Takayuki Mōri

Bisphenol A-induced apoptosis of cultured rat Sertoli cells

Bisphenol A (BPA) was examined for its effects on cultured Sertoli cells established from 18-day-old rat testes. We demonstrated that exposure of cultured Sertoli cells to BPA decreased the cell viability in a dose- and a time-dependent manner and that exposure to BPA brought about morphologic changes of the cells, such as membrane blebs, cell rounding, cytoskeletal collapse, and chromatin condensation or fragmentation, all of which conform to the morphologic criteria for apoptosis. Immunocytochemistry showed that active caspase-3, a major execution caspase, was expressed in round Sertoli cells positively labeled by the TUNEL method. Co-localization of active caspase-3 and aggregated actin fragments was also observed in the round Sertoli cells. Theses results suggest that BPA induces cell death of Sertoli cells by promoting apoptosis. Apoptosis-inducing cell death was observed in cells exposed to 150-200 microM BPA, while BPA at <100 microM had only slight cytotoxic effects on the cells.

Spermatozoa of the Shrew, Suncus murinus, Undergo the Acrosome Reaction and Then Selectively Kill Cells in Penetrating the Cumulus Oophorus

Abstract In the musk shrew, Suncus murinus (and other shrews), the cumulus oophorus is ovulated as a discrete, compact, matrix-free ball of cells linked by specialized junctions. In examining how they penetrate the cumulus, Suncus spermatozoa were observed to first bind consistently by the ventral face over the acrosomal region to the exposed smooth surface of a peripheral cumulus cell. This was apparently followed by point fusions between the plasma and outer acrosomal membranes. Thereafter, spermatozoa without acrosomes were observed within cumulus cells that displayed signs of necrosis, as did some radially neighboring cumulus cells linked by zona adherens and gap junctions. Eventually, penetration of spermatozoa as far as the perizonal space around the zona pellucida left linear tracks of locally necrotic cells flanked by normal cumulus cells. Based on these and previous observations, we conclude that the acrosome reaction in Suncus is always induced by cumulus cells, and that reacted spermatozoa penetrate the cumulus by selective invasion and killing of cumulus cells along a linear track. Loss of the acrosome also exposes an apical body/perforatorium that is covered with barbs that appear to assist reacted fertilizing spermatozoa in binding to the zona pellucida. Because fertilized eggs displayed no other spermatozoa within or bound to the zona, an efficient block to polyspermy must prevent such binding of additional spermatozoa.

Complementary DNA Cloning and Characterization of Rat spergen-2, a Spermatogenic Cell-Specific Gene 2 Encoding a 56-Kilodalton Nuclear Protein Bearing Ankyrin Repeat Motifs

Abstract Differential display in combination with a cDNA cloning approach were used to isolate a novel gene, spergen-2, which has an open reading frame of 1500 nucleotides and encodes a protein of 500 amino acids that contains ankyrin repeat motifs and a putative nuclear localization signal. Expression of spergen-2 is developmentally upregulated in testis. In situ hybridization revealed that spergen-2 mRNA is expressed in spermatocytes and round spermatids (steps 1–6). Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spergen-2 protein is predominantly expressed in nuclei of late spermatocytes (stages IX–XIV) and spermatids (steps 1–11), indicating the restricted expression of spergen-2 during spermatogenesis. In nucleoplasm of spermatogenic cell nuclei, spergen-2 tends to localize in the interchromosome space with relatively low DNA density. These findings indicate a potential role of spergen-2 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermatid differentiation.

Functional morphology of Gené's organ inHaemaphysalis longicornis (Acari: Ixodidae)

The structure and function of Genés organ, which secretes the egg wax, were investigated inHaemaphysalis longicornis. The hatching rates of eggs deposited before and after the prevention of the eversion of Genés organ were compared at the conditions of 30°C, 100% RH and soaking in liquid paraffin. The movement of Genés organ was observed, and the structures of this organ in unfed and ovipositing females were examined with both light and scanning electron microscopes.The hatching rates of eggs without contact with Genés organ were markedly lower (4.9%) than that of normal eggs (94.5%) even at the suitable condition for eggs (30°C, 100% RH). In contrast, the hatching rates of eggs soaked in liquid paraffin without contact of Genés organ were considerably higher (71.8%) than that of eggs without both Genés organ contact and soaking in liquid paraffin.When oviposition commenced, the camerostome of the female was widened by a downswing of the capitulum, the paired horns of Genés organ emerged through it, and the oviposited egg from the genital aperture was coated with egg wax. At the same time of retraction of the horns which grip an egg, the capitulum was swung up and the egg was loaded on the scutum.Genés organ included the outer and inner cuticles, and the outer one is highly folded like bellows when the horns of this organ were retracted into the haemocoel. The undeveloped glands lying in the peripheral region of Genés organ in the unfed stage commenced their enlargement at the time of the onset of feeding, and they became large tubular glands in the 1-day ovipositing female. The columnar glandular cells of the tubular glands extended their domed apical region into the lumen and contained numerous secretory granules during oviposition.These results suggest that the tubular glands secrete the egg wax which is indispensable for egg survival by serving as a waterproofing agent.

Sperm invasion of the oviducal mucosa, fibroblastic phagocytosis and endometrial sloughing in the Japanese greater horseshoe bat, Rhinolophus ferrumequinum nippon

SummaryIn the Japanese greater horseshoe bat, Rhinolophus ferrumequinum nippon, a vast number of spermatozoa invade the oviducal epithelial cells of the caudal isthmus and become concentrated in the endometrial lamina propria of the bicornuate horns. They are subsequently phagocytised and digested by fibroblasts. Then, finally, they become deposited in the uterine lumen together with a large quantity of secretory material from the endometrial connective tissue which, prior to this event, undergoes a massive infiltration by polymorphonuclear leucocytes.

Molecular Cloning of Rat Spetex2 Family Genes Mapped on Chromosome 15p16, Encoding a 23-Kilodalton Protein Associated with the Plasma Membranes of Haploid Spermatids

Abstract We used differential display in combination with cDNA cloning to isolate a novel rat gene, designated as Spetex2, that has an open reading frame of 582 nucleotides, encoding a protein of 194 amino acids. Spetex2 mRNA was highly expressed in testis and spleen, and its expression in rat testis was developmentally up-regulated. In situ hybridization revealed that Spetex2 mRNA was predominantly expressed in haploid spermatids at steps 1–13 within the seminiferous epithelium. A BLAST search against rat genome databases at the National Center for Biotechnology Information revealed that the Spetex2 gene is composed of four exons and is mapped to at least 18 loci in a cluster on rat chromosome 15p16, indicating that the genes occur as a repeated tandem array over a long stretch of genomic DNA. By immunocytochemical analysis with confocal laser-scanning microscopy, SPETEX2 protein was detected as a dot-like distribution on the cell periphery of haploid spermatids (steps 1–13) but was not observed in other spermatogenic cells. On the basis of these data, we hypothesize that SPETEX2 might be correlated with cell differentiation of spermaytids in rat testis.

Ultrastructure of receptaculum seminis in Haemaphysalis longicornis (Acari: Ixodidae) in relation to inserted endospermatophore

The receptaculum seminis, opening into the female genital tract, is found only in the metastriate ixodid ticks. An endospermatophore that has been inserted into the female genital aperture at copulation is first stored in the receptaculum seminis, where spermiogenesis is completed before the sperm ascend the oviducts. The receptaculum seminis consists of a simple cuticularized epithelium. Epithelial cells in sexually matured females develop during feeding and become active in secretion. Secretions discharged from epithelial cells are released into the lumen of this organ through the cuticle and may act on the wall of the inserted endospermatophore. The fact that resumption of spermiogenesis (spermateleosis) has already occurred before destruction of the endospermatophore just after copulation suggests that secretions from epithelial cells of the receptaculum seminis are not the trigger of spermateleosis, but a destructive agent of the endospermatophore wall. J Morphol 231:143–147, 1997.

Noradrenergic and cholinergic nerves in the uterus of the Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, change with reproductive cycle.

Abstract The pattern of uterine innervation by noradrenergic (NA) and acetylcholinesterase-positive (AChE) nerves in different reproductive stages of the adult Japanese long-fingered bats were investigated histochemically and immunohistochemically. In the non-pregnant bat, the uterine horn was supplied with abundant NA and AChE nerves. These two types of nerves were closely associated with the uterine arteries and myometrial smooth muscles. In the pregnant bat, NA and AChE nerves supplying the uterus did not degenerate much during hibernating period, but reduced markedly after arousal. In the postpartum bat, the density of nerves recovered progressively. The significant change in the innervation pattern of uterine NA and AChE nerves in the pregnant bats under and after hibernation, and in the postpartum bat must be considered in relation to the adrenergic and cholinergic controlling mechanisms on the uterine function that is matched for the unique reproductive cycle of this bat.

Electron microscopical and biochemical studies of the major pectoral muscles of bats

Abstract The major pectoral muscles (musculus pectoralis superficialis) of two species of bats (Miniopterus schreibersi fuliginosus and Rhinolophus ferrumequinum nippon) were studied by electron microscope and quantitative determination of myoglobin were carried out. 1. 2. In the pectoral muscles of both species, numerous mitochondria and lipid droplets occur between myofibrils. 2. 3. The average myoglobin concentration is 5.58 mg/g in M. s. fuliginosus and 1.83mg/g in R. f. nippon. 3. 4. It is suggested that these differences are related to the physiological properties of the muscles needed for different types of flight.

Regurgitation feeding of young in harvest mice, Micromys minutus (Rodentia: Muridae)

Regurgitation feeding of young is regarded as a behavior specific to the Canidae among mammals. In this study, we document regurgitation feeding by female harvest mice, Micromys minutus . We gave a composite ball made from sunflower seeds and small pieces of blue plastic to lactating females. Subsequently, some pieces of plastic were found in the digestive tracts of young with whom the mother had displayed regurgitation-like behavior. Regurgitation feeding started on the day of parturition and continued through weaning, but a switch existed in quality and quantity of behaviors related to regurgitation at ca. 9 days postpartum in this species.