Seasonal changes in the histochemical properties of the olfactory epithelium and vomeronasal organ in the Japanese striped snake, Elaphe quadrivirgata.

Seasonal changes in the histochemical properties of the vomeronasal and olfactory epithelia of the Japanese striped snake were examined in four seasons, viz. the reproductive, pre-hibernating, hibernating and post-hibernating seasons. In the vomeronasal and olfactory supporting cells, secretory granules were much more abundant in the hibernating season than in the other seasons. In the vomeronasal and olfactory receptor cells, the lipofuscin granules were much fewer in the post-hibernating season than in the other seasons. In histochemical studies with 21 lectins, several lectins stained the vomeronasal and olfactory epithelia (receptor cells, supporting cells and free border) more weakly in the hibernating season than in the reproductive season. However, all lectins stained both epithelia in the hibernating season after sialic acid removal in a similar manner as in the reproductive season after sialic acid removal. These lectin histochemical studies indicate that sialic acid residues in the vomeronasal and olfactory epithelia are more numerous in the hibernating season than in the reproductive season. The results suggest that during hibernation, the vomeronasal and olfactory receptor cells possibly undergo rapid cell turnover, and that during this time, the vomeronasal and olfactory epithelia are securely protected from pathogens by an innate immune defence system.

Lectin histochemical analysis of the olfactory bulbs in the barfin flounder (Verasper moseri).

Several lines of evidence have shown that the olfactory system of the fish contains the main and accessory olfactory systems. However, morphological data indicate that the accessory olfactory bulb, the primary centre for the accessory olfactory system, will not differentiate in the fish. Therefore, the fish olfactory bulb is supposed to engage in both main and accessory olfactory systems. To examine this possibility, we investigated the olfactory bulb of the barfin flounder (Verasper moseri) by histochemical examination using lectins. The olfactory bulb of the barfin flounder showed a laminar structure with four layers, and diffuse glomerular architecture was observed in the glomerular layer. Based on the expression patterns of sugar residues, the glomerular layer of the barfin olfactory bulb was largely divided into three portions. Heterogeneity in the lectin-binding pattern among olfactory glomeruli was clearly demonstrated by the fluorescent double-lectin staining. The results of this study suggest that the fish olfactory bulb contains both regions equivalent to the main and accessory olfactory bulbs, and they are subdivided into small subsets with different functions.

Distribution patterns of uterine glands and embryo spacing in the mouse.

To clarify the mechanism of implantation, relationship between positioning of the mouse embryo in the uterus and distribution of uterine glands along the long axis of the uterine horn was examined by three-dimensional remodelling of the uterine endometrium. There were two unique regions in the endometrium. Uterine glands were distributed widely from mesometrial to anti-mesometrial side in one region. It was localized from lateral to anti-mesometrial side in another. These different regions were alternately aligned throughout the uterine horn. The number and position of embryos was consistent with that of the latter region. This study suggests that the type of distribution of uterine glands is closely related to the positioning of the embryo in mice.

Upregulation of the expression of Fas antigen and Fas ligand in a human submandibular gland ductal cell line by okadaic acid.

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells. The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells.

Characterization of cytoplasmic dynein light-intermediate chain isoforms in rat testis.

We obtained three kinds of novel cytoplasmic dynein light-intermediate chain (LIC) isoforms from rat testis and brain by reverse transcription polymerase chain reaction (RT-PCR). The primers for RT-PCR were designed according to LIC-2 (LIC 53/55) (15). In one novel isoform, the 42 bp specific sequence named TDL was inserted between 1,106 and 1,107 nucleotides (nts) of LIC-2, whereas the 57 bp sequence corresponding to LIC-2 1,339-1,395 nts (BDL) was absent. The TDL and BDL regions were specifically digested with restriction enzymatic treatment and followed by subcloning of non-digested cDNA band in testis and brain, producing an isoform without TDL and BDL regions. BDL specific RT-PCR of testis cDNA followed by sequencing produced an isoform with two specific regions. By Northern blot hybridization using TDL and BDL specific antisense oligo DNA probe, 4.4, 3.5, and 2.0 kb of signals were detected. With both TDL and BDL probes, the 2.0 kb signal was intensely detected in testis, while the 4.4 kb was defected in brain. This indicates that TDL and BDL are derived from the same size of mRNAs. In situ hybridization method using these probes showed that all seminiferous epithelial cells, especially late pachytene spermatocytes, were positive, indicating that LIC 53/55 isoforms were coexpressed in these cells. These findings indicate that LIC 53/55 isoforms provide a variety of dynein subunits, and thus may regulate the dynein-dependent intracellular transport system.

Localization of cytoplasmic dynein light-intermediate chain mRNA in the rat testis using in situ hybridization.

Expression of cytoplasmic dynein light-intermediate chain mRNA in the rat testis was examined using in situ hybridization. The ribonucleotide probe, referred to the 5' end of open reading frame (6-515 nucleotids) of cytoplasmic dynein light-intermediate chain 53/55 (LIC-2) of the rat brain (Hughes et al., 1995. J. Cell Sci., 108: 17-24), was used. All spermatogenic cells were positive. Pachytene spermatocytes in later stages (after-stage VII) were the most intensely positive and round spermatids were also intense. These findings indicated that all spermatogenic cells may store the light-intermediate chain signal, and spermatocytes may produce it during later stages. The reaction in Sertoli cells was constant in intensity during the spermatogenic cycle, indicating that the light-intermediate chain mRNA signal may have no relation to the stage-dependent organelle transport, and that there may be post-translational regulation of the light-intermediate chain. In interstitium, only a few positive cells were observed. Northern blot hybridization demonstrated that one major band (2.0 kb) and two minor bands (4.4 kb and 3.5 kb) were detected in the testis, while one major band (4.4 kb) and one minor band (3.5 kb) were in the brain. This indicated that there are at least 3 isoforms in cytoplasmic dynein light-intermediate chain 53/55.

Monoclonal antibody TSd-1 is specific to elongating and matured spermatids in testis of common tree shrew (Tupaia glis).

The monoclonal antibody (MAb), named TSd-1, specific to spermatogenic cells of the common tree shrew (Tupaia glis) was established and characterized using immunohistochemistry and immunoblotting. MAb TSd-1 reacted with elongating and matured spermatids in a stage-dependent manner. TSd-1 recognized a 94 kilodalton (kDa) peptide in the plasma membrane and cytosol. Additionally, an extremely weak 107 kDa band was detected only in the cytosol. The reactions were not detected in round spermatids. In elongating Stage VI spermatids, the plasma membrane and the granular structure within the cytoplasm were intensely positive, and most intense after the appearance of new round spermatids in the lower layer (Stage I). The reactions were observed neither in the other organs of the common tree shrew nor in the testes of other animals, indicating that TSd-1 antigen is specific to the spermatogenic cells of the common tree shrew, and may act on elongating or matured spermatids.

Cloning and sequencing of the mouse cDNA encoding a phospholipid hydroperoxide glutathione peroxidase.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenoprotein, reduces the hydroperoxides of phospholipid, cholesterol, and cholesteryl ester in biomembranes. In this study, a full-length cDNA clone encoding the PHGPx was isolated from mouse testes using a RACE (rapid amplification of cDNA ends) technique. According to sequence analysis, the cDNA encodes a polypeptide of 197 amino acids (aa) that initiates the translation at ATG(145-147) and contains an inframe TGA selenocysteine codon. It also has selenocysteine insertion sequences in the 3'-UTR that are involved in the insertion of selenocysteine at an opal codon. Moreover, the mouse PHGPx contains the active-site residues Gln108 and Trp163 that interact with selenocysteine, and the N-terminal 27-aa residues that may act as a potential mitochondrial targeting signal. According to the deduced aa analysis, mouse PHGPx shares a high level of aa identity with pig (93.4%), human (92.9%), and rat (98%) PHGPxs. However, the PHGPx mRNA particularly showed a high degree of expression in testis. This suggests that the PHGPx in testis may have more than just an antioxidant function.

Testicular morphology of a greater Indian rhinoceros (Rhinoceros unicornis).

The testis of a greater Indian rhinoceros (Rhinoceros unicornis) was examined by naked eyes and light microscopy. The animal sampled was estimated to be 42 years old. Testis was ellipse-shaped and weighed 1,300 g. Although a number of elongated spermatids were distinguishable in some seminiferous tubules, the lumen of seminiferous tubules was closed and connective tissues conspicuously increased in amount in the intertubular space. These findings in testicular morphology of the animal may be due to ageing.