年森 清隆

We isolated the MC31 cDNA clone coding the antigen specifically recognized by the monoclonal antibody mMC31, and found that MC31 was identical to rat CE9. Therefore, this molecule is called MC31/CE9. MC31/CE9, a member of the immunoglobulin superfamily molecules, was localized on the rat sperm flagellar plasma membrane. We analyzed the expression and cellular localization of MC31/CE9 mRNA and protein in the adult rat testis by use of Northern hybridization, in situ hybridization, and immunohistochemical analyses. In the course of spermatogenesis, MC31/CE9 mRNA first appeared in type B spermatogonia. The mRNA signal intensity increased progressively to pachytene spermatocytes and remained constantly at a considerable level throughout the subsequent phases of spermatocytes and round spermatids, and then decreased gradually from step-11 spermatids to disappear in step-15 spermatids. On the other hand, MC31/CE9 protein expression showed a bimodal pattern. Immunohistochemical analysis for the MC31/CE9 protein revealed its most intense immunoreactivity on the flagella of step-8 to step-19 elongated spermatids. The cytoplasmic immunoreactivity of the MC31/CE9 protein also appeared in preleptotene to early pachytene spermatocytes and elongated spermatids, with particularly intense immunoreactivity in the Golgi complexes of zygotene and early pachytene spermatocytes (stage XIII to III) as well as step-8 to step-13 spermatids. Between these two phases, the MC31/CE9 protein proved undetectable in the cytoplasm of any spermatogenic cells. Sertoli cells and Leydig cells were devoid of MC31/CE9 mRNA and its protein. Therefore, the production of MC31/CE9 is thought to be posttranscriptionally regulated during spermiogenesis.

We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation.

We produced a monoclonal antibody, designated MC301, against the extract of testicular cells from 12-day-old rats. This age corresponds to the onset of meiosis during testis development. MC301 specifically recognized a 90-kDa glycoprotein, GP90-MC301, which was ubiquitously expressed in various tissues and localized predominantly in the Golgi area of epithelial cells. In adult testes, stage-specific intense expression of GP90-MC301 was shown in the cytoplasm of meiotic spermatogenic cells from the preleptotene to mid-pachytene stages. Immunoelectron microscopy demonstrated that the glycoprotein was localized in spermatocytes on protein synthesis-related orgahelles such as the Golgi apparatus, endoplasmic reticulum, and nuclear envelope. The plasma membrane of spermatocytes and the intercellular space surrounding the cells were also immunoreacrive. No specific immunoreactivity was found on the organelles in other testicular cells. A considerable amount of the glycoprotein was detected in the extracellular fluid of the testes. These results suggest that GP90-MC301 is produced mainly by spermatocytes in the testis and secreted into the surrounding intercellular space. The evidence for developmentally regulated expression of GP90-MC301 in the meiotic spermatogenic cells suggests a possible role for the glycoprotein in male germ cell meiosis.

We report the ultrastructural changes in acrosome morphology during the final steps of rat spermiogenesis, focusing on the relationship between the acrosome morphogenesis and the tubulobulbar complexes (TBC) development. During steps 18-19, the electron-lucent area in the dorsal cortex of the anterior acrosome gradually diminished, and finally, the acrosome became condensed and reduced its volume. Simultaneously with this tightening up of the acrosome, TBC developed from the head portion of late spermatids, protruding into the surrounding Sertoli cells. To investigate the incorporation of acrosomal contents into TBC, step 19 spermatids were stained by periodic acid-Schiff (PAS) reaction and by using the antiacrosomal monoclonal antibody mMN7. Both PAS-reactivity and the mMN7-immunoreactivity were found in the TBC, as well as in the acrosome. In addition, the acrosome projected into the TBC-like structure, and materials of a density similar to that of the acrosome were observed in the core of the TBC. These results suggest that the TBC eliminate excess acrosomal contents prior to spermiation.

Hsp70-2 is a unique member of the mouse 70-kDa heat shock protein family that is synthesized during meiosis in spermatogenic cells. Germ cells in male mice homozygous for a targeted mutation in the Hsp70-2 gene (Hsp70-2(-/-)) arrest in development and undergo apoptosis at the end of the pachytene spermatocyte stage of meiotic prophase, However, cells with a putative acrosome were present occasionally in histological sections of the testes of juvenile and adult Hsp70-2(-/-) mice. This study verified that acrosomes were present and investigated the relationship between acrosome formation and the process of meiosis. Histochemistry with the periodic acid-Schiff procedure and immunostaining with monoclonal antibody MN7 verified that acrosomes were present in Hsp70-2(-/-) mice, and electron microscopy showed that some of these cells had condensing nuclei characteristic of step 8-9 spermatids. The frequency of acrosome-containing cells in Hsp70-2(-/-) mice was less than 0.01% of that in wild-type mice. Propidium iodide staining and cytophotometry indicated that the average DNA content of nuclei in MN7-positive cells in Hsp70-2(-/-) mice was usually about twice, or occasionally the same as, that of nuclei in round spermatids of wild-type mice. Meiotic metaphase I and II chromosome spreads were observed in spermatogenic cells from Hsp70-2(-/-) mice but at a much lower frequency than in wild-type mice. These results indicate that not all pachytene spermatocytes in Hsp70-2(-/-) mice arrest in meiosis, but they may divide once or sometimes twice and begin acrosome formation and nuclear condensation. This demonstrates that some aspects of spermatid development can occur without the completion of meiosis in mice, as has been reported recently for Drosophila.

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.

Calmegin is a testis-specific Ca2+-binding protein that is homologous to calnexin. Recently, sperm from transgenic mice lacking calmegin have been shown to be infertile. To further characterize calmegin, we analyzed the precise stage of expression and the intracellular localization of this protein in germ cells during mouse spermatogenesis by an immunoperoxidase technique using the anti-calmegin monoclonal antibody TRA369. Light microscopic immunocytochemistry showed that calmegin appeared in early pachytene spermatocytes, with the highest expression in round and elongating spermatids, and disappeared in the maturation phase of spematids at step 15. Immunoelectron microscopy showed that selective localization was found at the endoplasmic reticulum membrane and the nuclear envelope of spermatogenic cells. During the maturation phase, a dramatic reduction in calmegin occurred in the endoplasmic reticulum of the spermatids, suggesting that the major function of calmegin has been completed by the time spermatids reach step 14. In addition, although the immunoreactivity was completely absent in the calmegin-deficient mutant mouse testis, ultra-structural analysis showed that mature sperm from the knockout mice were normal. This suggests that calmegin is not required for the morphogenesis of male germ cells. Thus, our results suggest that calmegin has a major role in mouse spermatogenesis, and also indicate that this protein would be useful as a maker molecule to study the functional role of the endoplasmic reticulum in the process of spermatid differentiation.

The acrosome reaction isa membrane fusion event that is prerequisite for sperm penetration through the zona pellucida. To elucidate the molecular mechanisms involved in membrane fusion,the expression and localization of Rab proteins, a subfamily of small GTPases that have been shown to play key roles in regulation of intracellular membrane traffic and exocytosis, were examined in rat testis and sperm. Reverse transcription polymerase chain reaction, immunoblot analysis, and immunofluorescence microscopy revealed that Rab3A protein, which is thought to be involved in regulation of exocytosis in neurons and endocrine cells, is associated with the sperm acrosome. The protein was undetectable in acrosome-free heads prepared by sucrose density gradient centrifugation. Immunogold electron microscopy performed on ultrathin cryosections provided further evidence that Rab3A protein is associated with the acrosomal membrane. Acrosome reaction assays revealed that synthetic peptide of the Rab3 effector domain inhibited acrosomal exocytosis triggered by calcium ionophore A23187 in a concentration-dependent fashion, suggesting that Rab3A acts as an inhibitory regulator in the acrosome reaction. In view of the putative role of Rab3A protein in membrane fusion systems, these results suggest that Rab3A could be involved in regulating the mammalian acrosome reaction by controlling the membrane fusion system in sperm. (C) 1999 Academic Press.

OBJECTIVE: This study was intended to investigate the temporal changes in heat shock protein 72 expression and microtubule-associated protein 2 disappearance in rat brain at 2 different ages after hypoxic-ischemic insult. STUDY DESIGN: Both 5-day-old and 14-day-old Wistar rats were subjected to unilateral common carotid artery ligation and hypoxia in 8% oxygen for 2 hours at 33 degrees C. Brain sections were examined sequentially for heat shock protein 72 expression at 0.5, 3, 6, 12, 24, 48, and 72 hours of recovery after hypoxia-ischemia and for microtubule-associated protein 2 disappearance at 0, 24, 48, and 72 hours of recovery and at 7 days of recovery after hypoxia-ischemia. Results of immunohistochemical staining for heat shock protein 72 and microtubule-associated protein 2 were used as markers for detection of early hypoxic-ischemic brain damage. Permanent neuronal damage was assessed with hematoxylin and eosin staining at 7 days after hypoxia. RESULTS: In 5-day-old rats microtubule-associated protein 2 expression was lost as early as 0 hours after hypoxia-ischemia in the cerebral cortex and hippocampus, with a peak at 48 hours after which expression recovered. Expression of heat shock protein 72 was detected in the ligated hemisphere at 0.5 hours after hypoxia-ischemia and peaked at 6 to 24 hours of recovery. In 14-day-old rats microtubule-associated protein 2 was stained in the cortex at 0 hours after hypoxia-ischemia but gradually disappeared in the cerebral cortex and hippocampus after 24 hours of recovery. The expression of heat shock protein 72 was not detected by 6 hours of recovery in the cerebral cortex and by 3 to 12 hours of recovery in the hippocampus, but heat shock protein 72 was persistently expressed in the cortex and hippocampus after 48 hours of recovery. Neuronal damage was significantly less in 5-day-old rats than in 14-day-old rats. CONCLUSION: In 5-day-old rats hypoxia-ischemia causes earlier changes in heat shock protein 72 and microtubule-associated protein 2 immunostaining results and causes less severe brain damage than in 16-day-old rats. (Am J Obstet Gynecol 1999;180:1254-62.).

The juvenile visceral steatosis mutant mice serve as an animal model of primary carnitine deficiency, classified as the sudden infant death syndrome. The defect in carnitine uptake was recently found to be due to a defect in the carnitine transporter gene. We herein report, for the first time, the characteristics of epididymal dysfunction in juvenile visceral steatosis mice. At 8-9 weeks of age, the epididymis was deformed and weight was significantly increased. Histologically, the duct of the proximal epididymis was dilated due to the accumulation of an unusually high level of spermatozoa. Spermatozoa were extravasated from the epididymal duct into the stroma. In contrast, the duct of the distal epididymis was constricted and contained no spermatozoa. Thus, the epididymal disorder causes obstructive azoospermia, leading to infertility. Copyright (C) 1999 Federation of European Biochemical Societies.

The fungicide carbendazim, a male reproductive system toxicant, affects the adult testis, while prepubertal animals are assumed to be unsusceptible to the chemical. The present study was conducted to re-evaluate the susceptibility of rat prepubertal testis (25-30 days old) to carbendazim based on the incidence of spermatid abnormalities, including nuclear enlargement (megaspermatids) and binucleate round spermatids. In control prepubertal rats treated orally with corn oil vehicle alone, seminiferous tubules containing magno- and/or binucleate round spermatids were often observed in the groups at stages II-III, IV-V and VI-VII. At 3.0 days after treatment with carbendazim (100 mg/kg), seminiferous tubules containing spermatids with identical abnormalities were significantly increased, including groups at all stages. Significant increases were also observed at stages IV-V and VI-VII at day 4.5, and VI-VII at day 7.5. In contrast, the frequency of spematids with these abnormalities was reduced in the groups at stages II-III and IV-V at day 7.5. These findings confirm that the prepubertal rat testis is susceptible to carbendazim during early spermiogenesis.

The acrosome plays an important role in fertilization. This study was designed to examine the role and behavior of a molecule, equatorin (the antigenic molecule of the monoclonal antibody mMN9), localized at the equatorial segment of the acrosome. In vitro fertilization (IVF) investigation was conducted to examine the role of this molecule, by assessing the effect of mMN9 in TYH medium (a modified Krebs Ringer bicarbonate solution) containing mMN9 at 0 (control), 25, 50, and 100 μg/ml. Under these conditions, the IVF investigation was divided into two experiments: 1) the zona pellucida (zona)-intact experiment, in which capacitated sperm inseminated cumulus-and zona-intact oocytes; and 2) the zona-free experiment, in which acrosome-reacted sperm inseminated zona-free oocytes. It was found that mMN9 did not affect sperm motility, zona binding, or zona penetration, but it significantly inhibited fertilization, reducing the rates of pronucleus and two-cell embryo formation in both the zona- intact and zona-free oocyte experiments. In addition, when judged at 5 h after insemination in the zona-intact experiment, nearly half of the unfertilized oocytes had accumulated sperm in the perivitelline space (perivitelline sperm), and concurrently we confirmed by electron microscopy the presence of many unreleased cortical granules preserved beneath the oolemma, indicating no occurrence of sperm-oocyte fusion. Confocal laser scanning light microscopy with indirect immunofluorescence demonstrated that equatorin was localized at the equatorial segment in both capacitated and perivitelline sperm (acrosome-reacted sperm). These results suggest that equatorin that is preserved at the equatorial segment is involved in the process of sperm-oocyte fusion in mice.

To investigate the contribution of vesicular transport to acrosome formation, we examined the effect of brefeldin A (BFA), a potent inhibitor of membrane traffic, on the ultrastructural changes of the organella in early spermatids. The experiments were done in an organ culture system of rat seminiferous tubules. After exposure to BFA, the fuzzy-coated projections of the endoplasmic reticulum (ER) cisternae directing toward the Golgi stack disappeared. The Golgi stacks were also disrupted, forming numerous small vesicles and tubular networks. The head-cap was fragmented in step 4-7 spermatids. We also investigated the effect of BFA on protein transport to the acrosome by using a monodonal antibody to the acrosomal protein MN7 (mMN7) as a model. After a 5-h exposure, the mMN7 immunoreactivity was absent from the developing acrosomic system in step 2-3 spermatids. These results indicate that BFA blocks the vesicular transport, causing an insufficient supply of acrosomal materials to the acrosome, which eventually leads to abnormal acrosome formation in early spermiogenesis.

We have previously shown that a 90-kDa intra-acrosomal antigen, MN7, is restricted to the anterior acrosomal region of mouse, rat, and hamster spermatozoa. The present study has examined the localization and the behavior of MN7 during sperm maturation in the epididymis of the guinea pig by immunoelectron microscopy. MN7 showed not only a specific localization in the apical segment of the guinea pig sperm acrosome, but also a distinct alteration during maturation, as follows. MN7 was exclusively found both at the dorsal matrix and on the outer acrosome membrane (OAM)/matrix-associated materials in the apical segment. MN7 was initially distributed throughout the electron-lucent dorsal matrix in immature sperm but, during maturation, became more restricted to the spherical bodies within the electron-lucent area. MN7 on OAM/matrix-associated materials was first distributed along the ventral margin and the small area posterior to the dorsal matrix but, during maturation, disappeared from the ventral margin and became restricted to the dorsal region. These results indicate that MN7 is a good tool for studying the stepwise maturation of epididymal spermatozoa.

Effects of a single, high dose of orally administered carbendazim (100 mg/kg) on acrosome formation in the early phases of spermiogenesis were examined by electron microscopy and immunocytochemistry up to day 7.5 post-treatment. No obvious abnormality of acrosome development was noted in the Golgi phase spermatids on day 1.5 post-treatment. On day 3, step 1 spermatids were seen in stage III seminiferous tubules. In stage V tubules at this post-treatment interval, direct connections between the trans-side saccules of the Golgi stacks and the outer acrosomic membranes were observed in step 5 spermatids. Similar direct connections between these two organelles were also observed in the advanced round spermatids in later stages at days 4.5 and 7.5. On day 4.5, step 1 and 3 spermatids were seen in stage V tubules. On day 7.5, round spermatids with various abnormalities of acrosome development were observed in stage VII tubules, in addition to the discontinuous and granular acrosomes reported previously. These features were not observed in testes of control animals. In the immunocytochemical analysis using an antibody mMN7 that recognizes a protein delivered from the Golgi apparatus to the acrosome, spermatids exposed to carbendazim showed various abnormal immunostaining patterns in the acrosomes. On the other hand, strong immunoreactivity was observed in the Golgi saccules connecting to the acrosomes. These results suggest that in testis treated with carbendazim acrosome development is impaired during the early phases of spermiogenesis, and material supply from the Golgi apparatus to the acrosome is perturbed, which is a possible cause of the abnormal development.

OBJECTIVE: Our purpose was to study the neuronal responses of heat shock protein-72 (a stress-inducible protein) and microtubule-associated protein-2 (a constitutive protein of the neuronal cytoskeleton) after hypoxia-ischemia and their relationship with permanent damage in the newborn rat brain. STUDY DESIGN: Seven-day-old rats were exposed to unilateral carotid artery ligation followed by 2 hours of hypoxia (8% oxygen/92% nitrogen) and then killed at time points ranging from 1 to 72 hours after injury. Brains were removed for immunohistochemical and routine staining. RESULTS: Heat shock protein-72 appearance and microtubule-associated protein-2 disappearance occurred from 1 hour after injury, mainly in the dentate gyrus of the hippocampal formation and the cerebral cortex. Such alterations reached maximal levels at 24 hours for both proteins. Microtubule-associated protein-2 staining recovered in almost all parts of the brain. However, the hippocampal CA3 showed a delay in the responses for both proteins, and microtubule-associated protein-2 did not recover the response to immunostaining. Histologic evaluation at 72 hours after hypoxia by routine methods showed predominant damage in the hippocampal CA3. CONCLUSION: Our results show that delayed responses of heat shock protein-72 and microtubule-associated protein-2 are related to a high incidence of neuronal cell loss in the hippocampal CA3 region.

The proper folding of newly synthesized membrane proteins in the endoplasmic reticulum (ER) is required for the formation of functional mature proteins. Calnexin is a ubiquitous ER chaperone that plays a major role in quality control by retaining incompletely folded or misfolded proteins. In contrast to other known chaperones such as heat-shock proteins, BiP and calreticulin, calnexin is an integral membrane protein. Calmegin is a testis-specific ER protein that is homologous to calnexin. Here we show that calmegin binds to nascent polypeptides during spermatogenesis, and have analysed its physiological function by targeted disruption of its gene. Homozygous-null male mice are nearly sterile even though spermatogenesis is morphologically normal and mating is normal. In vitro, sperm from homozygous-null males do not adhere to the egg extracellular matrix (zona pellucida), and this defect may explain the observed infertility. These results suggest that calmegin functions as a chaperone for one or more sperm surface proteins that mediate the interactions between sperm and egg. The defective zona pellucida-adhesion phenotype of sperm from calmegin-deficient mice is reminiscent of certain cases of unexplained infertility in human males.

The proper folding of newly synthesized membrane proteins in the endoplasmic reticulum (ER) is required for the formation of functional mature proteins. Calnexin is a ubiquitous ER chaperone that plays a major role in quality control by retaining incompletely folded or misfolded proteins. In contrast to other known chaperones such as heat-shock proteins, BiP and calreticulin, calnexin is an integral membrane protein. Calmegin is a testis-specific ER protein that is homologous to calnexin. Here we show that calmegin binds to nascent polypeptides during spermatogenesis, and have analysed its physiological function by targeted disruption of its gene. Homozygous-null male mice are nearly sterile even though spermatogenesis is morphologically normal and mating is normal. In vitro, sperm from homozygous-null males do not adhere to the egg extracellular matrix (zona pellucida), and this defect may explain the observed infertility. These results suggest that calmegin functions as a chaperone for one or more sperm surface proteins that mediate the interactions between sperm and egg. The defective zona pellucida-adhesion phenotype of sperm from calmegin-deficient mice is reminiscent of certa

The early testicular damage after acute cadmium (Cd) exposure and the prevention of Cd-induced testicular toxicity by the oral administration of disulfiram (DSF) at various times after Cd exposure in rats were studied. Although moderate enlargement in interstitial tissues was observed at the first 3 h after Cd injection, no morphological changes in the inside of seminiferous tubules were observed. At 6 h after Cd exposure, damage of both interstitial tissues and seminiferous tubules was observed. Rats received an intraperitoneal injection of DSF (0.5 mmol/kg) or an oral administration of DSF (0.5, 1.0 or 2.0 mmol/kg) 0.5, 1, 3 or 6 h after the subcutaneous injection of CdCl2 (26.7 mu mol (3 mg) Cd/kg). The oral treatment with DSF at dose levels of 0.5 mmol/kg (0.5 or 1 h), 1.0 mmol/kg (3 h), or 2.0 mmol/kg (3 h) prevented the increase in testicular lipid peroxidation and calcium and Cd concentrations and the decrease in testicular weight, which were observed at 7 d after Cd injection, as well as by the injection of DSF at 0.5 mmol/kg (0.5 h). The results at 59 d after Cd injection indicated that the testicular Cd concentrations and testicular weight after DSF treatment were changed in sterile rats, but not in fertile animals. These results indicate that Cd-induced testicular toxicity occurs 3-6 h after Cd injection, and that more effective chelation therapy with DSF for testicular toxicity by Cd is considered to arise from the oral administration of DSF at dose levels of more than 1.0 mmol/kg within 3 h after acute Cd exposure in rats.

We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis. (C) 1995 Wiiey-Liss, Inc.

OBJECTIVES: Although the physiologic and pathologic roles of endothelin-1 in reproduction have been investigated, little is known about human uterine tissue levels. We studied the levels of immunoreactive endothelin-1 and immunoreactive big endothelin-1 in human endometrium and myometrium during each menstrual phase. STUDY DESIGN: Materials were obtained at hysterectomy (endometrium, n = 33 myometrium, n = 27). We measured immunoreactive endothelin-1 and immunoreactive big endothelin-1 by radioimmunoassay and performed an immunohistochemical study of the tissue. Data were analyzed by the Mann-Whitney U test. RESULTS: We detected larger amounts of immunoreactive endothelin-1 and immunoreactive big endothelin-1 in the endometrium than in the myometrium throughout the menstrual, proliferative, and secretory phases. Endometrial immunoreactive endothelin-1 and immunoreactive endothelin-1 were significantly increased in the menstrual phase (endothelin-1 68.8 ± 23.3 pg/mg protein, n = 5, p &lt 0.005 big endothelin-1 45.2 ± 5.7 pg/mg protein, n = 5, p &lt 0.003) compared with the other phases (endothelin-1 30.7 ± 9.5 and 30.5 ± 14.0 pg/mg protein big endothelin-1 19.9 ± 6.7 and 24.1 ± 7.4 pg/mg protein). Immunohistochemistry demonstrated that the endometrial stromal cells were positive for antiendothelin monoclonal antibody only in the premenstrual and menstrual phases. CONCLUSION: Levels of immunoreactive endothelin-1 and immunoreactive big endothelin-1 are different in each type of uterine tissue and in each phase of the menstrual cycle. These changes may indicate some role of endothelin-1 in menstruation. © 1995, All rights reserved.

Mouse intra-acrosomal antigens, recognized by monoclonal antibodies, MN7 and MC41, were analyzed by SDS-PAGE and Western blotting. Both the MN7- and MC41-antigens were glycoproteins of 90kDa and 200 kDa, respectively. However, the two antigens exhibited different biochemical properties; the MN7-antigen was extracted only in the acidic condition (pH 3.0), while the MC41-antigen was detectable in both acidic and neutral conditions. The releasing process during the acrosome reaction was examined by immunoelectron microscopy. The MN7-antigen, which was initially distributed uniformly in the entire acrosome, gradually disappeared from the acrosome, being localized once at the cortex-medulla border region at the middle stage, and dispersed without close contact with the vesiculated membrane. By contrast, the MC41-antigen, which was initially restricted to the cortical region of the anterior acrosome, was localized on the electron-dense materials lining closely to the fenestrated membrane at the middle stage, and dispersed keeping in close contact with the vesicles. These findings indicate that the antigens originally segregated into different subdomains can be differentially released during the acrosome reaction.

Human neutrophil peptides (HNPs) 1, 2 and 3, also called defensins, have high antimicrobial activity and are cytotoxic to both normal and transformed human cells. The localization of HNPs 1-3 and their mRNAs in cells of normal human bone marrow and peripheral blood was investigated. Immunoreactivity for HNPs 1-3 appeared in the promyelocyte stage and increased with the maturation of the neutrophil series. HNPs 1-3 were absent from such mononuclear phagocyte systems as peripheral monocytes, alveolar macrophages, and Kupffer cells in the liver. HNP 1-3 mRNAs were detected in bone marrow cells by Northern blot analysis and in peripheral leukocytes by the reverse transcription-polymerase chain reaction (RT-PCR). HNPs 1-3 are restricted to cells of neutrophil lineage and seem to function as microbicidal agents in the neutrophil-mediated defense system.

The localization of an acrosomal protein was studied using a monoclonal antibody MN7 raised against mouse spermatozoa. MN7 specifically recognized the anterior acrosome of several mammalian (mouse, rat, hamster) spermatozoa fixed with paraformaldehyde. An immunoblot study with periodate treatment showed that MN7 recognized a carbohydrate region of a 90 kDa protein in an extract of mouse and rat cauda epididymal spermatozoa. The change in distribution of the MN7 antigen during acrosome development was investigated in the rat testis using the pre-embedding immunoperoxidase technique. The antigen first appeared in the proacrosomic granules of spermatids in steps 1-2. Small vesicles adjacent to the outer acrosomal membrane and the developing acrosomic system were immunoreactive during steps 4-7. The majority of the antigen was then redistributed to the head-cap portion during steps 8-18, and finally restricted to the anterior acrosome in the step 19-spermatid. These results suggest that the antigen is transported to the acrosome by way of the vesicles that originate from the Golgi apparatus during early spermiogenesis, and are then delivered to the final destination within the acrosome by the intra-acrosomal migration during late spermiogenesis.

This report documents ultrastructural changes in the postacrosomal region of the hamster and mouse sperm head at the initial stage of gamete interaction in vivo. Prominent structures were sequentially visualized: first, the periodic substructure crossbridging the postacrosomal sheath to the overlying plasma membrane, and then the small, electron-dense, granular structures lining the outer surface of the postacrosomal sheath. The periodic substructure became visible at the restricted region where the sperm plasma membrane was just prior to or in the act of detaching from the periodic substructure. The granular substances lined up along the external face of the postacrosomal sheath immediately after the detachment of the sperm plasma membrane, but before the complete degradation of the periodic substructure. These structural changes were completed before sperm nuclear decondensation started. The region where the granular structures were visualized was close to that of the antigen recognized by the monoclonal antibody MN13, which is supposed to be involved in the association of the periodic substructure with the overlying plasma membrane (TOSHIMORI et al., 1991). Based upon these results, we present the progress of events at the initial stage of gamete interaction.

A 37-year-old man with the characteristic clinical signs of Kartagener's syndrome, including bronchiectasia, chronic paranasal sinusitis, and situs inversus, was found to have immotile cilia and sperm. Electron microscopy demonstrated that the majority of cilia lacked dynein arms and radial spokes and that various defects of microorgans existed in the sperm. The most frequent defect was total defect of axoneme followed by defect of inner dynein arms in the sperm. These findings suggest an association between the structural abnormality of absent inner dynein arms and immotility of cilia and sperm in Kartagener's syndrome. © 1993, The Japanese Society of Internal Medicine. All rights reserved.

A monoclonal antibody (MC41) was produced that specifically recognizes a sperm acrosomal antigen of approximately 165000 dalton in the rat. Rat testis was examined using a pre-embedding immunoperoxidase technique to reveal the pathway of the MC41 antigen to the acrosome during spermiogenesis. The MC41 immunoreaction appeared in several organelles of spermatids in a stage-specific manner: (1) in the endoplasmic reticulum (ER) throughout spermiogenesis, (2) in the outer acrosomal membrane from steps 9 to 19, (3) as a weak immunoreaction in the vesicular structures in the acrosomal matrix from steps 11 to 17, and (4) as a strong immunoreaction in the acrosomal matrix especially at the terminal step of spermiogenesis (step 19). However, no immunoreaction was observed in the Golgi region throughout spermiogenesis. These results suggest that the pathway of the MC41 antigen leads firstly from the ER to the outer acrosomal membrane and secondly to the acrosomal matrix. This pathway does not involve the Golgi apparatus and is referred to as the " extra-Golgi pathway ".

MN9, a monoclonal antibody raised against mouse spermatozoa, specifically recognizes the equatorial segment of sperm head in several mammalian species, including humans. Colloidal gold-immuno-electron microscopy of mouse spermatozoa has shown that the antigen is localized in the space between the outer and inner acrosome membranes and on the acrosome membranes at the equatorial segment. Immunoblotting after electrophoresis of spermatozoa from the cauda epididymidis has identified two immunoreactive bands: 38 kDa and 48 kDa in mouse, and 48 kDa in rat. During spermiogenesis in rat, this antigen is transported to the equatorial segment via a unique pathway, first appearing in some cisternae of the endoplasmic reticulum and in the Golgi apparatus of spermatids at around step 3. The antigen can further be found on the vesicles at the trans-side of the Golgi apparatus, in the matrix of the head cap, and on the head cap membrane in step-4 to step-7 spermatids. The antigen appears to be concentrated at the equatorial segment during late spermiogenesis. Neither the (pro-)acrosomic granule nor the surrounding membrane are required in this pathway. This pathway can be termed the 'Golgi-head cap tract'.

Previously, we reported that the epitope of a 54-kDa sperm surface sialoglycoprotein on the flagellum is masked by sialic acid residues. The epitope is referred to as a hidden determinant or cryptodeterminant. This paper reports the manner in which the epitope is masked as evaluated qualitatively and quantitatively by means of SDS-PAGE/immunoblots (Western blot) and ELISA. Immunoblotting with four specific monoclonal antibodies to the 54-kDa sialoglycoprotein-T21 (IgM), MC71 (IgG1), MC81 (IgM), and MC91 (IgM)-demonstrated that not only IgM but also IgG antibody MC71 and the Fab fragment MC71 are masked. Quantitative evaluation with ELISA to compare the antibody titration curves of the masked and unmasked antigens on sialidase-treated and untreated sperm, respectively, indicated that sialidase caused the antibody-binding ability of the epitopes to increase to a different level for each antibody. There were 32-256-, 8-16-, 16-, and 2-4-fold increases in binding to T21, MC71, MC81, and MC91 antibodies, respectively. These results suggest that the antigen-masking through the cryptodeterminant does not depend upon the subtype or the molecular mass of the antibody, but upon the biochemical nature of the epitope region that is closely related to the sialic acid. The mechanism and physiological roles of the antigen-masking are discussed.

We identified a rat sperm flagellar surface antigen using an IgG1 monoclonal antibody (MC31) against rat epididymal sperm. Avidin-biotin-peroxidase immunohistochemistry demonstrated that the antigen was first expressed in the cytoplasm of early primary spermatocytes, then gradually became restricted to the principal piece of the sperm flagellum during spermatogenesis. However, when the sperm reached the corpus epididymidis, the antigen was expressed on the surface of both the principal piece and the midpiece of the flagellum. The epithelial cells of the epididymis were not stained with MC31. Immunogold electron microscopy showed that the antigen was present on the surface of the sperm flagellar plasma membrane. Immunoblotting of Triton X-100 extracts of epididymal sperm after one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions demonstrated that MC31 detected a major antigen of 26,000-28,000 daltons (26-28K). Two-dimensional isoelectric focusing and SDS-PAGE indicated that the 26-28K antigen had an isoelectric focusing point (pl) of 5.8-5.3; minor antigens were also detected from 26K (pl 5.8) to 35K (pl 5.0). These results indicate that the antigen recognized by MC31 is an acidic 26-35K protein that originates in the testis, is integrated into the sperm flagellar plasma membrane of the principal piece during spermatogensis, and then is expressed on the entire flagellar surface during epididymal transit.

In a survey of sperm antigens in the rat, a new intra-acrosomal antigen was found using a monoclonal antibody MC41 raised against rat epididymal spermatozoa. The MC41 was immunoglobulin G1 and recognized spermatozoa from rat, mouse and hamster. Indirect immunofluorescence with MC41 specifically stained the crescent region of the anterior acrosome of the sperm head. Immuno-gold electron microscopy demonstrated that the antigen was localized within the acrosomal matrix. Immunoblot study showed that MC41 recognized a band of approximately 165000 dalton in the extract of rat sperm from the cauda epididymidis. Immunohistochemistry with MC41 demonstrated that the antigen was first detected in approximately step-2 spermatids, and distributed over the entire cytoplasmic region of spermatids from step 2 to early step 19. The head region became strongly stained in late step-19 spermatids and then in mature spermatozoa. Distinct immunostaining was not found in the developing acrosome of spermatids throughout spermiogenesis. These results suggest that the MC41 antigen is a unique intra-acrosomal antigen which is accumulated into the acrosome during the terminal step of spermiogenesis.

Using a monoclonal antibody T21, we reported that a mouse sperm maturation-associated antigen sialoglycoprotein of 54000 daltons (54K sialoglycoprotein) was secreted at the distal caput to proximal corpus epididymidis and that the 54K sialoglycoprotein had a hidden determinant (cryptodeterminant), which could be eliminated by sialidase treatment (Toshimori et al. (1988): Histochemistry 90:195-200; (1990a): Biol Reprod 42:151-160; (1990b): Arch Histol Cytol 53:339-349). This study evaluated the mouse sperm susceptibility to phagocytosis by macrophage in vitro. Comparisons were made between sperm from the caput epididymidis (caput sperm) incubated in modified Krebs Ringer's solution (MKR) and caput sperm incubated in MKR containing cauda fluid, and between sialylated (sialidase-untreated) sperm from the corpus and cauda epididymidis (corpus/cauda sperm) and desialylated (sialidase-treated) corpus/cauda sperm. The results showed that macrophages were least actively engaged in phagocytosis for caput sperm incubated in MKR containing cauda fluid, and most active for desialylated corpus/cauda sperm. Incubation of caput sperm in MKR containing cauda fluid revealed that the 54K sialoglycoprotein in cauda fluid could be bound to the flagellar surface of caput sperm. These results together with previous findings strongly suggest that the 54K sialoglycoprotein bound to immature sperm during maturation in the epididymis is implicated in the protection of sperm from phagocytosis with the aid of sialic acid residues.

Monoclonal antibody MN13 raised against mouse spermatozoa specifically recognizes the postacrosomal region of the sperm head in several mammalian species. Colloidal gold-immunoelectron microscopy of demembranated mouse spermatozoa indicated that the antigen is associated with the outer layer of the periodic substructure apparently linking the postacrosomal sheath to the overlying plasma membrane. The antigen recognized by MN13 may contribute to the intimate association of the postacrosomal sheath with the overlying plasma membrane.

Using a monoclonal antibody T21, we reported that a mouse sperm maturation-associated antigen sialoglycoprotein of 54000 daltons (54K sialoglycoprotein) was secreted at the distal caput to proximal corpus epididymidis and that the 54K sialoglycoprotein had a hidden determinant (cryptodeterminant), which could be eliminated by sialidase treatment (Toshimori et al. (1988): Histochemistry 90:195-200 (1990a): Biol Reprod 42:151-160 (1990b): Arch Histol Cytol 53:339-349). This study evaluated the mouse sperm susceptibility to phagocytosis by macrophage in vitro. Comparisons were made between sperm from the caput epididymidis (caput sperm) incubated in modified Krebs Ringer's solution (MKR) and caput sperm incubated in MKR containing cauda fluid, and between sialylated (sialidase-untreated) sperm from the corpus and cauda epididymidis (corpus/cauda sperm) and desialylated (sialidase-treated) corpus/cauda sperm. The results showed that macrophages were least actively engaged in phagocytosis for caput sperm incubated in MKR containing cauda fluid, and most active for desialylated corpus/cauda sperm. Incubation of caput sperm in MKR containing cauda fluid revealed that the 54K sialoglycoprotein in cauda fluid could be bound to the flagellar surface of caput sperm. These results together with previous findings strongly suggest that the 54K sialoglycoprotein bound to immature sperm during maturation in the epididymis is implicated in the protection of sperm from phagocytosis with the aid of sialic acid residues. © 1991 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

An exhaustive anatomic dissection of all of the veins of the finger distal to the proximal interphalangeal joint was done. More than 3200 segments of veins were individually dissected out, measured, and recorded. From this survey new schematic diagrams have been drawn emphasizing the pertinent venous anatomy at the proximal, distal interphalangeal joints and eponychial levels. Suggestions are made for regions that are apt to have the largest vessels available for anastomosis. © 1991, American Society for Surgery of the Hand. All rights reserved.

The arterial architecture in the finger distal to the proximal interphalangeal joint was studied in sixty-seven cadaver fingers with the aid of an operating microscope. The course, frequency, location, and diameter of the dorsal nail fold artery and its anastomosis was recorded. Similar measurements were performed for the palmar anastomosis. The frequency of arterial tortuosity in the digital artery was compiled and a classification of its morphology devised. The characteristic of arterial tortuosity provides a degree of protection to these essential structures. Failure to recognize this facet of arterial anatomy may be one more factor contributing to our inability to successfully revascularize the distal finger in certain patients. © 1991, American Society for Surgery of the Hand. All rights reserved.

We have reported several aspects of a mouse sperm maturation-associated antigen, a 54,000 dalton (54K) sialoglycoprotein, of which epitope can be masked by sialic acid residues against a monoclonal antibody T21 (35-38). However, the producing cell of the 54K sialoglycoprotein has long been unclear. This report documents the origin of the 54K sialoglycoprotein based on morphological evidence using avidin-biotin peroxidase complex (ABC) immunohistochemistry and indirect immunoperoxidase-electron microscopy with a recently established monoclonal antibody MC81. Immunoblots of Nonidet P-40 extract of cauda epididymidis sperm after one-dimensional, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that MC81 recognized a major protein of 54,000 dalton (54K). The epitope for MC81 antibody was suggested to be present on a sugar chain of the 54K protein when assessed by periodate treatment. Immunoblots of sperm extracts separated by two-dimensional SDS-PAGE indicated that a major spot of 54K dalton was in acidic range less than 5 in the isoelectric focusing point. These results suggest that the antigen of MC81 is identical to that of T21. Immunohistochemistry with MC81 revealed that principal cells located at the distal caput to corpus epididymidis were immunoreactive. The region where the principal cells were first recognized by MC81 was more proximal than the region where luminal sperm were first stained. In addition, MC81 exclusively recognized the Golgi apparatus area at the supranuclear region of the immunoreactive principal cells. Sialidase-treatment prior to primary antibody (MC81) increased of the immunostaining intensity in all immunochemical studies examined. Together with the result of the previous part of this study, it is strongly suggested that the 54K sialoglycoprotein can be secreted by the principal cells at the distal caput to corpus epididymidis, with the epitope for MC81 masked by sialic acids.

This chapter summarizes the current knowledge of the ultrastructural aspects of gamete interactions. The events associated with fertilization are subjected to ultrastructural analysis by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) as phenomena that involve two single, highly specialized cells (the sperm and egg), and are of fundamental importance for the production of new individuals and the conservation of the species. The fertilization events are analyzed by reproductive biologists, improved existing methods, and developed new sophisticated techniques. These include in vitro fertilization (IVF) procedures and various cytochemical techniques using specific membrane probes, such as lectins, antibiotic filipin, and monospecific antisera. The chapter provides information to understand the relationship between the bioactive molecules and the cell structures. The significance and clinical application of IVF techniques such as zona-drilledova, in vivo information is required to compare such biological events as the fine structure of the gametes between in vivo and in vitro conditions, and between human and other species, to assess the mechanism. © 1990 Academic Press Inc.

The present study was performed to clarify the distribution of ANP-containing cells in the adult rat heart by immunostaining for ANP using antiserum against α-human ANP. ANP-immunoreactive cells were generally present in the atrial walls except for the sinoatrial node. In the ventricular walls, they were distributed in the impulse conducting system, particularly the left bundle branch, Purkinje fibers on the left side of the interventricular septum, and those in the false tendons in the left ventricle, while they were sporadically seen in the atrioventricular node and bundle of His. The immunoreactive cells contained specific granules that were positive for ANP. These findings demonstrate that ANP-containing cells are present in the atrial and ventricular walls. © 1988 Springer-Verlag.