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ABSTRACT. Spermatogenesis and acrosomal formation in the greater Japanese shrew mole, Urotrichus talpoides, were studied by light microscopy. On the basis of acrosomal changes, morphology of spermatid head, nuclear shape, appearance of meiotic figures, location of spermatid and period of spermiation, the cycle of the seminiferous epithelium was classified into 12 stages, and developing spermatids could be divided into 15 steps. The mean relative frequencies of stages from I to XII were 10. 9, 8.7, 9.8, 7.3, 8.5, 10.3, 12.5, 8.7, 5.8, 5.4, 5.1 and 7.1%, respectively. Similar to the case in the musk shrew, the spermatid nucleus of the greater Japanese shrew mole remained in the middle region of the seminiferous epithelium and only the acrosome extended towards the basement membrane. The elongation of the acrosome, however, was not prominent. The proacrosomal vesicle first appeared in stage II and then one large and round granule was seen in stage III. The acrosomal vesicle became flattened on the surface of the nucleus in stage IV. Spreading of the acrosomic system has been recognized from stage VII. In stage VII, spermiation occurred. In stage IX, the spermatid nucleus began to elongate. Elongation and condensation of the nucleus were clearly observed in stage X. In stage XII, pachytene spermatocytes divided into diplotene spermatocytes. In stage XII, meiotic figures and secondary spermatocytes were observed. KEY WORDS: cycle of the seminiferous epithelium, greater Japanese shrew mole, insectivore, spermatogenesis, spermiation.

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Summary: The multivesicular nuclear body (MNB) within the Sertoli cell nucleolus has been observed in the ruminant testis, but not in other mammalian species. Generally, the MNB is composed of vesicles, tubules and ribosome-like structures. This study has been conducted in order to clarify MNB formation during postnatal development. The testes were obtained from immature Shiba goats at 1, 2, 3, 4, and 5 months old, and from adults. They were fixed with 5% glutaraldehyde, postfixed with 1% OsO 4 , dehydrated in ethanol and embedded in Araldite-M. The serial cross-sections of seminiferous tubules were morphologically and morphometrically observed using light and transmission electron microscopy. In these Shiba goat testes, the MNB contained vesicles and tubules in various sizes, as well as ribosome-like structures. The volume of each Sertoli cell nucleus at each age (1, 2, 3, 4, 5 months old and adult) was about 269.3 mm 3 , 327.1 mm 3 , 361.3 mm 3 , 431.2 mm 3 , 525.0 mm 3 , and 760.4 mm 3 , respectively. The average number of vesicles per Sertoli cell nucleus was 0, 7.4, 11.1, 12.3, 15.5, and 32.7, respectively. At 1 month old, one or more nucleoli with fibrillar components were identified in the Sertoli cell nucleus. No MNB was observed. At 2 months old, a MNB first appeared, though it was underdeveloped and infrequently encountered. At this stage, a MNB, consisting of a small amount of vesicles and ribosomes, was located in the peripheral region of the nucleus. At later stages (3, 4, and 5 months old), MNBs gradually developed, increased in number, moved from the periphery to the central region of the nucleus, and associated with the nucleus to form a well-developed MNB. In the adults, the Sertoli cell nucleus displayed a welldeveloped and large-sized MNB situated in the central region.

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A critical regulatory role of hematopoietic stem cell vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT Ligand. However, endothelial-derived KIT Ligand is expressed in both a soluble and membrane-bound form, and not unique to bone marrow niches and is also systemically distributed through the circulatory system. Here we confirm that upon deletion of both the soluble and membrane-bound form of endothelial-derived KIT Ligand hematopoietic stem cells are reduced in mouse bone marrow. However, deletion of endothelial-derived KIT Ligand was also accompanied by reduced soluble KIT Ligand levels in blood, precluding any conclusion as to whether the reduction in HSC numbers reflect reduced endothelial expression of KIT Ligand within HSC niches, elsewhere in the bone marrow and/or systemic sKIT Ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion specifically of the membrane bound form of KIT Ligand also reduced systemic levels of soluble KIT Ligand although with no effect on stem cell numbers, implicating a hematopoietic stem cell regulatory role primarily of soluble rather than membrane KIT Ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT Ligand, hematopoietic stem cells were unaffected in bones with deletion of KIT ligand when implanted in mice with normal systemic levels of soluble KIT Ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow.

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2335 The stepwise lineage-commitment from hematopoietic stem cells (HSCs) in the bone marrow (BM) to T-lymphocyte-restricted progenitors in the thymus represents a paradigm for how distinct stages of lineage restriction from a multipotent to a lineage-restricted progenitor require different extrinsic cues. However, the commitment stage at which progenitors migrate from the BM to the thymus remains unclear. Previous studies demonstrated the existence of adult early thymic progenitors (ETPs) restricted to T lymphocyte and granulocyte-monocyte (GM) fates (Bell and Bhandoola, Nature 2008; Wada et al., Nature 2008). The challenge remains to couple this thymic T–GM restricted progenitor to candidate thymus seeding progenitors (TSPs) identified in the BM, such as common lymphoid progenitors, lymphoid-primed multipotent progenitors (LMPPs) and HSCs, which all also possess B cell potential. Using high resolution FACS and sensitive clonal assays, we have identified the most primitive ETPs in the neonatal thymus and demonstrate at the single cell level that rare Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs possess combined granulocyte-monocyte (GM), T and B lymphocyte but not megakaryocyte-erythroid (MkE) lineage potentials, identical to closely molecularly related thymus-seeding progenitors in the BM. Moreover, global molecular profiling demonstrates that neonatal Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs cluster closest to multipotent progenitors in the BM, rather than subsequent (DN1 and DN2) progenitor stages in the thymus. Finally, in support of Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs with combined T, B and GM lineage potentials, being the most multipotent progenitors in the thymus, phenotypic and functional studies demonstrate that the neonatal thymus is not seeded by HSCs or MPPs with MkE potential. These findings establish for the first time a distinct lineage commitment stage for the transition of T-lineage commitment from the BM to the remote thymus. Disclosures: No relevant conflicts of interest to declare.