Immunohistochemistry and immunocytochemistry of atrial natriuretic polypeptide in porcine heart

Summary Specific granules in porcine hearts were observed in atrial cardiocytes, Purkinje fibers, and transitional cells of the ventricle. These granule-containing cells were immunohistochemically stained by applying the avidin-biotin-peroxidase complex method using an antiserum against -human atrial natriuretic polypeptide (ANP). Immunoelectron microscopy of sections stained using the immunogold method indicated that these specific granules are storage sites of ANP. Furthermore, an impulse-conducting system consisting of immunoreactive cells was clearly distinguishable from nonimmunoreactive ventricular cardiocytes. We conclude that specific-granule-containing cells, i.e., ANP-producing cells, are located in both the atrial walls and the ventricular impulse-conducting system. The presence of ANP may be correlated with impulse conduction.     

Identification of the redox partners of ERdj5/JPDI,a PDI family member,from an animal tissue

ERdj5 (also known as JPDI) is a member of PDI family conserved in higher eukaryotes. This protein possesses an N-terminal J domain and C-terminal four thioredoxin domains each having a redox active site motif. Despite the insights obtained at the cellular level on ERdj5, the role of this protein in vivo is still unclear. Here, we present a simple method to purify and identify the disulfide-linked complexes of this protein efficiently from a mouse tissue. By combining acid quenching and thiol-alkylation, we identified a number of potential redox partners of ERdj5 from the mouse epididymis. Further, we show that ERdj5 indeed interacted with two of the identified proteins via formation of intermolecular disulfide bond. Thus, this approach enabled us to detect and identify redox partners of a PDI family member from an animal tissue.     

Changes in distribution and molecular weight of the acrosomal protein acrin2 (MC41) during guinea pig spermiogenesis and epididymal maturation

In this study, we examined the localization and characteristics of an intra-acrosomal protein, acrin2 (MC41), during guinea pig spermiogenesis and post-testicular sperm maturation in the epididymis, using the monoclonal antibody MC41. Immunoelectron microscopy demonstrated not only a specific domain localization of acrin2 in the apical segment of the guinea pig sperm acrosome, but also its dynamic behavior according to the spermatid differentiation and passage through the epididymis, as follows: acrin2 was exclusively localized in the membrane of the endoplasmic reticulum of early-stage spermatids but was not detectable in the developing acrosome until spermatids reached the maturation phase. In the final stage of spermiogenesis, acrin2 became localized in the outer acrosomal membrane (OAM)/matrix-associated materials both in the small region posterior to the dorsal matrix and along the ventral margin of the acrosomal apical segment. The acrosomal location of acrin2 in caput epididymidal sperm was almost identical to that observed in the final step spermatids, but during maturation it became progressively more restricted in area until on distal cauda epididymidal sperm it remained only in the dorsal region. In Western blot analysis, the MC41 antibody recognized a 165-kDa protein in the mature sperm extract. Furthermore, it was demonstrated that molecular weight reduction of the protein occurred during sperm passage through the epididymis. These findings indicate that acrin2 changes progressively in both distribution and size during development and maturation of the acrosome.     

Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia

Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3 ^?/?) mice were infertile, as previously reported by Ichikawa et al. (2009). To investigate the involvement of Galnt3 in spermatogenesis, we examined the differentiation of germ cells in Galnt3 ^?/? mice. Galnt3 mRNA was most highly expressed in testis, and Galnt3 protein was localized in the cis-medial parts of the Golgi stacks of spermatocytes and spermatids in the seminiferous tubules. Spermatozoa in Galnt3 ^?/? mice were rare and immotile, and most of them had deformed round heads. They exhibited abnormal acrosome and disturbed mitochondria arrangement in the flagella. At the cap phase, proacrosomal vesicles of various sizes, which had not coalesced to form a single acrosomal vesicle, were attached to the nucleus in Galnt3 ^?/? mice. TUNEL-positive cells were increased in the seminiferous tubules. The binding of VVA lectin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), in the acrosomal regions of spermatids and spermatozoa in Galnt3 ^?/? mice was drastically reduced. Equatorin is a N, O-sialoglycoprotein localized in the acrosomal membrane and is suggested to be involved in sperm–egg interaction. Immunohistochemical and Western blot analyses showed a drastic reduction in the reactivity with MN9 antibody, which recognizes the O-glycosylated moiety of equatorin and inhibits sperm–egg interaction. These findings indicate that deficiency of Galnt3 results in a severe reduction of mucin-type O-glycans in spermatids and causes impaired acrosome formation, leading to oligoasthenoteratozoospermia, and suggest that Galnt3 may also be involved in the process of fertilization through the O-glycosylation of equatorin.     

Filipin-sterol complexes in golden hamster sperm membranes with special reference to epididymal maturation

Summary The distribution of membrane filipin-sterol complexes (FSCs) was qualitatively surveyed on freeze-fracture replicas of spermatozoa from the male reproductive tract and ejaculates of golden hamster. In the head, the acrosomal plasma membrane showed the strongest filipin labeling on the principal segment, but it was absent in the quilt-like pattern areas. These latter were observed in both caput and corpus epididymal spermatozoa, but were absent in mature spermatozoa. The postacrosomal plasma membrane had few FSCs and both the outer and inner acrosomal membranes were always negative to filipin. The nuclear membrane of the principal segment was constantly filipinpositive. The nuclear membrane of the postacrosomal region had more FSCs than that of the principal segment, particularly in mature spermatozoa. Many linear, rod-like FSCs were observed on the postacrosomal nuclear membrane of mature spermatozoa, especially in the uterine spermatozoan samples. In the neck, the plasma membrane had only a few FSCs. The redundant nuclear membrane was slightly filipin-positive, while the membrane scroll of mature spermatozoa was heavily labeled. In the tail, the plasma membrane of both the middle and principal piece was moderately labeled.     

Appearance of an intra-acrosomal antigen during the terminal step of spermiogenesis in the rat

Summary 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.     

Behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro

In this study we examined the behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro by assessing the effect of its pertinent monoclonal antibody mMC101. Experiments were designed to assess the effect of mMC101 on sperm-zona pellucida binding, the acrosome reaction, zona pellucida penetration, sperm-egg fusion, and fertilisation in vitro. mMC101 did not affect sperm motility or primary and secondary binding to the zona pellucida, but significantly inhibited fertilisation of zona-pellucida-intact oocytes in a dose-dependent manner. In the presence of mMC101 at 100 microg/ml concentration in TYH medium, none of the oocytes developed to pronuclear stage by 5 h after co-incubation of the gametes, but the pronucleus formation rate recovered to some extent (45.3%) after 8 h, indicating a delay of early embryonic development. mMC101 also delayed and significantly suppressed zona pellucida penetration by sperm. Acrin 3 dispersed and did not remain on completely acrosome-reacted sperm. Although mMC101 did not influence the zona-pellucida-induced acrosome reaction, it significantly inhibited fertilisation when acrosome-reacted sperm in the presence of mMC101 inseminated zona-pellucida-free oocytes. However, fertilisation remained unaffected when acrosome-reacted sperm in the absence of mMC101 inseminated zona-pellucida-free oocytes even in its presence. Thus, acrin 3 appears to facilitate zona pellucida penetration and is also likely to be involved in sperm-oocyte fusion by modifying the sperm plasma membrane during the acrosome reaction.