Follicle-stimulating hormone (FSH; follitropin) is a pituitary glycoprotein composed of two posttranslationally modified subunits, which must properly assemble to be biologically active. FSH has been difficult to purify and to obtain in quantities sufficient for detailed biochemical studies. We have targeted FSH expression to the mammary gland of transgenic mice by using cDNAs encoding the bovine a and FSHP subunits and a modified rat f-casein gene-based expression system. Lines of bigenic mice expressing both subunits have been generated either by coinection of the subunit transgenes or by mating mice that acquired and expressed transgenes encoding an individual subunit. Up to 60 international units (15 ,sg) of biologically active FSH per ml was detected in the milk of the bigenic mice. These lines provide a model system for studying the post-transcriptional mechanisms that effect the expression and secretion of this heterodimeric hormone.Follicle-stimulating hormone (FSH; follitropin) is a member of the glycoprotein family of pituitary hormones, which includes thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and chorionic gonadotropin (CG). Like LH and CG, FSH is a gonadotropin and is composed of a common a subunit that is noncovalently linked to a hormone-specific P subunit (1, 2). FSH has been difficult to purify and to obtain in sufficient quantities for detailed biochemical studies (for a review, see ref. 3). The a and FSHI subunits are posttranslationally modified, and the nature and extent of such modifications can exert a profound effect on subunit assembly, secretion, and stability (4-6). Only heterodimers with appropriately glycosylated subunits exhibit significant biological and receptor-binding activity (5,7,8). Targeting FSH to the mammary gland of transgenic animals would, therefore, serve as a model system in which to study glycoprotein processing and secretion as well as a means to produce large quantities of FSH. A standardized source of recombinant FSH would be useful to both human and livestock fertilization programs to achieve the reproducible development of ovarian follicles.Several different milk protein-based constructs have been employed to express diverse heterologous proteins in the milk of a variety of transgenic animals (for reviews, see refs. 9-11). We have demonstrated previously that a -524/+490 minimal rat f3-casein promoter fragment can direct the expression of chloramphenicol acetyltransferase to the mammary gland (12). To determine whether the mammary gland could be used to secrete large quantities of a bioactive heterodimeric protein into milk, we have used a modified rat ,8-casein-based vector to target and express bovine FSH (bFSH) to the mammary gland and into the milk of transgenic mice. MATERIALS AND METHODSConstruction of the Transgenes. The FSH subunit cDNAs were obtained from Genzyme; a as a 730-base-pair (bp) EcoRI fragment and FSH(3 as a 560-bp EcoRI/BamHI fragment. The cDNA fragments were inserted into pUC19 (13) with the rat 13-casein -524/+49...

Lenses from normal Wistar rats and those from a Wistar strain with X-ray-induced cataract mutation were examined electron microscopically for morphological characterization and acid phosphatase (AcPase) localization. Ocular anlages of 11 through 18 days of gestation were included in the study. A late-separating or persistent lens stalk occasionally was seen in the mutant eyes at 13 days of gestation, but never was observed in eyes of normal animals. The first regular morphological lens abnormality, also observed at 13 days, consisted of an accumulation of unelongated fiber cells (fusiform cells) in the posterior of the mutant lens. Ultrastructurally, these cells contained increased amounts of polyribosomes and coagulated proteins, but lacked the microtubules characteristic of elongated fiber cells. Intercellular AcPase activity appeared in the lens epithelium of both strains of rat beginning at 13 days of gestation, but was sparse in the normal strain. Intercellular reaction product attributable to AcPase activity also was noted in persistent lens stalks. In all ages combined, 66% of the mutant lenses, but only 20% of the normal lenses displayed intercellular activity. The Golgi/GERL complex in the apical regions of the lens epithelial cells of both strains contained AcPase reaction product. Reactive coated vesicles assumed to be primary lysosomes were present in nearby cytoplasm or associated with the Golgi/GERL. It is possible that the elevated amounts of hydrolase activity found in the mutant lenses may play a role in the development of the cataract characteristic of this lens.

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Substandard ultrastructural preservation of mitochondria in rat lenses, resulting from glutaraldehyde fixation, prompted us to apply the technique of Minassian and Huang which involved the addition of 0.1% sodium azide (NaN3), an inhibitor of mitochondrial respiration, to the fixative. As had been found in other tissue types by these investigators, we determined that the presence of NaN3 greatly improved the ultrastructure of mitochondria in embryonic lenses. That the effect of NaN3 was not strictly osmotic was established by comparison of lenses fixed in azide solution with those fixed in non-azide solutions of comparable and higher osmolalities.

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