Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct

Female hamsters were mated shortly after the onset of oestrus. At 3 or 6 h after mating, the right oviduct was flushed in situ with 30, 90 or 180 microliters medium to remove spermatozoa from the lumen, leaving only those firmly attached to the isthmic mucosa of the oviduct. When eggs were recovered from oviducts at 20 h after flushing the majority were fertilized, indicating that the spermatozoa that were firmly attached to the mucosa were capable of detaching and ascending to the ampulla to fertilize eggs. Neither the time of flushing nor the volume of flushing medium had a significant effect on the percentage of spermatozoa that remained in the isthmus after flushing. These results suggest that there is no change in the surface of the oviduct mucosa that causes the release of spermatozoa from the caudal isthmus near the time of ovulation. When incapacitated spermatozoa were introduced into the oviduct, many of them attached to oviductal mucosa, while capacitated spermatozoa did not. This indicates that it is a change in the sperm surface, rather than the mucosal surface, that causes the release of spermatozoa, i.e. spermatozoa remain attached to the isthmic mucosa until they become capacitated and then detach and migrate to the ampulla to fertilize the eggs.     

Alpha-L-fucosidase from bull seminal plasma: its purification and acrosome reaction promoting property

Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.     

Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status

Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the head alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.     

Inner acrosomal membrane of mammalian spermatozoa: its properties and possible functions in fertilization

The inner acrosomal membrane (IAM) develops during the spermatid stage of differentiation as that portion of the Golgi-derived acrosome granule that tightly associates with the condensing sperm nucleus. In some mammalian species, an electron-dense proteinaceous material accumulates between the IAM and the nuclear envelope, collectively comprising the "perforatorium." Evidence, including its partial purification and its structural resistance to detergents and sonication, suggests that the IAM is an unusually resiliant membrane. Dense paracrystalline arrays of intramembranous particles, a lack of lectin-mediated receptor modulation, and its lack of participation in sperm-egg fusion suggest that the IAM lacks the same degree of fluidity as the egg surface plasmalemma. Observations using monoclonal antibodies, however, suggest that some specific antigenic modulations may be possible within the IAM. Its structural rigidity is of obvious mechanical value during sperm penetration through the zone pellucida. An additional role as a scaffold for putative zona lysin material remains controversial. Biochemical evidence suggests that acrosin, for example, is not entirely soluble and that some remains sperm-associated, depending on the conditions of acrosome disruption. Nevertheless, morphological studies do not agree on acrosin's specific localization to the IAM. Currently there is only very limited information concerning the localization of the other acrosomal enzymes to the IAM. Another possible role for the IAM in some species may be in recognizing the zona pellucida. Evidence for this derives from the observation that fucoidin, a fucose heteropolysaccharide, inhibits guinea pig sperm-zona binding, and bound fucoidin can be localized to the IAM and equatorial regions of the living acrosome-reacted spermatozoa. Finally, the IAM may have a role in early recognition/adhesion with the colemma.     

Multiple causes of pregnancy failure in hamsters precociously ovulated by human chorionic gonadotropin

Cycling adult female hamsters can be induced to mate and ovulate 24 h early by the injection of 20 IU human chorionic gonadotropin (hCG) at 1500 h on Day 3 (day before proestrus), but pregnancy is not established. Although there is evidence of decreased sperm transport in precociously ovulated females, this does not appear to be the primary cause of infertility. Reduced size and vascularity of corpora lutea (CL) in treated females suggests incomplete or failed CL activation. Control and hCG-treated females were killed by exsanguination under ether anesthesia at intervals for the first 5 days after mating. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, estradiol, and progesterone were measured by radioimmunoassay. Luteinizing hormone in treated animals was very high at 2200 h on Day 1 after mating (31 h after the hCG injection), due to endogenous release, and dropped below control levels thereafter. Follicle-stimulating hormone, by contrast, was significantly lower than controls at 2200 h on Day 1 and remained low until 2200 h on Day 3 after mating. Prolactin in treated animals was not different from that in controls, except for 1000 h on Day 4, when it showed a significant dip. Estradiol in treated animals was significantly higher than in controls at 2200 h on Day 1 (when LH was also high and FSH was low), and remained high at 1000 h and 2200 h on Day 2, dropping thereafter to control levels. Progesterone was initially at control levels but had dropped significantly by 1000 h on Day 2 and remained low for the next 24 h. These results suggest that pregnancy failure is due to inadequate activation of corpora lutea. This may be due to: 1) immaturity of follicles at the time of ovulation; 2) inappropriate timing of preovulatory events; 3) the luteolytic effects of high levels of LH or estradiol or both; 4) the low level of FSH in the early stages of corpus luteum development; or 5) a combination of the above. Abnormalities of prolactin secretion were not investigated in detail but cannot be ruled out at this time.     

Sperm motility initiation factor is a minor component of the Pacific herring egg chorion

Polyclonal antibodies were generated to the 105 kDa herring sperm motility initiation factor (SMIF) and used to explore the role of SMIF in sperm-egg interaction. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with SMIF antibodies, it was demonstrated that SMIF is present as a minor (4–7% of total chorion protein) component of the chorion. The major polypeptides in the chorion migrated at 117 kDa and in a grouping between 48–54 kDa, with other minor bands above and below. The only detectable glycosylated component was the 105 kDa band, which was resolved at two isoelectric points (8.22 and 8.31) after isoelectric focusing gel electrophoresis. Using antibodies to SMIF, fertilization was blocked, sperm motility was inhibited in vitro in the presence of solubilized SMIF and SMIF binding sites on sperm were localized. Lastly, SMIF was localized to the region of the herring egg that encircles the micropyle.     

Factor in urinary extracts from pregnant women that inhibit mouse oocyte maturation in vitro

Mouse oocyte maturation inhibitory factors, on the basis of inhibitory activity of spontaneous germinal vesicle breakdown (GVBD) of denuded mouse oocytes in culture, were extracted and partially purified by reversed-phase resin adsorption and Sephadex G-100 and G-50 column chromatographies from the urine of pregnant women. Denuded oocytes obtained from ovaries of ICR mice underwent spontaneous GVBD by cultivation for 3 h in modified Krebs–Ringer's buffered solution, while this spontaneous GVBD was found to be inhibited by adding the final preparation (U-D-4) of urine. The inhibition was dose dependent, ranging from 0.6 to 10 μg protein/ml medium. Oocytes treated with U-D-4 and resuspended in control medium resumed GVBD. The molecular mass of U-D-4 was estimated to be less than 2,000 Da with gel filtration. Ether treatment failed to extract inhibitory factor(s) from U-D-4 and pepsin treatment inactivated U-D-4, indicating that inhibitory factor(s) in U-D-4 are peptide-like substances. The inhibitory effect of U-D-4 on spontaneous GVBD was partially reversed in the presence of naloxone, a potent opioid antagonist. U-D-4s obtained from urine samples of pregnant women, nonpregnant women, and men showed the inhibitory effect on spontaneous GVBD; however, the activity of U-D-4 obtained from pregnancy urine was significantly more potent than those of the other urine samples. © 1993 Wiley-Liss, Inc.     

In vitro neurotoxicity of amyloid β-peptide cross-linked by transglutaminase

Transglutaminase catalyzes the intermolecular cross-linking of peptides between Gln and Lys residues, forming an -(-glutamyl) lysine bond. Amyloid -peptide, a major constituent of the deposits in Alzheimer disease, contains Lys16, Lys28, and Gln15 which may act as substrates of transglutaminase. Transglutaminase treatment of amyloid -peptide (1–28) and amyloid -peptide (1–40) yielded cross-linked oligomers. Transglutaminase-treated A retarded neurite extension of PC12 cells, and rat cultured neurons of hippocampus and septum, brain areas severely affected by Alzheimer disease, and subsequently caused cell death, whereas the transglutaminase-untreated counterparts did not show harmful effects. The transglutaminase-catalyzed oligomers of amyloid -peptide and their neurotoxicity may be involved in two characteristics in Alzheimer disease, neuronal degeneration and formation of the insoluble deposits.Abbreviations: AD – Alzheimer disease, A – amyloid -peptide, DMEM – Dulbecco's modified Eagle's medium, DMEM/F–12–1:1 mixture of DMEM and Ham's F–12 medium, FCS – fetal calf serum, HS – horse serum, PAGE – polyacrylamide gel electrophoresis, MTT – 3-(4,5-dimethylthiazol–2-yl)–2,5-diphenyltetrazolium bromide, NGF – nerve growth factor, TGase – transglutaminase.     

Photomicrobial sensors for selective determination of phosphate

A photomicrobial sensor consisting of immobilized Chlorella vulgaris and an oxygen electrode has been developed for selective determination of phosphate. When 40 mM phosphate was added to the sensor system, the photocurrent increased to a maximum under light irradiation with a response time of 1 min. The current increased with increasing phosphate concentration in the range 8–70 mM. Selectivity of the sensor was satisfactory. Good agreement was obtained between the phosphate concentrations in lake water determined by the photomicrobial sensor and by conventional colorimetry (correlation coefficient 0.96).     

Photomicrobial electrode for selective determination of sulphide

Summary A photomicrobial electrode, which uses the photosynthetic bacteria Chromatium sp. in conjunction with a hydrogen electrode, was developed for the determination of sulphide. The response time of the photomicrobial electrode was 5–10 min. A linear relationship was obtained between the current of the electrode and the sodium sulphide concentration below 3.5 mM. The minimum detectable concentration of sodium sulphide was 0.4 mM. Selectivity of the sensor is satisfactory. A good agreement was obtained between the photomicrobial electrode and the ethylene blue method (correlation coefficient: 0.90).