We investigated non-specific staining in a catalyzed reporter deposition (CARD) reaction and improved its blocking methods in supersensitive immunohistochemistry, based on our simplified catalyzed signal amplification (CSA) system (Hasui et al. 2002). In the CARD reaction using biotinyl tyramide, non-specific staining could be reduced by pretreatment with a casein solution or 3% bovine serum albumin (BSA)-phosphate buffer saline (PBS) with 0.1% Tween 20. In the CARD reaction using FITC-labeled tyramide, non-specific staining could be blocked by pretreatment with 0.3% BSA-PBS with 0.1% Tween 20 or 3% polyethylene glycol-PBS with 01% Tween 20. Thus, our new simplified CSA system features: 1) destruction of the endogenous peroxidase activity; 2) blocking of the nonspecific reaction of the primary antibody; 3) a primary antibody reaction; 4) blocking of the non-specific reaction of the polymer reagent by casein treatment; 5) a polymer reaction; 6) blocking of the non-specific reaction of CARD reaction by casein treatment; 7) a CARD reaction; and 8) detection of deposited tyramide. This new system proved useful for detecting an extremely low amount of antigen in the endogenous biotin-rich tissues such as the gastrointestinal tract and liver. By this method, the Ki67 antigen in the G1 phase cell cycle could be detected and a metabolic disorder of the Ki67 antigen was implicated in a carcinoid tumor in the stomach. We believe that this new simplified CSA system represents a new standard of supersensitive immunohistochemistry for use in light-microscopic investigation.

The arylhydrocarbon receptor (AhR) is a nuclear transcription factor mediating toxic effects of chemicals such as dioxins. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a member of polyhalogenated aromatic hydrocarbons family, exerts a wide-variety of toxic effects in a tissue- and species-specific manner including the reproduction process. Recently, AhR-mediated direct effects of TCDD on a cell-specific interaction with ovarian steroids have been shown. However, information regarding the effects of TCDD on the mouse implantation is limited. We therefore examined the expression and localization of AhR in the pregnant mouse uterus from 4 to 10 days of gestation (day 4 to day 10) using immunohistochemistry to investigate the effect of TCDD on uterine tissue during the peri-implantation period. Intense AhR expression was detected in the uterine vasculature throughout the periods examined. We also found that implanted blastocysts and their surrounding luminal epithelia and decidualized stroma expressed AhR on day 5. On days 6 and 7, persistent AhR expression was found in the transitional zone between the invading embryonic tissue and decidual tissue. On days 9 to 10, placental vasculature and spongiotrophoblasts displayed AhR immunoreactivity. The administration of TCDD on day 4 decreased the number of surviving implanted embryos on day 7 in a dose-dependent manner. This effect of TCDD was inhibited by the simultaneous administration of an AhR antagonist, alpha-naphthoflavone (alpha-NF). The spatio-temporal expression of AhR during the peri-implantation phase of the mouse uterus may indicate functional roles of this orphan receptor in fetomaternal interactions as well as substantiate the risk of exposure to chemicals such as dioxins during the reproductive period.

Spermatogonial stem cells (SSCs), having yet to possess decisive markers, can only be detected retrospectively by transplantation assay. It was reported recently that mouse gonocytes collected from DBA/2 and ICR neonates propagated in vitro. This cultured germ cell, named the germline stem cell (GS cell), produced functional sperm to make progeny when transplanted into recipient mouse testes. Here we show that GS cell lines can be established not only from neonatal testes but also from the testis of adult mice. We also confirmed that GS cells once transplanted into a host testis can be recovered to resume in vitro expansion, indicating that they are convertible mutually with SSCs in adult testes. Confocal laser microscopic examination showed GS cells resemble undifferentiated spermatogonia in the adult testis. This unique cell line could be useful for research in germ cell biology and applicable as a new tool for the genetic engineering of animals.

Questions persist regarding male germ stem cells and how they mature during the prespermatogenic period of testicular development. We successfully labeled the prespermatogonia with green fluorescence protein (GFP) by using Oct-4 enhancer/promoter. This study shows that GFP was specifically expressed in prespermatogonia, spermatogonia and spermatids that faithfully reproduce the endogenous expression of Oct-4. Histochemical analysis revealed that most of the TRA98-positive gonocytes are also positive for GFP. However, the frequency of GFP expressing cells out of TRA98 expressing cells decreased together with the maturation of gonocytes in the first week after birth. To compare the stem cell activity between GFP-positive and -negative populations, we performed a transplantation of sorted cells into testes from an individual population. Colonization efficiency of germ cells from a GFP-positive population resulted in a 30-fold increase in colonization compared with a GFP-negative population. Since the expression of Oct-4 in prespermtogonia correlates well with the stem activity, Oct-4 might be a crucial molecule in the stem cell property of spermatogonia but not in cell survival.

It has long been a mystery how fluid and migrating cells in the hepatic sinusoids reach lymphatic vessels in the portal tract. Here we describe previously-unknown channels that connect the space of Disse with the portal tract in the rat liver. Transmission electron microscopy was performed on livers injected with either horseradish peroxidase (HRP) or lipopolysaccharide, and scanning electron microscopy was carried out on livers macerated with KOH. Transmission electron microscopy revealed the presence of channels with collagen fibers traversing the limiting plate. A tracer study showed that HRP was in the channels as well as along inlet venules. Dendritic cells in the hepatic sinusoids or between hepatocytes of the limiting plate were also observed extending their pseudopodia through the channels in the limiting plate to the interstitial space of the portal tract. Scanning electron microscopy further showed that many channels (1-3microm in diameter) penetrated through the limiting plate independently of blood vessels and connected the space of Disse with the interstitial space of the portal tract. In addition, the portal tract possessed prelymphatic vessels that were lined with fibroblast-like cells and frequently contained dendritic cells. The initial segment of the portal lymphatic vessels opened to the interstitial tissue space. These results indicate that fluid and dendritic cells in the hepatic sinusoids probably pass through both the space of Disse and the channels traversing the limiting plate, enter the interstitial space of the portal tracts, and finally move from the prelymphatic vessels to the portal lymphatic vessels.

The formation and organization of a mammalian sperm head occurs through diverse cellular and molecular processes during spermiogenesis. Such cellular events include sequential changes in the nucleus and the acrosome-which is derived from the Golgi apparatus-in concert with prominent bundles of microtubules, the manchette. However, these complex processes are readily impaired by a variety of intrinsic and extrinsic factors, eventually causing various types of male infertility--such as teratozoospermia--which include the deformation of the acrosome and nucleus. In order to comprehend such idiopathic male infertility syndromes, it is important to clarify the mechanism involved in sperm head formation and organization. In addition to the manchette, two key structures in these events are the acroplaxome and the perinuclear theca. The acroplaxome forms the acrosome plate with periodic intermediate filament bundles of the marginal ring at the leading edge of the acrosome, and its nature has recently been characterized. The perinuclear theca, which is located in the perinuclear region in the sperm head, contains not only a cytoskeletal element to maintain the shape of the sperm head but also functional molecules leading to oocyte activation during fertilization. This review discusses recent developments regarding the formation and organization of the mammalian sperm head in relation to its relevant functions.

To understand the precise distribution of tripeptidyl peptidase I (TPP-I), a defect of which has been shown to induce late infantile neuronal ceroid lipofuscinosis, various tissues from rats and mice were analyzed using biochemical and immunohistochemical techniques. Western blot analyses showed that a protein band immunoreactive to anti-TPP-I appeared in tissue extracts of both animals at a molecular weight of approximately 47 kD. Protein levels of TPP-I differed among tissues; they were high in the rat brain, liver, stomach, kidney, thyroid and adrenal glands and in the mouse brain, stomach, kidney, and testis. The proteolytic activity of TPP-I was detectable; it differed in the tissues examined and did not always reflect the expression levels of the protein in the tissues. In particular, the TPP-I activity was low in the brains of both animals and high in the rat testis, although its protein levels were high in the former tissue and low in the latter. Double immunostaining showed the immunoreactivity for TPP-I to be well localized in granular structures of epithelial cells in renal tubules and the cerebral choroid plexus, both of which were also stained with lamp2, a lysosomal membrane protein marker, indicating that TPP-I is a lysosomal enzyme. The immunoreactivity was intense in F4/80-immunopositive macrophages/microglial cells located in various tissues including the thymus, spleen, liver, alimentary tract, and central nervous system. Although the immunoreactivity differed depending on the tissues and even within the same tissues between the species, it was detected in all tissues examined, especially in nerve cells, some types of endocrine cells, and oxyntic cells such as gastric parietal cells and bone osteoclasts. However, the immunoreactivity was faint and week in rat thyroid gland, although its protein level was high in the tissue. These lines of evidence suggest that TPP-I, a lysosomal serine proteinase, is widely distributed in rat and mouse tissues, although its expression levels vary among them.

We investigated the differential distribution of basement membrane type IV collagen a chains in the mouse brain by immunohistochemistry using a chain-specific monoclonal antibodies. Subendothelial basement membranes were found to contain alpha1 and alpha2 chains. Basement membranes surrounding smooth muscle cells on blood vascular walls were immunoreactive for alpha1 and alpha2 chains but not for alpha5 and alpha6 chains. Interestingly, the pia mater contained a thin basement membrane which was positive for alpha1, alpha2, alpha5, and alpha6 chains, suggesting that glia limitans superficialis coheres basement membranes containing [alpha1(IV)]2alpha2(IV) and [alpha5(IV)]2alpha6(IV) molecules. In contrast, capillaries always possessed thin basement membranes of [alpha1(IV)]2alpha2(IV) molecules. Cerebrospinal fluid is produced through filtration of blood at the choroid plexus, where two distinct basement membranes were detected by anti-al and anti-alpha2 antibodies. The subendothelial basement membrane appeared to consist of [alpha1(IV)]2alpha2(IV) molecules, whereas the subependymal basement membrane in the choroid plexus was strongly positive for alpha3, alpha4, and alpha5 chains, indicating that the filtering unit was composed of alpha3(IV)alpha4(IV)alpha5(IV) molecules. That the specific localizations of these molecules are shared by renal glomeruli and the choroid plexus leads us to hypothesize that the supramolecular network containing alpha3(IV) alpha4(IV)alpha5(IV) molecules may function as a permeability selective barrier.

The cortical peritubular interstitium of the normal kidney contains both fibroblasts and antigen-presenting dendritic cells. Characteristics of these interstitial cells were analyzed in an overnutrition model by electron microscopy after the cold-dehydration technique and immunohistochemistry for antigen-presenting cells. In control rats, fibroblasts and dendritic cells were clearly identified by electron microscopy on the basis of their distinct ultrastructures. Fibroblasts possessed slender cell processes, and contained an abundance of actin filament bundles occasionally anchoring to surrounding structures, whereas dendritic cells possessed irregularly-shaped cell processes with a clear cytoplasm and a paucity of actin filament bundles. In the experimental kidney from diabetic rats given a high cholesterol diet, the peritubular interstitium contained fibroblasts and vacuolated cells, and the extracellular matrices such as collagen bundles were distinctly increased compared with the control rat kidney. Immunohistochemical staining with OX6 and ED1 revealed that the peritubular interstitium in the control rat kidney contained dendritic cells, while that in the experimental rats was occupied by macrophages. The present study provides the first evidence indicating that overnutrition may dramatically affect the immune cells in nonlymphoid tissue.

We investigated the effects of the leptin and ciliary neurotrophic factor (CNTF) on gap junction formation between folliculo-stellate cells in the anterior pituitary glands of male rats. Thirty-day-old Wistar-Imamichi strain male rats were castrated, and 30 days later they received intraperitoneal injections of either human recombinant leptin or recombinant rat CNTF. They were divided into seven groups according to the injected materials: PBS as a control, either 0.04, 0.2 or 1.0 mg/kg leptin or either 0.004, 0.02, or 0.1 mg/kg CNTF. Five rats from each group were killed 1, 2, 3, 4 and 5 days after the injections, and the pituitary gland was removed from each rat. Then the specimens were prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence of gap junctions as the ratio of the number of gap junctions existing between folliculo-stellate cells per intersected follicle profile in electron photomicrographs. The administration of 1.0 mg/kg leptin and 0.1 mg/kg CNTF to castrated male rats increased the number of gap junctions between folliculo-stellate cells. These observations indicate that the formation of gap junctions within the anterior pituitary gland of male rats is under the influence of leptin and CNTF.