Geographical variation in the skeletal morphology of red jungle fowl.

1. The skulls and postcranial skeletons of the red jungle fowl (Gallus gallus) were compared osteometrically between the populations from North and South Vietnam, North and Central Laos and Southeast Bangladesh. The populations include the three subspecies of G. g. spadiceus, G. g. gallus and G. g. murghi and were sampled to reveal the geographical morphological variations among populations in G. gallus. 2. The morphometric characteristics of subspecies murghi could be clearly distinguished from those of the other subspecies using a canonical discriminant analysis. However, the size and shape of the skull of the gallus population from South Vietnam were not statistically different from that of the subspecies spadiceus from North Laos. The canonical discriminant scores also clearly indicated that there were morphological similarities in the skulls of the populations from North Laos and South Vietnam. 3. From the results, therefore, it is concluded that red jungle fowls do not exhibit high levels of osteometric variation between geographical localities at least within the Indochinese Peninsula. 4. This contrasts with previous studies which have described these subspecies as having various external morphological differences and have argued that zoogeographical barriers exist between the north and south areas of the Indochinese Peninsula.

Di(n-butyl) phthalate induces vimentin filaments disruption in rat sertoli cells: a possible relation with spermatogenic cell apoptosis.

Phthalate esters have been extensively used as a plasticizer of synthetic polymers. Previous studies have revealed that some phthalate esters including di(n-butyl) phthalate (DBP) induce spermatogenic cell apoptosis, although its mechanism is not yet clear. The present study describes that disruption of Sertoli cell vimentin filaments by DBP administration may relate to spermatogenic cell apoptosis. The present histopathological study revealed that a single oral administration of 500 mg/kg DBP caused progressive detachment and displacement of spermatogenic cells away from the seminiferous epithelium and sloughing of them into the lumen. Degenerative spermatogenic cells characterized by chromatin condensation were frequently observed in DBP-treated rats. Ultrastructurally, the degenerative spermatogenic cells were separated from their neighbours, and a collapse of Sertoli cell vimentin filaments was recognized in DBP-treated rats. Sertoli cell cultures showed the increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, vimentin filaments clearly showed a gradual collapse in DBP-exposed Sertoli cells in vitro. These in vivo and in vitro experiments indicate that DBP-induced collapse of Sertoli cell vimentin filaments may lead to detachment of spermatogenic cells, and then detached cells may undergo apoptosis because of loss of the support and nurture provided by Sertoli cells.

Phagocytosis plays an important role in clearing dead cells caused by mono(2-ethylhexyl) phthalate administration.

The role of phagocytosis in eliminating apoptotic spermatogenic cells caused by mono(2-ethylhexyl) phthalate (MEHP) was studied. Twenty-one-day-old C57Bl/6N male mice were given a single dose of 800 mg/kg MEHP in corn oil by oral gavage and sacrificed at 1, 3, 5, 7 and 9 days after initial exposure. At the same time, the role of phagocytosis in MEHP related apoptosis was examined using microinjection of annexin V into the seminiferous tubules of living mice. Results showed that mice treated with MEHP had a lower rate of testis weight gain (lower regression line) and a significant TUNEL-positive spermatogenic cell number compared to control. However, this incident was reversible, and the number of TUNEL-positive cells returned to normal after 9 days. Mice microinjected with annexin V and later treated with MEHP showed a large amount of TUNEL-positive cells compared to mice treated with MEHP only. This clearly proves that phagocytosis plays an efficient and highly important role in eliminating dead cells in the injured testis of mice treated with MEHP.

Disturbance of neural respiratory control in neonatal mice lacking GABA synthesizing enzyme 67-kDa isoform of glutamic acid decarboxylase.

To examine the role of GABA in the respiratory rhythm and pattern generation in neonatal mice, we analyzed the function of the respiratory control system of 67-kDa isoform of glutamic acid decarboxylase (GAD67)-deficient neonatal mice. In these mutant (GAD67-/-) mice, GABA levels in the brainstem were reduced to about 30% of those in wild-type (GAD67+/+) mice. In in vivo preparations, ventilatory parameters were analyzed by whole body plethysmography and electromyography of intercostal muscles. GAD67-/- mice exhibited abnormal respiratory patterns, i.e. irregular respiratory rhythm, and periodic gasp-like respiration followed by shallow breathing with short inspiratory duration and apnea. In in vitro GAD67-/- brainstem-spinal cord preparations, inspiratory C4 burst duration was shorter than that in GAD67+/+ preparations. Whole cell recordings revealed that activities of inspiratory neurons in the ventral medulla of GAD67-/- mice were characterized by a short depolarization period and a paucity of firing during the inspiratory phase. Superfusion of the in vitro GAD67-/- preparation with 10 microM GABA prolonged C4 burst duration and partly restored a normal pattern of inspiration, although the restoration was limited. These results indicate that reduced GABA levels during the perinatal period induce malfunction in the respiratory control system. We suggest that GABAergic transmission is not essential for basic respiratory rhythm generation but plays an important role in the maintenance of regular respiratory rhythm and normal inspiratory pattern in neonatal mice.

Vasa homolog genes in mammalian germ cell development.

Many vasa homologue genes to Drosophila vasa have been isolated in various animal species. They provide specific molecular probes to analyze the establishment and the differentiation of germ cell lineage. In mammals, the expression of VASA protein becomes detectable in PGCs at the late migrating stage. Interestingly, during spermatogenesis the intracellular localization of VASA protein is closely associated with the chromatoid body.

Decreased monocarboxylate transporter 1 in rat soleus and EDL muscles exposed to clenbuterol.

We hypothesized that a shift in muscle fiber type induced by clenbuterol would change monocarboxylate transporter 1 (MCT1) content and activity of lactate dehydrogenase (LDH) and isoform pattern and shift myosin heavy chain (MHC) pattern in soleus (Sol) and extensor digitorum longus (EDL) of male rats. In the clenbuterol-administered rats (2.0 mg x kg(-1) x day(-1) subcutaneously for 4 wk), the ratio of muscle weight to body weight increased in the Sol (P < 0.05) and the EDL (P < 0.01). Clenbuterol induced the appearance of fast MHC(2D) and decreased slow MHC(1) in Sol (13%) but had no effect on EDL. The MHC pattern of Sol changed from slow to fast type. Clenbuterol increased LDH-specific activity (P < 0.01) and the ratio of the muscle-type isozyme of LDH to the heart type (P < 0.05) in Sol. The LDH total activity of the EDL muscle was also increased (P < 0.05). Furthermore, MCT1 content significantly (P < 0.05) decreased in both Sol and EDL (27 and 52%, respectively). This study suggests that clenbuterol might mediate the shift of MHC from slow to fast type and the changes in the regulation of lactate metabolism. Novel to this study is the observation that clenbuterol decreases MCT1 content in the hindlimb muscles and that the decrease in MCT1 is not muscle-type specific. It may suggest that the genetic expressions of individual factors involving slow-type MHC, heart-type isozyme of LDH, and MCT1 are associated with one another but are regulated independently.

Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells.

To obtain a reliable molecular probe to trace the origin of germ cell lineages in birds, we isolated a chicken homolog (Cvh) to vasa gene (vas), which plays an essential role in germline formation in Drosophila. We demonstrate the germline-specific expression of CVH protein throughout all stages of development. Immunohistochemical analyses using specific antibody raised against CVH protein indicated that CVH protein was localized in cytoplasm of germ cells ranging from presumptive primordial germ cells (PGCs) in uterine-stage embryos to spermatids and oocytes in adult gonads. During the early cleavages, CVH protein was restrictively localized in the basal portion of the cleavage furrow. About 30 CVH-expressing cells were scattered in the central zone of the area pellucida at stage X, later 45-60 cells were found in the hypoblast layer and subsequently 200-250 positive cells were found anteriorly in the germinal crescent due to morphogenetic movement. Furthermore, in the oocytes, CVH protein was predominantly localized in granulofibrillar structures surrounding the mitochondrial cloud and spectrin protein-enriched structure, indicating that the CVH-containing cytoplasmic structure is the precursory germ plasm in the chicken. These results strongly suggest that the chicken germline is determined by maternally inherited factors in the germ plasm.

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development.

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.

Expression of the spermatid-specific Hsp70 antigen is conserved in mammals including marsupials.

The anatomical location of testes in mammals ranges from a location close to that observed in the embryo to a lower position usually involving a pendant scrotum. In scrotal mammals, the abdominal position of the cryptorchid testis, which elevates its temperature, is detrimental to spermatogenesis and causes infertility. Spermatocytes are sensitive but late spermatids are relatively resistant to thermal stress suggesting that the latter might be protected in some way. In general, most organisms express Hsp70 proteins, which play a crucial role in the protection of cells against thermal stress. We have found previously that the Hsc70t protein, a member of the Hsp70 family of proteins, is constitutively expressed in the late spermatids of mice. Here, we have utilized immunohistochemistry with anti-mouse Hsc70t antiserum to examine the expression of the spermatid-specific Hsp70 antigen in the testes of several mammalian species with different degrees of testes migration. Our data indicate that the antigen is conserved in the mammals including marsupials. We also examined whether antigens of Hsp70-related proteins were expressed in non-mammalian vertebrates including not only homoiothermal but also poikilothermal animals. The spermatid-specific Hsp70 antigens were not detectable in the testes of the animals examined. From results of immunohistochemistry with BRM22 monoclonal antibody which reacts broadly with Hsp70 family proteins, however, we revealed constitutive expression of antigens of Hsp70-related proteins in spermatogenic cells of the vertebrates. These results suggest that the expression of spermatid-specific Hsp70 protein may be involved in the developmental pathway during spermiogenesis in mammals rather than in thermotolerance.

The Hsp70 homolog gene, Hsc70t, is expressed under translational control during mouse spermiogenesis.

Hsc70t is a member of the Hsp70 family of genes and is constitutively expressed after meiosis in mouse spermatogenesis. Immunohistochemistry and in situ hybridization techniques were used to examine the precise localization of the Hsc70t product during the various stages of spermatogenesis. A rabbit antiserum raised againstthe mouse Hsc70t-lacZ fusion protein detected the Hsc70t protein in the late spermatid-enriched fraction after two-dimensional Western blot analyses. On histological sections, the protein appears in the cytoplasm of spermatids as they progress from step 9 to the final step of spermatogenesis. An antisense RNA probe generated from the 3' untranslated region of Hsc70t cDNA detected Hsc70t mRNA in late round spermatids from step 7 onward with the signal disappearing in spermatids at step 15. Thus, Hsc70t mRNA first appears after meiosis in haploid cells but is not translated effectively until these cells progress to the transcriptionally inactive stage which coincides with chromatin condensation. These results establish that the synthesis of Hsc70t protein is under strict translational control.

Epitopic regions for antibodies against tumor necrosis factor alpha. Analysis by synthetic peptide mapping.

The location of biologically relevant epitopes on human tumor necrosis factor alpha (hTNF-alpha) was evaluated by testing the immunoreactivity of anti-TNF-alpha antibodies against 149 sequential, overlapping octamer peptides. A goat polyclonal antibody raised against recombinant hTNF-alpha, which neutralizes hTNF-alpha biological activities, reacted with oligopeptides corresponding to hTNF-alpha residues 7-11, 17-23, 30-39, 42-49, 106-112, and 135-142. A possible assembled epitopic region (residues 25, 27, and 144) for neutralizing murine monoclonal antibodies designated 11D7G4 and 9C4G5 was deduced from the fact that they bound to tripeptides, mimicking a discontinuous epitope. These antigenic regions wer found to include of adjoin poorly conserved amino acids and they were located in the turns between beta-sheets on the surface of the molecule. Three of the sequential epitopic regions and an assembled region were closely related to the receptor binding sites proposed in several other studies. These antibodies appear to neutralize TNF-alpha activities by directly masking receptor binding sites.