Testicular development in cynomolgus monkeys.

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.

Morphometric examination for development of reproductive organs in male cynomolgus monkeys.

We previously reported on a histological classification of cynomolgus monkey testis into six grades (1, immature; 2, prepuberty; 3, onset of puberty; 4, puberty; 5, early adult; 6, adult) based on spermatogenesis development. In this investigation, the accessory reproductive organs from the same animals underwent histomorphometric examination, in addition to being examined histologically and weighed, to evaluate relationships between these parameters and the six grades. Seminiferous tubule diameter increased corresponding to the testicular maturity grade and was notably increased at grade 6. Beginning from grade 3, increases in the areas of the ductus epididymis were noted, and reserved sperm was visible in the lumen. In the prostate, the glandular lumen area per unit area showed an increase beginning from grade 3 but no clear differences between grades 4 and 6; advanced development of epithelial height was observed at grade 6. In the seminal vesicle, development of the epithelial cell layer was markedly increased at grade 6. It was concluded that development of the male accessory reproductive organs began after reserved sperm was observed in the lumen of the ductus epididymis (grade 3) and that these organs were developed notably when the testis reached sexual maturity (grade 6).

Src homology 2 (SH2)-containing 5'-inositol phosphatase localizes to podosomes, and the SH2 domain is implicated in the attenuation of bone resorption in osteoclasts.

c-Src plays an important role in bone resorption by osteoclasts. Here, we show using wild-type and ship(-/-) osteoclasts that Src homology 2 (SH2)-containing 5'-inositol phosphatase (SHIP) appeared to negatively regulate bone resorption activated by c-Src. SHIP was found to localize to podosomes under the influence of c-Src, and the presence of either the amino-terminal region comprising the SH2 domain or the carboxyl-terminal region was sufficient for its localization. Although SHIP lacking a functional SH2 domain was still found in podosomes, it could not rescue the hyper-bone resorbing activity and hypersensitivity to receptor activator of nuclear factor-kappaB ligand in ship(-/-) osteoclasts, suggesting that the localization of SHIP to podosomes per se was not sufficient and the SH2 domain was indispensable for its function. Cas and c-Cbl, known to function in podosomes of osteoclasts, were identified as novel proteins binding to the SHIP SH2 domain by mass spectrometric analysis, and this interaction appeared to be dependent on the Src kinase activity. These results demonstrate that c-Src enhances the translocation of SHIP to podosomes and regulates its function there through the SH2 domain, leading to an attenuation of bone resorption.

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells.

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.