Genital morphology of female goblin spiders (Arachnida: Araneae: Oonopidae) with functional implications

Spider genital morphology usually provides the best characters for taxonomy. Furthermore, functional genital morphology helps to understand the evolution of complex genitalia and their role in the context of sexual selection. The genital systems of most haplogyne spider families are poorly investigated with respect to their morphology. The present study investigates the female genitalia of the oonopids Oonops pulcher, Oonopinus kilikus, and Pseudotriaeris sp. by means of light microscopy and SEM. The male palps are briefly described. Females of O. pulcher store spermatozoa in an anterior and a posterior receptaculum (PRe). The genitalia resemble the primitive dysderoid genitalia supporting the hypothesis that the subfamily Oonopinae contains more basal oonopids. In O. kilikus, the anterior receptaculum is reduced to a sclerite. Spermatozoa are stored in a PRe. The receptacula of Pseudotriaeris sp. are reduced to sclerites. Spermatozoa in the uterus internus indicate that fertilization happens there or in the ovary. The anterior sclerite might serve females to lock the uterus during copulation as suggested for other gamasomorphines. The male palp of O. kilikus is simple, whereas the palps of O. pulcher and Pseudotriaeris sp. appear more complex. Complicated structures on the palp of Pseudotriaeris sp. indicate that males exert copulatory courtship.     

Sperm dumping in a haplogyne spider

The present study shows that females of Silhouettella loricatula (Arachnida: Araneae: Oonopidae) manage to process sperm in an unusual and previously unknown way. The male ejaculate consisting of spermatozoa and globular secretion is enclosed in a secretory sac. This may avoid the mixing of sperm from different males and at least severely limit sperm competition. The process of sperm enclosure occurs within the female's sperm storage site (receptaculum) as the ejaculate is not surrounded by a sac inside the male's sperm-transferring organs (palpal bulbs). The secretion forming the sac is produced by glands adjoining the receptaculum. The possibility that globular secretions in the male palpal bulbs partly contribute to the sac cannot be ruled out completely. It is suggested that in S. loricatula , the main function of sperm enclosure in a sac is enabling females to dump the ejaculate of a male. The present study represents the first report on sperm dumping in the family Oonopidae. During five first and three second copulations in the laboratory, the dumping of a sac was observed. One dumped sac was sectioned and contained spermatozoa. Two couples were flash-fixed with liquid nitrogen early during copulation, which revealed the mechanism of the sac dumping. By muscle contractions, the receptaculum is bent backwards and the sac moved into the genital opening. The actual sac dumping occurs most probably in cooperation with the male, which moves his pedipalps rhythmically during the entire copulation. Extensions and furrows on the emboli suggest that they may additionally be used as copulatory courtship devices. The enclosure of sperm from the current male in secretion takes place during or immediately following copulation as all mated females sacrificed after copulation had a new sac containing spermatozoa in the receptaculum. Dumping sperm of a previous male during the next copulation may allow females to bias sperm precedence.     

Genital morphology of the haplogyne spider <Emphasis Type="Italic">Harpactea lepida</Emphasis> (Arachnida,Araneae, Dysderidae)

Female Harpactea lepida possess a single genital opening leading into a diverticulum. This diverticulum shows no secretory layer. It continues posteriorly into a receptaculum which is associated with gland cells. In the two already described dysderids, Dysdera crocata and D. erythrina, the bilobed spermatheca lies anteriorly to the diverticulum. Gland cells are associated with the spermatheca and the diverticulum. In H. lepida, the sclerotized genital structures lie dorsally to the diverticulum and consist of a posterior and an anterior part. The posterior part shows a lamella extending laterally to sclerites functioning as muscle attachments. The anterior part has two roundish structures. A hollow stalk-like sclerite functioning as muscle attachment extends towards anterior. The posterior and the anterior part of the sclerotized genital structures fit together. A narrow uterine valve connecting the uterus externus with the diverticulum forms between them. It may be opened by muscles as also suggested for D. erythrina. In H. lepida, spermatozoa embedded in secretion are found in the diverticulum and the receptaculum. There is no evidence that they are stored under different conditions like in D. erythrina. Additional spermatozoa are found in the uterus externus of H. lepida which could be an indication for internal fertilization. Spermatogenesis occurs in cysts in the testes of male H. lepida. In the vasa deferentia, the ductus ejaculatorius and the palpal bulb, the spermatozoa are embedded in homogenous secretion. The palpal bulb has a distal extension bearing a crown-like structure. The embolus is situated at the base of the extension. In memoriam of Konrad Thaler.     

Comparative immunoreactivity of mouse and hamster sperm proteins recognized by an anti-P26h hamster sperm protein

The binding of the spermatozoon to the zona pellucida is a species-specific phenomenon. We have previously shown that the binding of hamster sperm to the homologous zona pellucida involves a sperm 26-kDa glycoprotein, the P26h, originating in the epididymis. In order to establish to what extent this sperm protein is involved in the species-specific recognition of the egg's extracellular coat, we have compared the inhibitory properties of anti-P26h antibodies in a sperm-zona pellucida assay using hamster and mouse gametes. Anti-P26h IgGs inhibit, in a dose-dependent manner, gamete interactions in both species, although in a less efficient manner in the mouse than in the hamster. While anti-26kDa Fab fragments are as efficient as the intact IgG to inhibit hamster sperm-zona pellucida binding, they have no effect on mouse gamete interaction. ELISA, Western blot, and immunohistochemical experiments have been performed in order to characterize the mouse antigen(s) recognized by the anti-P26h antiserum. ELISA and Western blots showed that this antiserum recognized two proteins on mouse spermatozoa that are less reactive than the hamster P26h. These antigens are localized in the acrosomal region of epididymal spermatozoa of both species. These results indicate that the hamster P26H involved in zona pellucida interaction has certain unique epitopes, while others are common to the sperm of both species. © 1995 Wiley-Liss, Inc.     

Temperature preference and tolerance of hybrid carp (female grass carp,Ctenopharyngodon idella x male bighead,Aristichthys nobilis)

Synopsis This study examined the effects of various pH values on sperm motility times of white suckers,Catostomus commersoni, from two artificially acidified lakes in the Experimental Lakes Area (ELA), northwestern Ontario. White sucker sperm were active (motile) in water at all pH values tested (3.0–7.0). The maximum sperm motility time was 76.9 seconds and the minimum time was 51.9 seconds. Sperm motility times of fish from Lake 302N were similar to those of fish from Lake 223 at all pH values except pH 5.0 and 5.5. At these two pH values, sperm motility times of Lake 223 fish were longer than those of Lake 302N fish. In both lakes sperm motility at ambient lake pH levels (Lake 223 + 5.1, Lake 302N + 6.3) were similar to those at pH 7.0. Motility times of all fish tested were within the range of time during which fertilization normally occurs, indicating that reproductive failures in Lake 223 were probably not caused by impairment of sperm motility times.     

Male reproductive traits, semen cryopreservation, and heterologous in vitro fertilization in the bobcat (Lynx rufus)

There is limited information on bobcat ejaculate traits and sperm cryopreservation and fertilizing ability. Bobcats were electroejaculated under general anesthesia in November (autumn) and April (spring), and endocrine and sperm traits were characterized. Testosterone (mean ± SEM: 0.90 ± 0.15 ng/mL) was not different between sampling times, but cortisol (average: 13.95 ± 1.73 μg/dL) was significantly higher in April. Average number of spermatozoa was 10.0 ± 3.4 × 10⁶ sperm/ejaculate, with values being significantly higher in April. Sperm motility (average 55.7 ± 5.8% motile sperm) was not different between sampling times. The proportion of normal spermatozoa in the ejaculate (average: 14.7 ± 2.1%) was significantly higher in April, but the percentage of spermatozoa with intact acrosomes (average: 43.7 ± 3.8%) was significantly higher in autumn. Spermatozoa were cryopreserved in a Tes-Tris-based diluent (TEST) or Biladyl, both containing 20% egg yolk and 4% glycerol. Diluted sperm were loaded into straws, refrigerated using a programmable thermoblock with a dry chamber, frozen in nitrogen vapors, thawed, and incubated in F-10 medium with 5% fetal bovine serum for up to 3 h. After cryopreservation in TEST, there were about 50% motile sperm upon thawing, and survival was high during incubation post-thaw. Cryopreservation in Biladyl led to similar results, but motility decreased substantially during incubation post-thaw. Bobcat spermatozoa fertilized domestic cat oocytes matured in vitro. Fertilization rates were higher for sperm collected in April and cryopreserved in TEST (46%) than for those cryopreserved using Biladyl (<3%). Fertilized oocytes cleaved in culture, and some (27%) reached the morula stage. This study has allowed us to gain further baseline information on bobcat reproduction, explore sperm cryopreservation conditions, and show that fertilizing capacity can be tested using in vitro-matured cat oocytes. These results will be important for future conservation efforts.     

Fate of sperm organelles during early embryogenesis in the rat

The report is part of a continuing study in which we employ monoclonal antibodies to membrane domains and internal organelles of rat spermatozoa in order to trace events during maturation, capacitation, fertilization, and early development. In the present study, we have used immunocytochemistry at the light and EM levels to localize one antibody, 5A5, to the fibrous sheath and a second, 3D5, to the outer mitochondrial membrane. Antibody 5A5 does not stain the fibrous sheath of spermatozoa of rodents other than the rat, while 3D5 can be localized to the outer mitochondrial membrane of rat, hamster, and mouse spermatozoa. In order to follow these antibodies during fertilization and early embryogenesis, we developed a method to stain internal components of zygotes and early embryos. Our findings suggests that the fibrous sheath disappears prior to the first cleavage and that mitochondria can be detected up to the 2-cell stage in mouse and the 4-cell stage in rat. © 1994 Wiley-Liss, Inc.     

Inhibition of the vacuolar H(+)-pump with bafilomycin A1 does not induce acrosome reaction or activate proacrosin in mouse spermatozoa

Acrosomal protease activation is regarded as an important event triggered by acrosomal reaction and leading to sperm passage through zona pellucida. Mammalian acrosome has an internal acid pH that probably helps to maintain inactive proenzymes that otherwise could be precociously activated and prevent normal fertilization. In this work, we have studied the effect of bafilomycin A1, a potent and specific inhibitor of vacuolar H(+)-pump (V-ATPase), on acrosome reaction and proacrosin activation. We used the pH-sensitive probe Lysotracker Green DND-26 to monitor qualitatively intra-acrosomal pH in cauda epididymal mouse spermatozoa. Our results showed that loss of Lysotracker label induced by bafilomycin A1 (acrosome alkalinization) did not induce acrosome reaction or proacrosin activation. We also developed a new technique for imaging the acrosome, and for evaluating the acrosome reaction, in live mouse spermatozoa using Lysotracker DND-26. These results showed that the V-ATPase is a key regulator of mammalian acrosome pH, and that acrosome alkalinization is not the only prerequisite to activate proacrosin under in vivo conditions. Our results suggest that acrosome alkalinization and acrosome reaction are two processes that could be independently regulated during mammalian sperm capacitation.     

Functional properties of caltrin proteins from seminal vesicle of the guinea pig.

Caltrin proteins from seminal vesicle content of the guinea pig bind with great specificity to different regions of the spermatozoa. Indirect immunofluorescence studies with polyclonal antibodies showed that caltrin I binds to the head, on the acrosomal cup, while caltrin II binds on the principal tail and the neck. No fluorescence was detected either in the midpiece or in the post-acrosomal area of the head when sperm were exposed to either of the caltrins. Calcium-induced hyaluronidase release, which occurs during the acrosomal reaction, was dramatically inhibited by caltrin I (approximately 85% inhibition). Caltrin II was less effective in preventing the enzyme release (approximately 50% inhibition). Chemical modification of the structure modified the biological activity of the two caltrins. Reduction and carboxymethylation of the cysteine residues diminished the inhibitory activity on 45Ca2+ uptake and reduced the ability of the proteins to react with their antibodies. Removal of the carbohydrate portion by chemical deglycosylation transformed the inhibitor proteins into enhancers of calcium uptake into the spermatozoa. Caltrin proteins from the guinea pig appear to play the same physiological role as bovine caltrin, regulating specifically calcium transport across the spermatozoal membranes related with the acrosome reaction and hyperactivation process. The dual behavior of caltrins to inhibit or enhance Ca2+ uptake enables them to fulfill this function. Nevertheless, molecular mechanisms different from those described for bovine caltrin seem to be involved in the control of the functional activity of the guinea pig caltrins.     

Characterization of a cDNA clone coding for human testis membrane cofactor protein (MCP,CD46)

Membrane cofactor protein (MCP) is a complement regulatory protein that acts as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. We have previously reported the characterization of a functional MCP molecule on the acrosomal membrane. This protein migrated as a single band with a molecular weight of 40,000 Da, which is 10,000–20,000 Da smaller than the known MCP molecules, and is devoid of N-and O-linked sugars. We have proposed that the difference in molecular weight resulted from the lack of sugars. To investigate if this is due to the absence of glycosylation sites, we have characterized a cDNA clone from a human testis cDNA library. This cDNA corresponds to a peculiar MCP form previously described, which is characterized by the presence of the serine/threonine/proline-rich exon C (STP^C) and the cytoplasmic tail known as CYT², and we conclude that the absence of mature oligosaccharide of the sperm MCP cannot be totally attributed to a defect of N- and O-glycosylation sequences but rather reflects an alteration of the mechanisms of glycosylation in spermatozoa. The presence of functional MCP on the acrosomal membrane, as well as the other complement regulatory protein, decay-accelerating factor, strongly suggests that these proteins may act concomitantly to protect the acrosome-reacted spermatozoa from the attack of the complement present in the female genital tract. © 1993 Wiley-Liss, Inc.