Megumi Moriya

Spermatozoan response to the toad egg matured after removal of germinal vesicle

Abstract The germinal vesicle (GV) was removed from toad oocytes at various times after treatment with progesterone, and enucleated eggs were inseminated under conditions that ensured fertilization of nucleated control eggs. The eggs enucleated before the initiation of GV break-down did not show genuine cleavage. Cytological examinations revealed, however, that spermatozoa enter the eggs enucleated even before the hormone treatment, without subsequent formation of pronuclei or DNA synthesis. The same lack of response was observed when several detergent-pretreated sperm were injected into enucleated eggs. When GV material was injected back into enucleated oocytes, the injected spermatozoa underwent transformation and DNA synthesis, although in variable degrees, in the egg cytoplasm. It is concluded that the egg becomes fertilizable independently of the GV during the hormone-induced maturation process. After entering the egg, however, spermatozoa require GV material for their participation in the developmental process.

MICROINJECTION OF TOAD SPERM INTO OOCYTES UNDERGOING MATURATION DIVISION

Spermatozoa of Bufo bufo japonicus were briefly treated with Triton X‐100 to remove their plasma membrane, and were injected into oocytes at various stages of maturation division. All the sperm injected into mature coelomic eggs transformed into pronuclei and synthesized DNA, as a normally fertilizing sperm does. The sperm injected into oocytes at the germinal vesicle (GV) stage did not show any change as long as the GV remained intact. In the oocytes which were induced to mature by progesterone, the injected sperm displayed characteristic features in synchrony with those of the resident female nucleus. These included the formation of several sperm‐derived chromosomes in association with multipolar spindles in the oocytes from the stage of the germinal vesicle breakdown to the first polar spindle; the appearance of swollen, vesicular nuclei without concomitant DNA synthesis in those at the stage of the first polar body emission; and the reappearance of the condensed chromosomes with giant spindles in those at the stage of the second meiotic metaphase. Pricking of these last oocytes induced the formation of several male pronuclei and DNA synthesis. These results prove that the injection of detergent‐treated sperm employed here provides an excellent means of studying the cytoplasmic state that regulates nuclear behavior.

Immuno-electron microscopic localization of calmodulin and calmodulin-binding proteins in the mouse germ cells during spermatogenesis and maturation

When extracts of mouse testis were Western-blotted against a monoclonal antibody which reacts with calmodulin in the presence of Ca2+, all calmodulin was associated with the macromolecules of molecular weight above 50 kDa. Immuno-electron microscopy of testes using this antibody indicated that calmodulin is localized at higher density in the nucleus and cytoplasm of germ cells during the developmental phase between pachytene and round spermatid, showing the highest level just before meiotic divisions. There was no special association of calmodulin to any organelles in these cells. Extremely low levels of calmodulin occurred in spermatogonia and other testicular tissue cells. Calmodulin decreased dramatically as spermatids underwent metamorphosis, becoming detectable only at the perinuclear space of sperm heads. Further relocation to the postacrosomal region occurred during sperm transit to the cauda epididymis. Immunodetection after the calmodulin overlay on ultrathin sections revealed a sharp increase of calmodulin immunogold deposits in the nuclei of spermatids accompanying their condensation. The results indicate that some calmodulin-binding proteins, but not calmodulin itself, accumulate in the nuclei during the final steps of spermiogenesis.

Immunohistochemical localization of the calcium/calmodulin-dependent protein phosphatase, calcineurin, in the mouse testis: its unique accumulation in spermatid nuclei

Immunohistochemical localization of a calmodulin-dependent protein phosphatase, calcineurin, was studied in the mouse testis in relation to previous observations showing that calmodulin is unusually rich in spermatogenic stages from mid-pachytene spermatocytes to elongating spermatids. The antibodies raised against calcineurin from scallop testis reacted with subunit B, but not subunit A, of calcineurin isoforms from mouse brain and testis. Indirect immunofluorescence using these antibodies on the mouse testis revealed positive reactions only in the nuclei of round or elongating spermatids: calcineurin started to accumulate in nuclei from the acrosomal cap phase, peaked at the initial stage of nuclear elongation, and decreased thereafter. There was almost no signal in the cytoplasm; spermatogenic cells at other stages, including spermatogonia, spermatocytes, mature sperm, and other somatic cells in the seminiferous tubules were totally negative. Immuno-electron microscopy gave the same result, on the basis of measuring the density of immunogold particles. These results suggest a role for calcineurin in remodeling of the nuclear chromatin in metamorphosing spermatids.

Immunoelectron Microscopic Localization of Sperm-specific Nuclear Basic Proteins during Spermatogenesis in Anuran Amphibians

Antisera were raised in rabbits against sperm‐specific nuclear basic proteins (SPs) of Bufo japonicus and Xenopus laevis. The localizations of these proteins in spermatogenic cells were then studied by electron microscopy with colloidal gold labeled antibodies as probes. The numbers of gold particles counted on ultra‐thin sections of cells at various spermatogenic stages were corrected for the density per unit area, on the basis of areas determined with a digitizer. No grains were deposited during early nuclear elongation stages. Grains appeared on nuclei at the beginning of chromatin granulation, and their density increased first gradually and then sharply at the last step of spermiogenesis. Recalculation of grain counts according to the estimated nuclear volumes of Bufo spermatogenic cells also indicated a sharp increase in the amount of SPs per nucleus in the last step of spermiogenesis. No significant localization of grains in the cytoplasm was observed at any stage of spermatogenesis.

Heart formative factor(s) is localized in the anterior endoderm of early Xenopus neurula

To elucidate the mechanisms of early heart morphogenesis in Xenopus laevis, we examined the effect of endoderm on heart morphogenesis in the early Xenopus neurula. Explants of anterior ventral (presumptive heart) mesoderm from early neurula were cultured alone or in combination with endoderm dissected from various regions. Heart formation was scored by an original heart index based on morphology. These explant studies revealed that anterior ventral endoderm plays a critical role in heart morphogenesis. Furthermore, we found that it was possible to confer this heart-forming ability on posterior ventral endoderm by the injection of poly(A)+ RNA from stage 13 anterior endoderm. These results imply that the heart formative factor(s) is localized in the anterior endoderm of the early neurula and that at least part of this activity is encoded by mRNA(s).

Structural analysis of functionally different smooth muscles.

SummaryThe ultrastructure of the longitudinal and circular muscle cells of the guinea pig stomach which show different contractile responses was compared. The extracellular space within the muscle bundles is about 12.1% in the longitudinal layer and about 4.4% in the circular layer. Nexuses were consistently found in the circular muscle layer but not in the longitudinal muscle layer. Numbers of both mitochondria and microtubules per unit area of smooth muscle cell are larger in the longitudinal than in the circular muscle. Cellular area occupied by sarcoplasmic reticulum is about 4.7% in the longitudinal muscle, 2.3% in the circular muscle. The numbers of caveolae are almost the same in both tissues. The most distinct difference between the two types of smooth muscle is the appearance of the thick filaments. The circular muscle cell contains approximately 50 thick filaments per 0.5 μm2 of cytoplasmic area, while the longitudinal muscle cell has only about 25 filaments which were usually much thinner than those of the circular muscle. These results indicate that the contractile apparatus itself is different in longitudinal and circular smooth muscles of the guinea pig stomach.

Function of desiccate in gustatory sensilla of drosophila melanogaster

Desiccate (Desi), initially discovered as a gene expressing in the epidermis of Drosophila larvae for protection from desiccation stress, was recently found to be robustly expressed in the adult labellum; however, the function, as well as precise expression sites, was unknown. Here, we found that Desi is expressed in two different types of non-neuronal cells of the labellum, the epidermis and thecogen accessory cells. Labellar Desi expression was significantly elevated under arid conditions, accompanied by an increase in water ingestion by adults. Desi overexpression also promoted water ingestion. In contrast, a knockdown of Desi expression reduced feeding as well as water ingestion due to a drastic decrease in the gustatory sensillar sensitivity for all tested tastants. These results indicate that Desi helps protect insects from desiccation damage by not only preventing dehydration through the integument but also accelerating water ingestion via elevated taste sensitivities of the sensilla.

Immunohistochemical detection of calmodulin and calmodulin-dependent protein kinase II in the mouse testis.

We reported previously that in mouse testis calmodulin-dependent protein phosphatase (calcineurin) is localised in the nuclei of round and elongating spermatids (Cell Tissue Res. 1995; 281: 273-81). In this study, we studied the immunohistochemical localisation of calcium/calmodulin-dependent protein kinase (CaM kinase II) using antibodies against CaM kinase IIgamma from chicken gizzard and specific antibodies raised against the amino acid sequence Ileu480-Ala493 of this enzyme, and compared it with the distribution of calmodulin. Indirect immunofluorescence was most concentrated in early spermatocytes and localised in the outermost layer of seminiferous tubules where the calmodulin level was relatively low. Measurements of immuno-gold particle densities on electron micrographs revealed that CaM kinase II is transiently increased in the nucleus of zygotene spermatocytes. These observations suggest the involvement of CaM kinase II in the meiotic chromosomal pairing process. An extremely high concentration of calmodulin in spermatogenic cells undergoing meiosis may not be directly related to activation of calmodulin-dependent kinases and phosphatases.

Purification and Partial Characterization of a Lectin-like Protein from the Sea Algae Laminaria diabolica that Induces Fertilization Envelope Formation in Sea Urchin Eggs

Abstract When unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus were treated with the extracts from the sea algae Laminaria diabolica, fertilization envelope formation (FEF) was observed in 1.5 to 2 min without subsequent cleavage. The molecular mass of the FEF-inducing protein purified from the Laminaria extracts was estimated by gel-filtration to be 120 kDa, and it induced a 100% FEF at a concentration of 525 ng/ml (4.4 nM). The FEF activity of the purified protein was completely inhibited by GlcNAc, less effectively by GlcN, and not at all by various other sugars and amino-sugars, suggesting its nature as a lectin. Normal FEF and cleavage were observed when eggs were inseminated in the presence of GlcNAc. Immunohistochemical observations using antibodies against purified protein revealed its specific localization in the cells lining the wall of mucilaginous lacunae and in secretory products in the lumen. The purified protein did not activate the pretreated with verapamil or diltiazem, while ionomycin, a Ca2+-ionophore, did induce activation of the similarly pretreated eggs. These results indicate that the Laminaria lectin triggers a very initial step in the cascade of the egg cortical reactions leading to FEF, independently of those induced by sperm.