We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.

A subset of early-onset familial Alzheimer's disease (FAD) 1 is inherited as an autosomal dominant trait, and presenilin (PS) 1 and 2 genes in addition to ß-amyloid precursor protein (APP) gene were identified as the causative genes. PS1 is mapped to chromosome 14 (1,2), while PS2 is mapped to chromosome 1 (3-5). These PS1 and PS2 genes encode multispanned transmembrane proteins showing high degrees of homology (4,5).Both proteins are located predominantly in the endoplasmic reticulum (ER), and partly in the Golgi apparatus and other compartments (6-8), but their physiological functions remain unclear. More than 50 different mutations have thus far been identified in PS1 (9), while only two mutations have been found in PS2 (10). The residue at position 141 (Asn) in PS2, which is conserved in human and mouse PS1 and PS2, is substituted by Ile (N141I) in the Volga German kindred. Another missense mutation (M239V) in PS2 has been found in Italian FAD families (5).Although the pathogenetic mechanism how Alzheimer's disease (AD) is developed by PS mutations remains unknown, mutations of PS1 and PS2 are known to have similar effects on the production of amyloid ß-protein (Aß) 42, the initially deposited Aß species in senile plaques (11)(12)(13)(14): While Aß42 is normally secreted in much lower quantities than Aß40, these aforementioned mutations induce elevation of the Aß42 levels in cultured cells and transgenic mice (15)(16)(17)(18)(19)(20). It has also been reported that in primary neuronal cultures derived from PS1-knockout mouse embryos, Aß secretion was remarkably decreased, concomitant with the accumulation of the C-terminal fragment of APP (21). The mutation in the two particular Asp residues in the PS1-transmembrane domains induced a profound decrease in the Aß production and an increase in the levels of the C-terminal fragment of APP (22). These observations indicate that PS1 may have a direct or indirect role in the γ -cleavage of APP.We previously reported that the mutant PS2 transgenic mice showed increases in the Aß42 levels in the Tris-saline (TS)-soluble fractions in an age-dependent manner during 2 to 8 months of age (20). On the other hand, a series of Aß quantitation studies of autopsied human tissues has clearly shown that Aß42 already accumulates to a significant extent in the TS- In the present study, we sought to obtain further insight into the effects of mutant PS2on the Aß levels in the TS-insoluble, guanidine hydrochloride-solubilized fraction of the mouse brain, and to characterize the intracellular compartmentalization of insoluble Aß. EXPERIMENTAL PROCEDURESTransgenic mice -The heterozygous PS2 transgenic mice used in this study were from the previously established lines W2 (wild-type PS2 transgenic mice), and M1 and M2(N141I mutant PS2 transgenic mice) (20). Each line of transgenic mice was backcrossed to the C57BL/6J strain, and those mice carrying the PS2 transgene were selected using a transgene-specific PCR assay (20). Littermates without PS2 transgenes were used as the nontransge...

The N1411 missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken~-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five-to 15-fold higher than that of endogenous mouse PS2 mRNA. The amyloid /3-protein (A/3) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the A/342 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of A~342content in the brain.

A member of the mitogen-activated protein kinase superfamily, MAK, has been proposed to have an important role in spermatogenesis, since Mak gene expression is highly restricted to testicular germ cells. To assess the biological function of MAK, we have established MAK-deficient (Mak ؊/؊ ) mice. Mak ؊/؊ mice developed normally, and no gross abnormalities were observed. Spermatogenesis of the Mak ؊/؊ mice was also intact, and most of the mice were fertile. However, Mak ؊/؊ male-derived litter sizes and their sperm motility in vitro were mildly reduced. These data show that function of MAK is not essential for spermatogenesis and male fertility. Spermatogenesis consists of three major stages: (i) a selfrenewing stage of spermatogonia (stem cells) by mitosis, (ii) a meiotic division stage of spermatocytes, and (iii) a morphological maturation stage during which haploid spermatids become mature spermatozoa. To understand the molecular mechanisms of mammalian spermatogenesis, research approaches using other cellular systems or experimental results from other species (for example, invertebrate systems) sometimes provide useful information. Since stem cells from other types of tissues also self renew and proliferate like spermatogonia, it is quite possible that similar mechanisms control the cellular events of both reproductive and other stem cells. Indeed, similar molecular properties have been described for different stem cell systems. For example, both steel factor and its receptor, c-kit, is crucial for hematopoiesis and spermatogenesis (9). Furthermore, hematopoietic stem cells and spermatogonial stem cells express integrin molecules on the cell surface and can be isolated based on expression of specific integrins (12,19,22). Meiosis is a process unique to the germ lineage cell; however, all eukaryotes undergo meiotic cell division in special circumstances. It has been shown that molecules critical for meiotic recombination in yeast also exist in mammals and have similar functions (reviewed in reference 6).Identification and characterization of specific molecules expressed within the testis is another approach to understanding spermatogenesis at a molecular level. Many testicular proteins have been identified and partially characterized (reviewed in reference 7). A serine-threonine kinase, MAK (male germ cell-associated kinase), is one such molecule. It was originally identified by weak cross-hybridization with a tyrosine kinase gene, v-ros (16). Since the expression of MAK protein was shown to be highly restricted in testicular germ cells at and after meiosis, it has been strongly speculated that MAK plays an important role(s) in cellular processes of spermatogenesis (10,13,16).To assess the function of MAK in spermatogenesis and male reproductive physiology, we generated Mak Ϫ/Ϫ mice by homologous recombination in embryonic stem (ES) cells and characterized their reproductive processes, including spermatogenesis and fertility. MATERIALS AND METHODS Establishment of Mak knockout ES cells and mice.A DNA fragment c...

The mammalian fertilization process takes place in a complex microenvironment within the female genital tract. A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), has been identified in oviductal fluid from various mammalian species, including humans. Although OGP is widely believed to be involved in the process of mammalian fertilization, including spermatozoon function and gamete interactions, based on experimental results obtained in vitro, its physiological significance remains controversial. The present study established OGP gene-null ( ogp (-/-)) mice, and primarily characterized their reproductive properties to study the physiological function(s) of OGP. Results obtained from studies using an in vivo or in vitro system showed that the fertility of ogp (-/-) females was within normal limits. These results indicate that OGP is not essential for the process of in vivo fertilization, at least in mice.

One of the most characteristic clinical features in cutaneous leishmaniasis is the development of nodules followed by ulcerations at the site of infection. Leishmania amazonensis-infected mice show similar ulcerative lesions. Leishmania-infected severe combined immunodeficiency (SCID) mice, however, have been shown to develop nonulcerative nodules. In the present study, the roles of T cells in ulceration were examined using SCID mice in cell reconstitution experiments. After development of nonulcerative nodules, SCID mice were inoculated with splenocytes from either Leishmania-infected or naive immunocompetent mice, resulting in ulceration in all mice. When naive splenocytes were depleted of CD4 ؉ , CD8 ؉ , or B220 ؉ cell populations and the remaining cells were injected into Leishmania-infected SCID mice after the development of nodules, only SCID mice inoculated with splenocytes depleted of CD4 ؉ cells did not show ulceration. The evidence obtained in this study clearly shows that the CD4 ؉ cell population is indispensable for ulceration in leishmaniasis lesions of SCID mice.

The prophylactic effect of FK463, a new water-soluble echinocandin-like lipopeptide with inhibitory activity against 1,3-␤-D-glucan synthase, against Pneumocystis carinii infection was investigated with the severe combined immunodeficient (SCID) mouse model. Treatment with FK463, pentamidine, and saline only was performed for 6 weeks from the day after the SCID mice were inoculated intranasally with infected lung homogenates. FK463 at 0.2 or 1.0 mg/kg of body weight, pentamidine at 4 mg/kg, or saline was subcutaneously administered daily into the backs of the SCID mice. The effects of the drugs were evaluated by detection of P. carinii cysts in mouse lung homogenates by toluidine blue O staining, lung histology, and PCR amplification of a P. carinii-specific DNA fragment from the lungs. P. carinii cysts were detected in the lungs of all mice administered saline. In contrast, no cysts were detected in mice administered both doses of FK463 and pentamidine. A specific DNA fragment was amplified from all mice administered saline and at least half or more of the mice administered FK463 and pentamidine. These results indicate that FK463 acts on cyst wall formation but not on trophozoite proliferation and is extremely effective in preventing P. carinii-associated pneumonia. These results suggest that FK463 is potentially useful as a prophylactic agent against P. carinii infection.Pneumocystis carinii is an opportunistic pathogen, and P. carinii-associated pneumonia (PCP) is a frequent cause of morbidity and mortality in immunocompromised patients with and without AIDS. Since the first report of pentamidine by Ivady and Paldy in 1958 (9), several effective chemotherapeutic regimens have become available for the treatment and prophylaxis of PCP. However, conventional therapy such as that with trimethoprim-sulfamethoxazole or parenteral pentamidine is often complicated by adverse reactions in AIDS patients that may require termination of the therapy, and the mortality rate for first episodes of PCP is still 5 to 20% (8). Therefore, special attention is focused on the treatment and prophylaxis of PCP for the current management of human immunodeficiency virus infection (2,5,15).Since the development of alternative drugs that do not cause adverse reactions is necessary, a new strategy to develop an anti-P. carinii drug that interacts with a target not found in other eukaryotic cells has been attempted (4). Such a drug might overcome the adverse reactions caused by conventional chemotherapeutic regimens which act on fungi as well as mammalian cells. This strategy involves selective inhibition of the biosynthesis of important structural elements in the fungal cell. On the basis of this strategy, echinocandins and pneumocandins, inhibitors of the synthesis of 1,3-␤-glucan, a major surface component of fungi including P. carinii, have been developed as potential anti-P. carinii drugs (1, 23). Iwamoto et al. (10, 11) isolated water-soluble echinocandin-like lipopeptides produced from Coleophoma empetri and reported that they are ...

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.