INSECT MOLECULAR BIOLOGY, Issue 1 2005
M. Hatakeyama
Abstract Germline transformation using a piggyBac -derived vector is feasible in the sawfly, Athalia rosae. A previously generated transgenic line carrying green fluorescence protein (GFP) genes as reporters was successfully maintained and preserved without consecutive rearing. Sperm taken from males that were frozen directly in liquid nitrogen and stored at ,80 °C for a year were microinjected into mature unfertilized eggs dissected from female ovaries. A fraction of the sperm-injected eggs was fertilized and developed into diploid females, and all of them expressed GFP. Haploid male progeny from these females segregated into GFP-positive and GFP-negative individuals in a ratio of 1 : 1 indicating heterozygosity of the parental females. The GFP genes were stably inherited staying at the location where they were originally integrated. [source]

Characterization of human sperm N -acetylglucosaminidase

INTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2008
S. L. Perez Martinez
Summary N -acetylglucosaminidase (NAG) is particularly active in mammalian spermatozoa and appears to be involved in fertilization. Although it is assumed that this enzyme is acrosomal, previous results from our laboratory suggest the presence of NAG at the sperm plasma membrane level. The present study attempted to analyse the subcellular distribution of this enzyme in human spermatozoa. Sperm were incubated under different conditions and NAG activity measured in the soluble extracts and cell pellets using a specific fluorometric substrate. A significant proportion of NAG activity was released when sperm were incubated in culture medium, suggesting a weak association with the plasma membrane. This location was confirmed by western blot analysis of plasma membrane fractions and immunofluorescence on non-permeabilized sperm, which showed a positive signal mainly on the acrosomal domain. The distribution of NAG activity between plasma membrane and acrosome was analysed after cell disruption by freezing and thawing. Triton X-100 stimulated sperm and epididymal NAG activity but not the enzyme obtained from other sources. In addition, biotinylated human recombinant NAG was able to bind to human sperm. Finally, after sperm incubation under capacitating conditions, NAG total activity increased and the sperm enzyme lost its ability to be stimulated by Triton X-100. The possible connection of these results with sperm maturation, capacitation and NAG participation in primary binding to the zona pellucida, was discussed. [source]

Use of the Sperm-Class Analyser® for objective assessment of human sperm morphology

INTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2003
C. Soler
Summary The Sperm-Class Analyser® was validated for assessing morphometric parameters of the head and midpiece of unwashed and washed human ejaculated spermatozoa from volunteers providing a wide range of semen quality. A higher proportion of sperm could be assessed (86% fresh semen and 75% washed sperm) if Hemacolor staining was used rather than DiffQuik (80 and 73%) or Papanicolaou (78 and 68%). Different stains employed different fixatives and the area, length, width and perimeter of the sperm head was significantly larger for washed sperm stained by Hemacolor and DiffQuik. Acrosomal area ranged from 48 to 51% of the sperm head area and this percentage was larger for washed sperm stained with DiffQuik. Sperm at the end of the slide, distant from the initial semen droplet, were larger in area and perimeter than those at that site or in the middle. The high precision and reproducibility of the equipment required assessing only 50 sperm on the slide. Far greater variation was found in head width, relative acrosomal area and midpiece width between different slides prepared from the same ejaculate, highlighting the inherent variability within the ejaculate and smear preparation, and requiring more than one slide to be assessed. [source]

A preliminary report on the implication of RT,PCR detection of DAZ, RBMY1, USP9Y and Protamine-2 mRNA in testicular biopsy samples from azoospermic men

INTERNATIONAL JOURNAL OF ANDROLOGY, Issue 1 2002
A. Friel
In this study, reverse transcription,polymerase chain reaction (RT,PCR) was optimized to analyse the presence of DAZ, RBMY1, USP9Y, protamine-2, SRY and actin messenger RNA (mRNA) in testicular cells of men suffering from idiopathic azoospermia. All samples (n=28), including five controls, showed normal expression of actin, SRY and USP9Y. Sperm was not recovered from eight patients after testicular biopsy. Of these, four patients showed altered mRNA levels for the fertility genes, DAZ, RBMY1 and protamine-2. One patient, who was previously shown to be azoospermia factor region (AZF)b deleted, lacked RBM mRNA and presented with reduced amplification of protamine-2 mRNA. This correlated with previous studies, which proposed that RBM expression is exclusive to AZFb and that the lack of testicular RBMY1 mRNA results in suppressed spermatogenesis. Two patients were each lacking DAZ mRNA but did show expression of RBMY1 mRNA at a reduced level, suggesting that there might be residual spermatogenesis in the absence of DAZ expression. Protamine-2 mRNA was detected in one patient and was absent in the second patient. Finally, one patient lacked DAZ, RBMY1 and protamine-2 mRNA. The 19 remaining azoospermic patients presented with normal expression patterns for each of the fertility genes studied. This study demonstrates that the expression of spermatogenesis-specific genes varies in azoospermia. The study of the expression of such genes in a larger number of patients might be useful in characterizing and identifying subpopulations of azoospermic men. [source]

Sperm morphology in the black coral Cirrhipathes sp. (Anthozoa, Antipatharia)

INVERTEBRATE BIOLOGY, Issue 3 2008
Elda Gaino
Abstract. Male polyps of the antipatharian Cirrhipathes sp., collected along the coral reef of Siladen Island (Sulawesi, Indonesia), were studied in order to gain an insight into the reproductive biology. Spermatocysts (maximum size 120 ,m) are located within the primary gametogenic mesenteries and are separated by mesenteric cell cytoplasmic extensions. Sperm, maturing along radial rows, have a fairly round shape and contain a series of electron-dense vesicles in the apical nuclear region. A single mitochondrion flanks the nucleus. A peculiar cup-like electron-dense body, edged with regularly spaced electron-dense granules, is interposed between the nucleus and the tail, and delimits a central region that includes two centrioles. Cross-sections of the cup-like body reveal that the distal centriole has a pericentriolar system, consisting of nine arms arranged in a radial pattern. Each arm branches into three processes that are connected to the electron-dense granules. Indirect evidence of spawning is derived from the accumulation of sperm in the gastric cavity. This process takes place through the lysis of the cells bordering the mesenteries. Intact cells of this bordering layer appear to be involved in the phagocytosis of non-expelled gametes. [source]

The Presence of a Glycosyl Phosphatidylinositol-Anchored ,-Mannosidase in Boar Sperm

IUBMB LIFE, Issue 6 2000
Masako Kuno
First page of article [source]

Inhibitory effect of osmotic concentration, potassium and pH on motility of the sperm of the North American burbot Lota lota maculosa

JOURNAL OF FISH BIOLOGY, Issue 1 2007
M. D. Zuccarelli
Seminal plasma factors maintaining North American (NA) burbot Lota lota maculosa sperm quiescent were examined. Sperm were diluted into buffered saline solutions of various compositions and motility assessed. After 1 h in these solutions at 10° C, aliquots of the suspension were diluted with tap water and motility again assessed. Dilution of sperm in an incubation solution containing Ca2+ in the absence of K+ initiated sperm motility resulting in low motility when sperm were subsequently diluted in tap water. Incubation solutions with osmolalities >200 mOsm kg,1 and containing 12·5 mM K+ prevented the onset of sperm motility and were associated with maximal sperm motility upon dilution in tap water. Sperm maintained at lower osmolalities exhibited limited motility upon dilution in tap water indicating interdependence between K+ and osmolality in maintaining sperm quiescent in the presence of Ca2+. Sperm kept in incubation solution at pH values < c. 7·5 for 1 h demonstrated reduced motility when subsequently diluted in tap water. That motility of sperm was pH sensitive was further indicated by CO2 inhibition of motility. Therefore, NA burbot sperm are probably maintained in an immotile state, yet with potential for motility, by combination of high K+, osmolality and possibly pH. The results from this study differ from published information on sperm quiescence in the temporally and geographically distinct Eurasian burbot Lota lota lota. [source]

The development of haddock and Atlantic cod sperm cryopreservation techniques and the effect of sperm age on cryopreservation success

JOURNAL OF FISH BIOLOGY, Issue 2 2004
R. M. Rideout
Three cryoprotectants [dimethyl sulphoxide (DMSO), propylene glycol (PG) and glycerol], two diluents (sucrose- and saline-based), two sperm collection times, two freezing rates and three times between thaw and activation (0, 30 and 60 min) were tested in order to develop a protocol for the cryopreservation of sperm of haddock Melanogrammus aeglefinus and Atlantic cod Gadus morhua. The faster freezing rate resulted in extremely low post-thaw motility in comparison to the slower freezing rate, which was successful for sperm from both gadids. In both cases, the use of PG resulted in significantly higher post-thaw sperm motility-recovery indices than with DMSO or glycerol, which did not differ significantly from one another. Diluent had no effect on post-thaw sperm motility for Atlantic cod or haddock. Sperm collected at the end of the spawning season tended to have reduced post-thaw motility compared to that collected 2 weeks after the start of spawning. A 30 min delay between thaw and activation of haddock and Atlantic cod sperm resulted in a significant decrease in sperm motility. When PG was used as cryoprotectant, sperm motility continued to decrease between 30 and 60 min post-thaw. With DMSO or glycerol as cryoprotectant, motilities were already very low after 30 min post-thaw and did not decrease any further after 60 min. Cryoprotectant, diluent and time between thaw and activation had no effect on mean or maximum sperm swimming speeds for either Atlantic cod or haddock sperm. Fertilization success for haddock eggs, like sperm motility, was higher with PG-frozen sperm than DMSO- or glycerol-frozen sperm. These results constitute the first reported successful cryopreservation of haddock sperm and improve on previous methods used to cryopreserve sperm from Atlantic cod. [source]

Variations of sperm release in three batches of zebrafish

JOURNAL OF FISH BIOLOGY, Issue 2 2004
J. R. Kemadjou Njiwa
By collecting and counting the number of sperm released during separate matings in three batches of zebrafish Danio rerio, aged 3,4, 4,5 and 5,6 months, males were observed to release sperm before the female started laying their eggs. After the female left the nest, the number and motility of sperm and life span of sperm of younger fish were higher than those of older fish in water samples collected under the nest and at the surface of the tank. Sperm were released in the form of sperm trails laid on the nest surface, subsequently active spermatozoa left the trails and moved in the water for several minutes. Sperm trails consisted of bands of viscous material in which the sperm were embedded. In most cases eggs were not laid directly over the sperm trail, suggesting that sperm may contact the eggs after the latter are released into the water. In all the three tested groups there was no significant difference (P > 0·05) between the number of sperm collected on some portions of the acetate sheets which lined the nest ceiling. This result demonstrated that the greater activity of younger fish accelerated the sperm dispersal in water. Male sperm duct glands, seminal vesicles, known to secrete mucosubstances are probably involved in the production of sperm trails. The possible influence of insemination on the mating style of zebrafish is discussed. [source]

MORPHOLOGY, LIFE HISTORY, AND MOLECULAR PHYLOGENY OF STSCHAPOVIA FLAGELLARIS (TILOPTERIDALES, PHAEOPHYCEAE) AND THE ERECTION OF THE STSCHAPOVIACEAE FAM.

JOURNAL OF PHYCOLOGY, Issue 6 2004

The phenology, life history, ultrastructure of reproductive structures, and molecular phylogeny using rbcL and rDNA (5.8S, internal transcribed spacer 2, and partial 26S) gene sequences of Stschapovia flagellaris, endemic to the northwestern Pacific Ocean, were studied. This species was first classified in the order Delamareales together with Delamarea, Coelocladia, and Cladothele. Those three genera, however, were later transferred to Dictyosiphonales, whereas the systematic position of Stschapovia remained unclear. At Abashiri, Hokkaido, Japan, the species regenerated by forming a new erect thallus from a perennial crustose holdfast or by presumably parthenogenetic development of eggs released from the erect thallus. There was no alternation of generations. In winter, the monoecious erect thallus formed reproductive structures (i.e. plurilocular antheridia and oogonia) in the thickened part of the thallus. Sperm had a chloroplast with an eyespot and a long anterior and short posterior flagellum. Eggs contained numerous disc-shaped chloroplasts, physodes, and vacuoles. Neither sexual attraction of the presumptive sperm by eggs nor their sexual fusion was observed. Molecular phylogenetic analyses revealed the closest phylogenetic relationship between Stschapovia and Halosiphonaceae, and they grouped with Phyllariaceae and Tilopteridaceae (Tilopteridales s. s.). Stschapovia and Tilopteridaceae have several important morphological similarities: chloroplasts lacking pyrenoids, lack of sexual reproduction despite the release of obvious sperm, occurrence of monoecious gametophytes, and similarity in the early developmental pattern of the erect thallus. In conclusion, we propose the establishment of the new family Stschapoviaceae to accommodate Stschapovia and the placement of the family in the order Tilopteridales together with Tilopteridaceae, Halosiphonaceae, and Phyllariaceae. [source]

ARE SPERM LIMITING IN THE SEA?

JOURNAL OF PHYCOLOGY, Issue 2000
M. L. Berndt
The reproductive success of marine species with external fertilization depends on environmental conditions during gamete release. There is special interest presently in whether water motion causes sperm limitation under natural conditions. We investigated gamete release of Fucus vesiculosus from an exposed shore to ascertain: 1) when gametes are released during the tidal cycle, 2) when fertilization occurs, and 3) what the natural sperm:egg ratios are. Water samples were collected and concentrated over five minutes every half hour off Pemaquid Point, ME from three replicate sites within each of two locations using a pump-filter device. Immunofluorescence microscopy revealed that gamete release occurred only on the two calmest spring tides. Sperm became present in the water column at the same time as oogonia (30 min,1 h prior to high tide [HT]) and reached peak concentration at exactly HT. The sperm:egg ratio was 76:1 on 8 Oct 1999 and 21:1 on 8 Nov 1999 at exactly 30 min prior to HT and dropped sharply after HT. Gametes continued to be collected for several hours after HT but analysis of pronuclear position in aceto-iron-hematoxylin stained eggs revealed that all fertilization occurred at approximately HT. We modelled the total number of days when reproduction was possible using these results and wind and wave data from the National Data Buoy Center. Our research provides evidence that gamete release by F. vesiculosus occurs at slack HT on calm days and that sperm are not a limiting factor in fertilization for this species. [source]

Improved Cryopreservation of Sperm of Paddlefish (Polyodon spathula)

JOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 4 2006
ÁKos Horváth
Experiments were performed to improve protocols for sperm cryopreservation of paddlefish (Polyodon spathula), a species for which there has been limited study. The first experiment was conducted to investigate the effects of two extenders (modified Tsvetkova's extender: mT and modified Hanks' balanced salt solution: mHBSS) in combination with methanol (MeOH) and dimethyl sulfoxide in two concentrations (5 and 10%) on the postthaw motility and fertilization rates of cryopreserved sperm. The highest postthaw motility (85 ± 5%) was observed when sperm were frozen using mT extender with 10% MeOH as cryoprotectant. Extenders (P = 0.0018) and cryoprotectants (P = 0.0040) each had a significant effect on the postthaw motility of paddlefish sperm. The highest fertilization (80 ± 3%) was found when eggs were fertilized with sperm frozen with mT extender in combination with 10% MeOH. However, there was no significant difference among fertilization rates when MeOH was used as a cryoprotectant in either concentration or in combination with either mT or mHBSS extenders. In the second experiment, 4000 eggs were fertilized with the pooled contents of five straws of thawed sperm (total volume of 1.25 mL) using mT extender in combination with 5% MeOH, and hatch rates as high as 79 ± 5% were observed. A third experiment was also conducted to clarify the role of MeOH concentration; however, no significant difference was found among fertilization and hatch rates when either 5 or 10% MeOH was used as a cryoprotectant. These results suggest that MeOH is a safe and reliable cryoprotectant for freezing of paddlefish sperm and obtaining viable postthaw sperm for consistent fertilization and hatch rates. Further, this experimental protocol is relatively simple and applicable for commercial hatchery production of paddlefish. [source]

The Effects of Osmolality, Cryoprotectant and Equilibration Time on Striped Bass Morone saxatilis Sperm Motility

JOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 3 2003
Shuyang He
Four experiments were designed to evaluate the effects of osmolality, cryoprotectant, and equilibration time on striped bass sperm motility. In the first experiment, solutions of NaCI or KCI with osmolalities ranging from 0 to 700 mmol/kg were tested on sperm activation. Over 60% of the sperm were activated by isotonic NaCI and KCI solutions with a treatment osmolality of 350 mmol/kg. Sperm remained motile until osmolality increased to 600 mmol/ kg. In the second and third experiments, Extenders 1, 2 and 3 with osmolalities of 350, 500, and 600 mmol/kg, respectively, were tested. Sperm samples stored in Extender 2 showed significantly higher (P 0.01) sperm motility after 10 min of exposure as well as greater (P < 0.01) post-thaw motility when compared to samples stored in Extenders 1 and 3. In the fourth experiment, two trials were carried out to evaluate the effects of cryoprotectant and equilibration time. In the first trial, methanol with a concentration of 5% and 10% yielded the highest (P < 0.05) sperm motility prior to freezing at all equilibration times examined. However, 5% DMSO yielded the highest (P < 0.01) post-thaw motility (38 ± 3.6%). DMSO with concentrations of 10% and 15% resulted in 17 ± 2.3% and 6 ± 1.0% post-thaw motility, respectively. Both methanol and DMA, at all concentrations tested, resulted in less than 10% post-thaw motility. In the second trial, four DMSO concentrations with three different equilibration times were examined. We observed a significant (P < 0.001) interaction effect between DMSO concentration and equilibration time. Post-thaw motility was significantly greater (P < 0.01) with a concentration of 5% DMSO at all equilibration times examined, compared to 1.25, 2.5, and 10% DMSO. An average post-thaw motility of 40 ± 2.9% was achieved after 10 min equilibration using 5% DMSO. [source]

Morphology and reproduction of Nipponomysella subtruncata (Yokoyama), a galeommatoidean bivalve commensal with the sipunculan Siphonosoma cumanense (Keferstein) in Japan

JOURNAL OF ZOOLOGY, Issue 4 2001
J. Lützen
Abstract The shell and anatomy of Nipponomysella subtruncata is described. The bivalve is attached singly or in groups of up to nine on Siphonosoma cumanense, a burrowing intertidal sipunculan in south-west Japan. The species is a protandrous hermaphrodite. Specimens 1.4,2.5 mm long are males, which between 2.1 and 3 mm in length reverse sex and remain females. Reproduction peaks in summer and the annual number of clutches is small. Ripe oocytes, 84,88 ,m diameter, are spawned into the suprabranchial cavity where they develop into 107-,m-long straight-hinged veligers. Following a planktotrophic period of unknown duration, the c. 360-,m-long spat normally settle upon and attach to the shells of larger, predominantly female, individuals. At a length of 1,1.6 mm they detach again and live separately thereafter. Sperm are transferred in spermatophores and stored within paired, mushroom-shaped receptacles located posteriorly in the female's suprabranchial cavity. The receptacles first appear in large males or in specimens in the process of reversing sex. Stored sperm probably survive long enough to fertilize more than one clutch. The anatomy of Nipponomysella is characteristic of the Montacutidae, and is of especial interest because of the unique structure of the sperm receptacles. [source]

Reproductive biology of the onychophoran Euperipatoides rowelli

JOURNAL OF ZOOLOGY, Issue 4 2000
Paul Sunnucks
Abstract The reproductive biology of the ovoviviparous peripatus Euperipatoides rowelli was investigated from field collections and laboratory cultures. The sexes have different demographics. The frequency distribution of individual weight is essentially L-shaped in females, but closer to normality for males: thus the sexes must exhibit different patterns of growth and/or mortality. Males are generally much smaller and rarer than females. The primary sex ratio seems to be 1:1 with equal investment in the sexes, while the tertiary ratio is highly female-biased. Logs with fewer individuals tend to be male-biased while well-populated logs tend to be female-biased. Males mature at 15,30% of the bodyweight of mature females. The weight frequency distribution of males without developed sperm in their tracts is strongly skewed to the lower weights, while that of males with sperm is more normally distributed, indicating that sperm production occurs as soon in life as possible. Males mature in their first year of life, if growth rates in culture may be extrapolated to the wild. In contrast to this rapid maturity in males, females may mature as late as their second or third years. Most mature females, and many prior to maturity, carry sperm in their spermathecae. After maturity, there is an approximately linear relationship between body mass and number of developing embryos. Reproduction in E. rowelli is significantly seasonal despite high individual variance, with a major bout of parturition in November,December (summer). A female can harbour one developed and one undeveloped batch of embryos in each uterus. Excesses of developed embryos in one uterus are counterbalanced by deficits of undeveloped ones, indicating that females can use their paired reproductive tracts independently. Individual females in culture can experience episodes of parturition approx. 6 months apart without re-mating, thus gestation may be 6 months or more. Sperm in spermathecae remain capable of vigorous swimming for at least 9.5 months. [source]

Early male reproductive advantage, multiple paternity and sperm storage in an amphibian aggregate breeder

MOLECULAR ECOLOGY, Issue 6 2003
J. A. Tennessen
Abstract We tested whether the order in which males encounter females affects reproductive fitness in spotted salamanders (Ambystoma maculatum). Using mating chambers in the field, we allowed one male access to a female before a second male. We then used four microsatellite markers in paternity analyses of the resulting larvae. First males sired a significantly larger number of offspring than second males, suggesting that male reproductive success is greatly enhanced by early arrival at breeding ponds. Multiple paternity was common among clutches, and frequently larvae were assigned to unidentified males that had not been in the chambers. Sperm from these males had either been stored by females for a year or obtained more recently at other breeding sites. [source]

Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2010
Sebastian Canovas
Sperm mediated gene transfer (SMGT) could provide the opportunity to carry out transgenesis on a mass scale using spermatozoa as vectors for exogenous DNA. However, the efficiency of sperm-mediated DNA transfer is still questionable, and the mode of transmission to the egg has not yet been well understood. Our aim was to investigate the capacity of bovine spermatozoa to carry exogenous DNA and its relationship to sperm functionality. We studied these parameters using flow cytometry to measure viability (necrosis and apoptosis) and capacitation status, computer-assisted semen analysis (CASA) to measure motility parameters and in vitro fertilization (IVF) to assess fertilizing capacity. Furthermore, we studied the effect of capacitation status on interaction with exogenous DNA, and the role of heparin supplementation in this process. Bull spermatozoa showed a high capacity to bind DNA quickly and reached a maximum after 30,min, with approximately half of the DNA-bound spermatozoa being viable. Incubation with exogenous DNA induced a decrease in sperm viability and motility and increased the proportion of apoptotic cells, but did not affect the cleavage rate in IVF assay. Heparin increased high-lipid disorder and the number of sperm with DNA bound (viable and dead). In conclusion, this study shows that live spermatozoa can bind exogenous DNA with a slight negative effect in some parameters of sperm function that in our opinion, would not drastically compromise fertility. Mol. Reprod. Dev. 77: 687,698, 2010. © 2010 Wiley-Liss, Inc. [source]

Superoxide dismutase content and fatty acid composition in subsets of human spermatozoa from normozoospermic, asthenozoospermic, and polyzoospermic semen samples

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003
J. Calamera
Abstract Human ejaculated sperm comprised discrete subsets of spermatozoa, with different degrees of maturation. These subpopulations can be isolated through density gradient centrifugation. Sperm from the lowest density layer show the highest content of docosahexaenoic acid and sterols, and produce the highest levels of reactive oxygen species. The main objective of this study was to determine the superoxide dismutase (SOD) content and fatty acid composition of subsets of spermatozoa isolated from normozoospermic, asthenozoospermic, and polyzoospermic semen samples. Four sperm fractions (1,4) were obtained using ISolate gradient centrifugation. Morphology, motion parameters, SOD content, and fatty acid composition were assessed in the original samples and their fractions. Overall, sperm from normozoospermic samples had higher SOD content than those of asthenozoospermic or polyzoospermic samples. Once fractionated in subsets, the sperm SOD content decreased significantly (P,<,0.0001) from fraction 1 (top) to 4 (bottom) in all three groups of samples. Fatty acid content as well as the oxidation coefficient followed the same pattern, decreasing from fraction 1 to 4 (F1,F4). Normo- and polyzoospermic samples showed similar amounts of fatty acids, while asthenozoospermic samples mostly revealed increased levels. Normozoospermic samples displayed the lowest unsaturated fatty acid (UFA)/SOD ratio. Spermatozoa from astheno- and polyzoospermic samples, two common seminal pathologies, showed higher UFA and lower SOD content than normal sperm, therefore exhibiting a higher susceptibility to peroxidative damage. F4 from all groups, containing the most mature spermatozoa, displayed the lowest polyunsaturated fatty acid and SOD content of all subsets, suggesting that excessive SOD activity as well as abundant peroxidative targets may both be deleterious to sperm function. Mol. Reprod. Dev. 66: 422,430, 2003. © 2003 Wiley-Liss, Inc. [source]

Marked sperm dimorphism in the ground beetle Scarites terricola: a novel type of insect sperm polymorphism

PHYSIOLOGICAL ENTOMOLOGY, Issue 4 2009
KÔJI SASAKAWA
Abstract. Sperm polymorphism describes the phenomenon of male ejaculates containing two or more distinct types of sperm. In insects, four types of sperm polymorphism are recognized in species from the orders Diptera, Hemiptera, Hymenoptera, Lepidoptera and Coleoptera. The present study describes dimorphic sperm of the ground beetle Scarites terricola (Coleoptera: Carabidae) as a novel type of sperm polymorphism in insects. Sperm from the spermatophore and male seminal vesicles are examined at the light-microscopic level, and both display marked dimorphism. One type has sperm formed into bundles, in which the head of numerous spermatozoa are ,glued' together, with tails free-moving. The other type are free as single spermatozoa and have a disproportionately large-sized head and an elongated tail. Both types are motile in Ringer's solution. The adaptive and phylogenetic importance of these findings is discussed. [source]

Sperm transfer during mating in the pharaoh's ant, Monomorium pharaonis

PHYSIOLOGICAL ENTOMOLOGY, Issue 3 2006
D. ALLARD
Abstract Sperm transfer in the pharaoh's ant Monomorium pharaonis (L.) is studied by making longitudinal sections through the gasters of mating pairs fixed in copula. Sperm is transferred inside a spermatophore similar to those found in two other ants, Diacamma sp. from Japan and Carebara vidua. Sharp teeth-ridges are present on the penis valves and, during copulation, these teeth make contact with a thick and soft cuticular layer covering the bursa copulatrix. This ensures an attachment long enough for the successful transfer of the spermatophore to the right position inside the female oviduct. The thick cuticle also protects the queen from serious damage by the male's sharp claspers. After a first successful copulation, sperm is still present inside the male's seminal vesicles, suggesting that males can mate multiply. Additional experiments, where single, initially virgin males are presented to several virgin females, confirm this. [source]

Effect of Carbohydrates on the Ability of Bull Sperm to Bind to Bovine Oviduct Epithelial Cells

REPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2009
Y Kon
Contents In the present study, we investigated the effect of various carbohydrates on the ability of bovine spermatozoa to bind to the bovine oviduct epithelial cells (OECs). We also examined the fertilization competence and motility of spermatozoa that bind to OECs in the presence of carbohydrates. Frozen-thawed spermatozoa were incubated with OECs, with and without various carbohydrates. The sperms were then divided into two fractions: OEC-binding sperms (B-sperm) and non-OEC binding sperms (NB-sperm). The fertilization rate, ability to bind the zona pellucida, and membrane integrity of the spermatozoa as determined using a hypo-osmotic-swelling test (HOST) were lower in NB-sperm than in the unseparated spermatozoa (control). The motility of the B-sperm was maintained for a longer time than that of the control spermatozoa. The addition of N -acetyl- d -glucosamine (GlcNAc, 5 mm) to the sperm-OEC mixture increased the number of B-sperm. D -mannose (5 mm) and D -fucose (5 mm) had no effect on the number of B-sperm. The motility of B-sperm, which bound to OECs in the presence of GlcNAc, however, was not maintained. When either OECs or the spermatozoa were treated with GlcNAc prior to sperm-OEC co-incubation, only sperm-side treatment enhanced sperm-OEC binding, but B-sperm motility was not maintained. The motility of spermatozoa incubated with GlcNAc was lower than that of controls. These results indicate that GlcNAc enhances sperm binding to OECs, probably via sperm surface modification, but does not promote increased sperm survival. [source]

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm,Egg Binding, Fertilization and Embryo Development

REPRODUCTION IN DOMESTIC ANIMALS, Issue 6 2008
RF Gonçalves
Contents Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 × 105 frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events. [source]

Moderate Seasonality in Testis Function of Domestic Cat

REPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2007
S Blottner
Contents Adult male domestic cats are known to produce sperm throughout the year, although sexual activity is influenced by geographical location. In the northern hemisphere, feral domestic cats reproduce usually between January and July. Thus, seasonality in testicular activity might be suggested. The aim of the present study was to investigate gametogene and endocrine activity of cat testis throughout the entire year. Testes and epididymides (n = 10,12 per month) were collected after castration. Spermatogenesis was quantified by assessment of testicular sperm per testis and by flow cytometric analysis of the cells with different DNA content. Sperm from cauda epididymis were evaluated according to motility and morphological integrity. Testicular testosterone concentration was determined by enzyme immunoassay. Testis mass and sperm production varied moderately throughout the year. Significant seasonal variations were observed in the proportion of cells in the G2/M phase of cell cycle (p = 0.004) and the meiotic transformation (ratio of haploid : tetraploid cells; p = 0.021). Changes in testicular testosterone concentration were more pronounced and showed periods with high (spring) and significantly reduced testosterone levels (autumn). A marked seasonal alteration (p < 0.001) with a peak in March was assessed in the percentage of progressively motile sperm. The proportion of morphological intact sperm was also significantly higher in spring compared with winter time (p < 0.001). In conclusion, the study suggests moderate seasonal changes in quantity of sperm, more pronounced annual variation in hormone production and a distinct seasonal influence on functional sperm parameters in domestic cat. [source]

Effect of Insemination,Ovulation Interval and Addition of Seminal Plasma on Sow Fertility to Insemination of Cryopreserved Sperm

REPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2007
M Abad
Contents In swine, the use of frozen-thawed (FT) sperm for artificial insemination (AI) is limited because of poor sow fertility, possibly associated with a post-thaw capacitation-like status resulting in fewer fully viable sperm. Sow fertility to AI with FT sperm may improve with deeper deposition of sperm within the female tract, insemination very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We performed two experiments to examine these suggestions. In experiment 1, 122 multiparous Yorkshire sows received 600 IU equine chorionic gonadotrophin at weaning and 5 mg pLH 80 h later to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after pLH injection. Thereafter, sows were assigned on the basis of parity to a single AI of FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with 10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO. All semen doses were adjusted to 3 × 109 live cells and deposited into the cervix. Experiment 2 employed 99 multiparous crossbred sows and repeated the treatments of experiment 1 except that all FT inseminations were intrauterine. In both experiments, farrowing rates were lower (p < 0.01) following FT inseminations with no effect of time of insemination or of supplemental SP. In experiment 1, litter size was smaller following FT insemination (p < 0.05), but no effect on litter size was evident in experiment 2. Supplemental SP had no effect on litter size in either experiment. The lack of effect of either SP or timing of FT insemination on sow fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more than just cryocapacitation. [source]

Evaluation of Fertilizing Potential of Frozen-thawed dog Spermatozoa Diluted in ACP-106® using an In Vitro Sperm,Oocyte Interaction Assay

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2007
RCS Cardoso
Contents The aim of present study was to evaluate frozen canine semen with ACP-106® (Powder Coconut Water) using an in vitro sperm,oocyte interaction assay (SOIA). Ten ejaculates from five stud dogs were diluted in ACP-106® containing 20% egg yolk, submitted to cooling in a thermal box for 40 min and in a refrigerator for 30 min. After this period, a second dilution was performed using ACP-106® containing 20% egg yolk and 12% glycerol. Samples were thawed at 38°C for 1 min. Post-thaw motility was evaluated by light microscopy and by using a computer aided semen analysis (CASA). Plasma membrane integrity and sperm morphology/acrosomal status were evaluated by fluorescent probes (C-FDA/PI) and Bengal Rose respectively. Moreover, frozen-thawed semen was analysed by a SOIA. Subjective post-thaw motility was 52.0 ± 14.8% and it was significant higher than the total motility estimated by CASA (23.0 ± 14.8%) because this system considered the egg yolk debris as immotile spermatozoa. Although normal sperm rate and acrosomal integrity evaluated by Bengal Rose stain was 89.6 ± 3.1% and 94.3 ± 3.1%, respectively, post-thaw percentage of intact plasma membrane was only 35.1 ± 14.3%. Regarding SOIA, the percentage of interacted oocytes (bound, penetrated and bound and/or penetrated) was 75.3%. Using regression analysis, it was found significant relations between some CASA patterns and data for SOIA. In conclusion, the freezing-thawing procedure using ACP-106® was efficient for maintain the in vitro fertility potential of dog spermatozoa. [source]

Studies on the Effect of Supplementing Boar Semen Cryopreservation Media with Different Avian Egg Yolk Types on in Vitro Post-thaw Sperm Quality

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2006
R Bathgate
Contents Fertility after insemination of cryopreserved boar semen is currently below that of fresh semen. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using an adapted Westendorf method in which the chicken egg yolk was replaced by either duck or quail egg yolk. The different composition of the yolk types, particularly the amount of cholesterol, fatty acids and phospholipids, were thought to potentially afford a greater level of protection to sperm against damage during freezing and thawing. Sperm frozen in medium containing chicken egg yolk displayed higher motility immediately after thawing, but there was no difference in the motility of sperm frozen with different types of egg yolk 3 or 6 h after thawing and maintenance at 37°C. Sperm frozen in media containing chicken or duck egg yolk had a higher proportion of intact acrosomes immediately after thawing than sperm frozen in medium containing quail egg yolk, but 6 h after thawing and maintenance at 37°C the sperm that had been frozen in medium containing chicken egg yolk had a higher proportion of intact acrosomes than the sperm frozen in media containing duck or quail egg yolk. Analysis of the composition of the different yolk types showed that the basic components of the yolks were similar, but the ratios of fatty acids and phospholipid classes differed. Duck egg yolk had more monounsaturated fatty acids (MUFA) than chicken egg yolk, which had more MUFA than quail egg yolk. Duck egg yolk contained more phosphotidylinositol (PI) than chicken or quail egg yolks and quail egg yolk contained more phosphotidylserine than either chicken or duck egg yolks. The differences in post-thaw motility and acrosome integrity of boar sperm when frozen in media containing the different types of egg yolk may be due to the variation in composition. [source]

The Use of HOS Test to Evaluate Membrane Functionality of Boar Sperm Capacitated in vitro

REPRODUCTION IN DOMESTIC ANIMALS, Issue 6 2002
D Lechniak
Contents The functional and structural integrity of sperm membrane are crucial for the viability of spermatozoa. The commonly used staining test (eosin + nigrosin) for assessing sperm membrane measures only its structural integrity. The hypoosmotic swelling test (HOS) originally developed for human sperm (Jeyendran et al. 1984) has been also applied to several species of domestic animals (bull, pig, horse, dog). The test enables to evaluate the functional status of the sperm membrane. The principle of HOS is based on water transport across the sperm tail membrane under hypoosmotic conditions. It has previously been used to assess the semen quality (Revell and Mrode 1994), to analyse fertilizing capacity (Rota et al. 2000; Perez-Llano et al. 2001) and also to detect viable, immotile cells for ICSI (Intra-cytoplasmic sperm injection) in human (Zeyneloglu et al. 2000). There are two procedures commonly used for sperm capacitation in the pig-sperm washing and incubation before insemination (Nagai 1994). Capacitation involves several changes like removing molecules coating the sperm head membrane, changes in membrane fluidity and intracellular ion concentration (Green and Watson 2001). Thus the membrane integrity as well as functionality may be affected as shown by Harrison (1996). The aim of the present study was to analyse changes in sperm membrane integrity after in vitro capacitation by use of the HOS test. [source]

Intraperitoneal Insemination in Mammals: A Review

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2002
JL Yaniz
Contents This review focuses on factors associated with the development of intraperitoneal insemination in mammals. Findings to date indicate that fertility improves as the sperm cell concentration rises, but that the optimal sperm number differs in each species. Sperm washing before intraperitoneal insemination favours fertility. Peritoneal fluid shows a variable effect on spermatozoa, depending on the hormonal status of the female. The optimal time for insemination appears to be just prior to ovulation. The technique may be performed either through the abdominal or the vaginal wall. Verification of sperm deposition in the proximity of the ovaries improves fertility rates. Although associated with some risk of infection and an immune reaction against spermatozoa, the intraperitoneal technique rarely gives rise to severe anaphylactic shock, peritonitis, adhesion formation and the production of anti-sperm antibodies and these complications may be prevented by adequate sperm pretreatment and antibiotic therapy. The success of intraperitoneal insemination in humans, with results comparable with those of intrauterine insemination in the treatment of infertility, suggest the potential use of this technique in domestic mammals, especially in those in which intrauterine insemination poses practical difficulties. Some of the methods applied in human intraperitoneal insemination, such as confirming the position of the needle in the peritoneal cavity, and sperm pre-treatments might also improve results in domestic species. Conversely, the use of the animal model should help to develop some aspects of this technique in humans. [source]

ORIGINAL ARTICLE: In situ Reconstruction of Humoral Immune Response Against Sperm: Comparison of SCID and NOD/SCID Mouse Models

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2009
Beata Grygielska
Problem, Comparison of two types of immunocompromised mouse strains (SCID and NOD/SCID) for production of human antisperm antibodies (AsA). Method of study, Human peripheral blood lymphocytes (PBL) were grafted to mouse peritoneal cavity and sensitized with natively glycosylated and N-deglycosylated sperm extracts. Results and conclusion, NOD/SCID mice inoculated with hu-PBLs exhibited higher AsA titres with a tendency for greater sperm agglutination than human AsA raised in SCID mouse model. A comparison between ,native' and deglycosylated sperm extracts revealed higher agglutination titres by sera induced with the latter ones. Inhibitory effect of human polyclonal AsA in sperm penetration assay, however, produced opposite results to that for agglutination. Western immunoblotting was used to evaluate reactive sperm antigens prior to and after in situ sensitization showing multiple bands different from positive reactions brought by original sera of in vivo primed AsA-positive males. It seems that in situ generated AsA recognized sperm entities different from those revealed by blood donor's sera samples. [source]

REVIEW ARTICLE: Mechanism of Prolonged Sperm Storage and Sperm Survivability in Hen Oviduct: A Review

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2008
Shubash C Das
A unique property of the avian oviduct is to store sperm for a prolonged period. The sperm storage tubules (SST) are located in the utero-vaginal junction of the oviduct, where sperm can be stored and survived for a few weeks after insemination or natural mating. The immune system in the oviduct is essential to prevent tissue infection by various microorganisms, and it may also affect the fate and survivability of sperm in the oviduct. Anti-sperm immunoresponses including infiltration of leukocytes may be induced in the vagina of the oviduct. Sperm that will participate in fertilization may be selected by these immunoresponses. However, sperm stored in the SST may be protected from the immunoresponse by SST structures and transforming growth factor ,, whose expression is increased during sperm storage in the SST. In this review, the mechanism of sperm survivability with reference to the regulation of anti-sperm immunoresponses in hen oviduct is emphasized. [source]