Mice that express enzymatically inactive cathepsin L exhibit abnormal spermatogenesis.

The finding of large, stage-specific changes in secretion of procathepsin L by rat Sertoli cells has led to the hypothesis that this proenzyme promotes the survival, replication, or differentiation of spermatogenic cells. Experiments described herein used a mouse model to test this hypothesis. To prove that mice are appropriate for this purpose, we first demonstrate that mature mouse Sertoli cells express cathepsin L mRNA in the same stage-specific manner as rat Sertoli cells and they also secrete procathepsin L. To test whether catalytically active cathepsin L is required for normal spermatogenesis, we examined the testes of 110- to 120-day-old furless mice, which express catalytically inactive cathepsin L. Morphologic examination of testes of furless mice revealed both normal and atrophic seminiferous tubules. Enumeration of atrophic tubules in furless and control mice demonstrates that lack of functional cathepsin L results in a 12-fold increase in seminiferous tubule atrophy. To determine whether lack of functional cathepsin L affects the production of male germ cells in apparently normal, nonatrophic tubules, we compared numbers in control and furless mice of preleptotene spermatocytes, pachytene spermatocytes, and round spermatids per Sertoli cell. Results demonstrate that the lack of functional cathepsin L causes a 16% reduction in formation of preleptotene spermatocytes and a 25% reduction in differentiation of these cells into pachytene spermatocyte. These results suggest that procathepsin L either directly or indirectly has two distinct functions in the testis. This proenzyme prevents atrophy of seminiferous tubules and promotes the formation of preleptotene spermatocytes and the differentiation of these meiotic cells into pachytene spermatocytes.

Molecular basis of a novel rhino (hr(rhChr)) phenotype: a nonsense mutation in the mouse hairless gene.

The hairless and rhino mutations are autosomal recessive allelic mutations that map to mouse Chromosome 14. In general, the rhino phenotype is a more severe manifestation of the hairless phenotype. In both hairless and rhino mice, the hair begins shedding in a cephalocaudal pattern within 7 days after birth, and never regrows due to a series of irreversible cellular events. The hairless mutation closely resembles the human disease known as papular atrichia (MIM 209500). Recently, this disease was linked to Chromosome 8p12, the human homolog of hairless was cloned and mapped to the same locus, and mutations have been identified in several different families. In order to gain insight into the pathophysiology of disease in papular atrichia, we sought to utilize mouse mutations as in vivo model systems. In this study, we report the identification of a homozygous nonsense mutation in the coding region of the hr gene in a hairless mouse captured on a chicken farm in the Midwestern United States. To reflect the place of identification of this new mutation at the hr locus, we have designated this allele hr(rhChr) using the laboratory code Chr (Christiano).

Molecular basis for the rhino Yurlovo (hr(rhY)) phenotype: severe skin abnormalities and female reproductive defects associated with an insertion in the hairless gene.

In 1989, mice bearing mutations at the hr (hairless) locus were first proposed as a model for the human hair growth disorder papular atrichia, since in both these mice and in corresponding patients, a complete hair loss develops due to disintegration of the normal follicle structure into dermal cysts and so-called utriculi. Recently, the human hairless gene was characterized, and pathogenetic mutations were found to be associated with a recessively inherited form atrichia with papular lesions; however, the functions of hr gene remain unclear. Allelic mutations in the murine hairless gene represent a potentially powerful tool to elucidate the role of the hairless gene protein product in hair follicle physiology. In 1980, several naked animals were discovered in a breeding colony of B10.R109/Y mice maintained in the Laboratory of Experimental Biological Models (L.E.B.M., Yurlovo, Moscow District, Russia). By cross breeding with hairless HRS/J hr/hr mice, this mutation was shown to be allelic with hairless. Here, we describe the molecular basis of the hr(rhY) mutation in mice, which consists of a 13 bp insertion in exon 16 of the hr gene. Histological evaluation of Yurlovo mouse skin revealed some differences as compared to the hairless and rhino mutations, with the formation of dermal megacysts being the most specific peculiarity of the Yurlovo mutation. These results, together with previous studies of hr(rhY)/hr(rhY) mutant mice, suggest that the rhino Yurlovo (hr(rhY)) mutation represents a third and potentially more severe variation of the hairless phenotype.

Rhino-9J (hr(rh9J)): a new allele at the hairless locus.

A spontaneous mutation arose in the MRL/MpJ-Fas(lpr)/Fas(lpr) colony in which mice lost hair after completion of the first hair cycle. Hair loss was progressive from head to tail. As these mice aged, they developed wrinkled skin and long curved nails. Histologically, affected skin developed comedones (pilary cysts), deep dermal cysts, and dermal foreign body granulomas associated with rupture of the follicles. The alopecic epidermis was acanthotic. The mutation was autosomal recessive. Crosses with RHJ/Le +/hr(rhJ) (rhino-J) mutant mice yielded affected offspring with the same phenotype, verifying the presence of a new allele of rhino, a mutation at the hairless (hr) locus on mouse chromosome 14.

Fibre optic confocal imaging (FOCI) of keratinocytes, blood vessels and nerves in hairless mouse skin in vivo.

Fibre optic confocal imaging (FOCI) enabled subsurface fluorescence microscopy of the skin of hairless mice in vivo. Application of acridine orange enabled imaging of the layers of the epidermis. The corneocytes of the stratum corneum, the keratinocytes in the basal layers and redundant hair follicles were visualised at depths greater than 100 microm. Cellular and nuclear membranes of keratinocytes of the skin were visualised by the use of acridine orange and DIOC5(3). Imaging of the skin after injection of FITC-dextran revealed an extensive network of blood vessels with a size range up to 20 microm. Blood cells could be seen moving through dermal vessels and the blood circulation through the dermal vascular bed was video-taped. The fluorescent dye 4-di-2-ASP showed the presence of nerves fibres around the hair follicles and subsurface blood vessels. Comparison was made between images obtained in vivo using FOCI and in vitro scanning electron microscopy and conventional histology. FOCI offers the potential to study dynamic events in vivo, such as blood flow, skin growth, nerve regeneration and many pathological processes, in ways which have not previously been possible.

Detection of thymine dimers in suprabasal and basal cells of chronically UV-B exposed hairless mice.

An immunocytochemical method was developed to study induction and removal of DNA damage in specific cell populations in the epidermis of hairless mice during chronic ultraviolet (UV) exposure. Identification of mouse suprabasal cells was performed with an immunoperoxidase stain. This stain was shown not to affect the fluorescent nuclear stains, used to reveal DNA and DNA damage. In skin cells from hairless mice irradiated daily with 1500 J/m2 UV-B for 11 consecutive days, cyclobutane thymine dimers accumulated in epidermal cells and reached a maximum level after 3 d. Thereafter dimer levels dropped to a lower, more constant level. So epidermal cells in vivo, both suprabasal and basal cells, remove dimers effectively, in contrast to cultured rodent cells, which display hardly any repair in genomic DNA. Dimer content in suprabasal cells was higher than that in basal cells, but initially the patterns of induction and removal of dimers in both cell types were rather similar. At days 4-11, however, after the drop in dimer content, the amount of dimers in basal cells prior to UV exposure was almost as low as that in non-exposed cells. The results presented here suggest important roles for both UV-induced DNA repair and cell proliferation in protecting epidermal cells against the mutagenic and carcinogenic effects of UV.

UVA-induced ultrastructural changes in hairless mouse skin: a comparison to UVB-induced damage.

In this ultrastructural study, albino hairless mice were irradiated with long-wavelength ultraviolet (UVA) (340-400 nm) thrice weekly for 32 weeks for a cumulative dose of 8000 J/cm2. Biopsies were taken from these mice, from age-matched unirradiated controls, and from mice irradiated with UVB for 20-30 weeks with a cumulative dose of approximately 6-9 J/cm2. The most striking UVA-induced changes were 1) elastic fiber hyperplasia without evidence of fiber disintegration, 2) a large increase in randomly deposited microfibrils; 3) massive duplication of vascular basement membrane; 4) extensive endothelial cell damage; and 5) collagen fibers with smaller diameters but without apparent damage. By contrast, after UVB, the hyperplastic elastic fibers frequently appeared to be degraded. Microfibrils were only moderately increased and remained in an organized array. Also, unlike with UVA, the epidermal basement membrane was duplicated whereas that of the vessels was mainly spared. Collagen fibers showed evidence of dissolution. Thus, ultrastructural features provide further evidence that skin damage induced by UVA can be dissimilar to that induced by UVB.

Vascularization of corneas of hairless mutant mice.

During the course of experiments examining the immunobiology of corneal transplants, the corneas of athymic, nude mice (nu/nu) were found to contain blood vessels that extended through the entire superficial stroma into the centermost portion of the cornea. The presence of corneal vessels was not related to the immunodeficient condition of the nude mouse since corneas from the severe combined immunodeficiency (SCID) mutant mouse strain were avascular and indistinguishable from corneas obtained from immunocompetent BALB/c mice. Furthermore, Langerhans cells were not found to accompany the blood vessels in the corneas of any of the nude mice examined. Corneal vascularization that was similar to that seen in the nude mouse was found in the cuthymic, hairless mutant mouse strain (SKH1; hr/hr). Although vascularization of the corneal stroma was associated with the heritable loss of hair, the genes responsible for hair loss in these two mutant mouse strains reside on different chromosomes. Understanding the processes involved in either promoting or preventing corneal vascularization may have significant impact in preventing corneal allograft rejection and in controlling inflammatory diseases of the corneal surface. The two mutant mouse strains described here may serve as valuable tools for such investigations.

The hairless mouse ear for in vivo studies of skin microcirculation.

The homozygous (hr/hr) hairless mouse ear was introduced in 1980 by Eriksson and coworkers as a model for in vivo studies of the skin microcirculation. Herein we expand on this work, presenting results of in vivo microvascular parameter measurements and morphologic studies in the intact ear. The in vivo measurements include microvascular diameter, RBC velocity, capillary density, and the frequency and amplitude of arteriolar vasomotion. In connection with the in vivo studies, a detailed anatomic description of the overall and vascular anatomy is given. Additionally, the preparation techniques for carrying out these in vivo and morphologic studies in the mouse ear are presented in detail.

The characterization of squamous cell carcinoma induced by ultraviolet irradiation in hairless mice.

Squamous cell carcinomas (SCCs) induced by ultraviolet irradiation in hairless mice were characterized according to their growth, gross appearance and light and transmission electron microscopic features. SCCs arose directly from irradiated skin (ab initio) or progressed from pre-existing epidermal tumours and lesions. SCCs could be graded using guidelines established for human tumours. SCCs comprised 60.8% of the tumours examined. Of these, 35.6% were designated as grade 1, 27.7% as grade 2, 7.9% as grade 3 and 28.7% as grade 4. Spindle cell tumours suspected of being SCCs were included in grade 4. Grades 1, 2 and 3 could not be distinguished on the basis of growth and gross appearance. Those arising ab initio presented as either red, ulcerated lesions or as raised, white, verrucose lesions. Grade 4 SCCs that arose ab initio presented as rapidly growing, red, spherical lesions. Those that arose from pre-existing tumours or lesions had no characteristic appearance, and variable growth. Light microscopically, grade 4 SCCs with an obvious point of origin from epidermis or other epidermal tumours, and putative grade 4 SCCs without such a point of origin, were characterized commonly by spindle cells, pleomorphic giant or multinucleated cells and individual cell reticular fibres. Ultrastructurally, spindle cells, although poorly differentiated, were distinct from flibroblastic proliferations and had few tonofilaments or desmosomes, and were inconsistently surrounded by basal lamina-like material. On the basis of these characteristics, and despite inconclusive positivity with immunoperoxidase staining for keratin and prekeratin, it was concluded that these spindle cell tumours were most probably of identical squamous cell origin.(ABSTRACT TRUNCATED AT 250 WORDS)

UVB-induced collagen changes in the skin of the hairless albino mouse.

Biochemical techniques have been used to measure ultraviolet-B (UVB)-induced changes in dermal collagen composition. Hairless albino mice were irradiated dorsally with a daily dose of 62 mJ/cm2 UVB for 12, 24, 30, and 36 weeks. Nonirradiated controls were housed under identical conditions. Additional groups were irradiated for similar periods and kept for a further 6-24 weeks without irradiation. Skin samples were taken from dorsal and ventral (nonirradiated) surfaces and types I and III collagen were quantified densitometrically after cyanogen bromide digestion and polyacrylamide gel electrophoresis. Type III collagen was expressed as a percentage of the total types I and III collagen and the ratio of dorsal/ventral type III (D/V III) was determined for each mouse. The ratio increased significantly in irradiated animals whereas it decreased in the corresponding period in control animals. In irradiated mice withdrawn from UV exposure the ratio D/V III tended to revert to control levels. These data are in agreement with those of our previous human studies, which showed an increase in type III collagen in sun-exposed skin when compared with covered sites.