Nobuhiko Okura

Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages

Leptospira interrogans is a spirochaete responsible for a zoonotic disease known as leptospirosis. Leptospires are able to penetrate the abraded skin and mucous membranes and rapidly disseminate to target organs such as the liver, lungs and kidneys. How this pathogen escape from innate immune cells and spread to target organs remains poorly understood. In this paper, the intracellular trafficking undertaken by non‐pathogenic Leptospira biflexa and pathogenic L. interrogans in mouse bone marrow‐derived macrophages was compared. The delayed in the clearance of L. interrogans was observed. Furthermore, the acquisition of lysosomal markers by L. interrogans‐containing phagosomes lagged behind that of L. biflexa‐containing phagosomes, and although bone marrow‐derived macrophages could degrade L. biflexa as well as L. interrogans, a population of L. interrogans was able to survive and replicate. Intact leptospires were found within vacuoles at 24 h post infection, suggesting that bacterial replication occurs within a membrane‐bound compartment. In contrast, L. biflexa were completely degraded at 24 h post infection. Furthermore, L. interrogans but not L. biflexa, were released to the extracellular milieu. These results suggest that pathogenic leptospires are able to survive, replicate and exit from mouse macrophages, enabling their eventual spread to target organs.

Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract

The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm(2). Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract.

Enhanced Expression of Tyrosine Kinase Receptor A in Germ Cells of Rat Testis with Acute Experimental Testicular Torsion

Objective and Methods: To examine the involvement of neurotrophic factor receptors in the testis with acute experimental testicular torsion, the expression of tyrosine kinase receptors (trk) A and B, and p75 nerve growth factor receptor (NGFR) were studied in the rat testis with ischemia/reperfusion (I/R) injury, using Western blotting and immunohistochemistry. Apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Results: There was a significant increase in TUNEL-positive reaction in spermatogonia in the seminiferous tubules in rat testes after testicular torsion. Western blot analysis showed that trk A expression reached a significant peak at 12 h after reperfusion (p < 0.01), as compared to sham-operated controls, whereas trk B was not increased in the testis after I/R. Constitutive expression of p75 NGFR was at or below the level detectable by Western blot analysis, and it remained unchanged in the testis after I/R. Immunohistochemistry demonstrated that after I/R trk A expression was increased in spermatocytes and spermatids in the seminiferous tubules, in contrast to the basal location of the TUNEL-positive reaction. Immunoreactivity of trk B was seen mainly in the interstitial cells in the sham-operated testis, and its localization was not changed after I/R. Conclusion: It is postulated that trk A and B, but not p75 NGFR, are involved differently in the survival of testicular cells during acute experimental testicular torsion. In particular, increased trk A seems to be related to germ cell survival following I/R.

Morphological and functional reconsideration of the cytoplasmic bridges which connect male germ cells in snails

Cytoplasmic bridges (CB) between male germ cells of three fresh-water snails have been examined by electron microscopy, using ultrathin sections and freeze-fracture replicas prepared by ordinary methods and after use of filipin for indicating the presence of membrane cholesterol. The bridge plasma membrane, which was formerly considered to be smooth, was found in these snails to be corrugated. The corrugations were periodically parallel and oriented parallel to the axis of the bridge. The mature bridges showed very low densities of intramembranous particles. No filipin-sterol complexes formed on either the P face or the E face of the bridge plasma membrane, in contrast to plasma membranes elsewhere. The numbers of corrugations in each CB varied with the species. The membrane corrugations overlie bundles of electron dense fibers measuring approximately 30 nm in diameter and 60 nm in center-to-center distance, fitting into convexities of the plasma membrane. The present observations lead us to the necessity of reconsidering the morphological and functional aspects of cytoplasmic bridges in vertebrate as well as in invertebrate germ cells.

Acrosome reaction of fowl sperm: Evidence for shedding of the acrosomal cap in intact form to release acrosomal enzyme

The aim of this study was to determine the site of enzyme release from the acrosome and the fate of the acrosomal cap during the process of acrosome reaction (AR) in fowl sperm. Gelatin substrate coverslips with halos were subjected to scanning electron microscopy to determine the site from which acrosomal proteolytic enzyme was released to form a halo around the acrosome of individual sperm. Aliquots of sperm treated with solubilized inner perivitelline layer (IPL) containing 5 mmol CaCl(2) were simultaneously subjected to fluorescent staining with fluorescein isothiocyanate-labeled peanut agglutinin and scanning electron microscopy to evaluate AR of sperm and to examine the status of the acrosomal region, respectively. Inside the halos, a gelatin-free (proteolyzed gelatin) layer was found extending some distance around the acrosome of sperm. All of the sperm showing the formation of halos on gelatin had a single circular opening around their subacrosomal rod at the base of the acrosomal cap. Interaction of sperm with solubilized IPL in the presence of 5 mmol CaCl(2) resulted in 41.4 ± 1.8% of the sperm to undergo AR, as evaluated by fluorescein isothiocyanate-labeled peanut agglutinin. Similarly, as observed using scanning electron microscopy, 38.2 ± 2.3% of the sperm treated with solubilized IPL plus 5 mmol CaCl(2) had exposed subacrosomal rod. In all sperm examined, no sign of disruption of the acrosomal membrane was found in the apical region of the acrosome. Rather, the acrosomal caps were found intact detached from the acrosomal region of the sperm, indicating that AR of fowl sperm resulted in the intact removal of the acrosomal cap. Based on these experimental observations, we suggest that the process of AR in fowl sperm is unique; the release of the acrosomal proteolytic enzyme may occur through a single circular opening formed at the base of the acrosomal cap and the acrosomal cap is detached in intact form from the posterior acrosomal region of the sperm.

Maturational changes in the survivability and fertility of fowl sperm during their passage through the male reproductive tract

The objective of this study was to examine whether domestic fowl (Gallus domesticus) sperm undergo maturation in their capacity for survival and fertilization in the male reproductive tract. Sperm collected from the testis, epididymis and the proximal, middle and distal vas deferens were simultaneously stored in vitro in minimum essential medium (MEM) at 39°C for 0, 3 and 6h, and at 4°C for 24 and 48h. Sperm membrane integrity was measured using the dual fluorescent stain SYBR-14/propidium iodide (PI). Aliquots of sperm from the various sites were subjected to artificial insemination (AI) into the uteri of hens to assess the duration of sperm survival in the oviduct and to determine the fertility status of the sperm. Testicular sperm exhibited a very low capacity to survive under in vitro liquid storage conditions, irrespective of the storage temperature used, and in the oviduct, and they had a low ability to fertilize the ovum. On the contrary, sperm from the distal vas deferens had a higher survival rate during in vitro storage periods, a longer life span in the oviduct, and high fertility. Survival and fertilizing capacity of the sperm recovered from the testes increased gradually (P<0.05) from the testes to the distal vas deferens. In conclusion, we suggest that fowl sperm may undergo functional maturation through a process of gradual changes in their survival and fertilization capacities during their passage through the successive parts of the male reproductive tract.

Plumping fluid added to storage medium increases twofold the functional life span of fowl spermatozoa in vitro at 4°C

Abstract 1. The objective of this study was to examine whether addition of plumping fluid (PF) to Lakes solution (LS) for storage of fowl spermatozoa in vitro at 4°C can prolong survival and improve the quality of spermatozoa. 2. In experiment 1, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 10%, 25%, 50% and 75% (v:v) PF for 0.5, 24, 48, 72, 96 and 120 h at 4°C. After the end of each storage period, spermatozoa were evaluated for their viability, mobility and penetrability. Viability was determined using SYBR-14 and propidium iodide (PI) staining. Mobility was assessed using an Accudenz assay. Penetrability was assessed using spermatozoa-inner perivitelline layer (IPL) interaction assay. 3. In experiment 2, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 25% and 50% (v:v) PF for 0.5, 24, 48 and 72 h at 4°C, and then fertility of the spermatozoa was evaluated using intravaginal artificial insemination (AI) in hens. 4. Storage of spermatozoa in LS alone resulted in loss of viability, mobility, penetrability and fertility within 48 h. In contrast, no loss of viability and penetrability was observed for the spermatozoa stored for 48, 96, 72 and 48 h in LS containing 10%, 25%, 50% and 75% (v:v) PF, respectively. In particular, fertilising capacity was not lost for the spermatozoa stored in the presence of 25% or 50% PF in LS for 48 and 24 h, respectively. 5. In conclusion, these findings demonstrated that in vitro exposure of fowl spermatozoa to PF during hypothermic storage in LS prolonged spermatozoa survival. A 25% (v:v) level of inclusion of PF in LS may be effective for the improvement of viability, penetrability and fertilising ability of the stored spermatozoa.