe matings for at least 2 months. Ac.3 males were either infertile or rarely transmitted the transgene to their offspring The infertile males mated, produced copulatory plugs, and had seminal vesicle weights and testosterone levels within the normal range. However, they produced significantly fewer spermatozoa and had lower testis weights than controls. Although the mitotic and meiotic phases of spermatogenesis appeared normal by histological criteria, condensing spermatids were missing from most tubules, and multinucleated cells were present in the lumen of seminiferous tubules and in the epididymis. We hypothesize that boar proacrosin which fails to reach the acrosome is activated in these transgenic mice, and that its proteolytic activity disrupts spermatogenesis during spermatid formation. © 1996 Wiley-Liss, Inc.     

Hyperplasia and tumours in lung, breast and other tissues in mice carrying a RAR beta 4-like transgene.

Transgenic mice were generated which express a truncated nuclear retinoic acid receptor beta (RAR beta), closely resembling the natural isoform RAR beta 4, under the control of the MMTV promoter. The transgene was expressed in salivary gland, testis, lung and mammary tissue in two different lines. At approximately 11-14 months virtually all the transgenic mice showed hyperplasia of the lung alveolar epithelium with an excess of type II pneumocytes. Hyperplasia of the mammary alveoli and terminal ducts was also seen in some females. Salivary glands and some sebaceous glands were hyperplastic in most male transgenic mice, but only rarely in females or in non-transgenics. Primary benign and malignant tumours were more numerous in transgenic mice than in controls, with a total of 23 in 43 mice versus two in 33 non-transgenic animals. Treatment with dexamethasone to increase transgene expression resulted in exaggerated versions of the above phenotypes. Overexpression of RAR beta 4 therefore appears to predispose various tissues to hyperplasia and neoplasia, and this by contrast to the RAR beta 2 isoform, which has tumour suppressor activity. A survey of ratios of RAR beta 4:RAR beta 2 expression in human lung tumour cell lines showed an increase compared with normal lung tissue, suggesting that RAR beta 4 may play a similar role in human tumorigenesis.     

Diverse roles for sex hormone-binding globulin in reproduction

Sex hormone-binding globulin (SHBG) transports androgens and estrogens in blood and regulates their access to target tissues. Hepatic production of SHBG fluctuates throughout the life cycle and is influenced primarily by metabolic and hormonal factors. Genetic differences also contribute to interindividual variations in plasma SHBG levels. In addition to controlling the plasma distribution, metabolic clearance, and bioavailability of sex steroids, SHBG accumulates in the extravascular compartments of some tissues and in the cytoplasm of specific epithelial cells, where it exerts novel effects on androgen and estrogen action. In mammals, the gene-encoding SHBG is expressed primarily in the liver but also at low levels in other tissues, including the testis. In subprimate species, Shbg expression in Sertoli cells is under the control of follicle-stimulating hormone and produces the androgen-binding protein that influences androgen actions in the seminiferous tubules and epididymis. In humans, the SHBG gene is not expressed in Sertoli cells, but its expression in germ cells produces an SHBG isoform that accumulates in the acrosome. In fish, Shbg is produced by the liver but has a unique function in the gill as a portal for natural steroids and xenobiotics, including synthetic steroids. However, salmon have retained a second, poorly conserved Shbg gene that is expressed only in ovary, muscle, and gill and that likely exerts specialized functions in these tissues. The present review compares the production and functions of SHBG in different species and its diverse effects on reproduction.     

Cell type-specific expression of the human renin gene.

We have previously produced transgenic mice carrying the human renin gene, whose expression is regulated in a tissue-specific manner. In the present study, we further characterized expression of the transgene. Northern blot analysis showed that the human renin gene is expressed in the kidney but not in the liver of two lines of transgenic mice with 10 and 50 copies of the transgene, suggesting that the integrated copy number of the human renin gene does not influence the dominant-renal expression pattern. Immunohistochemical study using a monoclonal antibody specific for human renin demonstrated that expression of human renin in the transgenic mouse kidney is confined to the epithelioid juxtaglomerular cells. Transfection experiments indicated that the chloramphenicol acetyltransferase fusion gene containing the 3-kb upstream sequences of the renin gene is activated only in human epithelioid embryonic 293 cells derived from kidney but not in human HepG2 cells from liver. These findings suggest that transfer of the cloned renin gene into mice and in vitro cultured cell lines can give rise to cell type-specific expression.