Inhibition of the human sperm acrosome reaction by proteinase inhibitors

The analogue of the second messenger cAMP, dibutyryl cAMP (dbcAMP), was shown to induce the human sperm acrosome reaction to the same extent as calcium ionophore A23187, providing preliminary evidence for the involvement of the adenylate cydase system in the acrosome reaction (AR) of human spermatozoa. Using the human synchronous acrosome reaction system, proteinase inhibitors were tested for their effect on the dbcAMP-induced human sperm acrosome reaction. The proteinase inhibitor 4′-acctamidophenyl4-guanidinoben-zoate (AGB), an inhibitor of proacrosin activation and of acrosin, when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, significantly (P < 0.01) inhibited the acrosome reaction at final concentrations of 1 × 10^?4 M to 1 × 10^?6 M in comparison to dbcAMP treatment alone. At concentrations less than 1 × 10^?6 M, no significant inhibitory effect was seen. Similarly, para-aminobenzamidine (pAB), also an inhibitor of proacrosin activation and of acrosin, significantly (P < 0.01) inhibited the dbcAMP-induced acrosome reaction at final concentrations of 1 × 10-4 M to I × 10-6 M when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, in comparison to stimulation by dbcAMP alone. However, at concentrations less than 1 × 10^?6 M, no significant (P > 0.05) inhibitory effect was seen. These results indicate that a serine proteinase, most likely acrosin, has a role in the human sperm acrosome reaction and suggest that the enzyme functions after the involvement of the adenylate cyclase system.     

Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS

Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP-106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.     

Functional morphology of the reproductive system of Galathea intermedia (Decapoda: Anomura)

Spermatophore formation in Galathea intermedia begins in the proximal part of the vas deferens. The contents subsequently form a spermatophoric ribbon, the so-called "secondary spermatophore," in its distal part. A strongly muscular ductus ejaculatorius is present in the coxa of the fifth pereiopod which builds up pressure for the extrusion of the spermatophoric ribbon. After extrusion, the ribbon is caught by the first gonopod, while the second gonopod dissolves the matrix of the ribbon. During copulation the spermatophores are randomly placed on the sternum of the female, near the genital opening, by the fifth pereiopods of the male. Subsequent ovulation of the female via the genital opening, an active process accomplished through muscular activity, results in fertilization of the eggs by the exploding spermatophores. External intersexes are characterized by both male and female external sexual characters, but in all individuals only male gonads are present. No trace of a female reproductive system could be detected. Thus, these external intersexes are exclusively functional males.     

The young sperm gambit

Thermodynamic and oxidative damage to the condensed nuclear DNA of spermatozoa may result in age-related deterioration that results in zygote death. By definition, any such spermatozoan-age effects are direct (i.e. nonheritable) and, if they occur, females may be selected to avoid such costs. This paper examines the potential of spermatozoan-age effects to provide alternative explanations for several reproductive phenomena.     

Semen collection and evaluation in free‐ranging Brazilian rattlesnakes (Crotalus durissus terrificus)

Two hundred‐ninety species of reptiles are estimated to need urgent action for conservation, with at least 113 threatened species worldwide. The International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species includes 80 species of snakes, with six native Brazilian species, a number likely to be an underestimation. Some authors believe that assisted reproduction would be an important tool to improve reproduction in captivity of some reptiles. An efficient technique for semen collection and evaluation is an important step in development of protocols for cryopreservation of semen or artificial insemination in snakes, contributing to the conservation of endangered species. Although these techniques are important, some basic semen parameters are described for four of the ～2,900 snake species in the world. The Brazilian rattlesnake (Crotalus durissus terrificus) was chosen as a model for semen collection in snakes because it is found quite often in Sao Paulo State. Semen was collected once from each animal by the same investigator during the mating season of this species in Brazil. After antiseptic cleansing of the skin around the cloaca, the snakes were injected subcutaneously with a dose of 15 mg/kg of 1% solution of lidocaine around the cloaca. Semen was collected with ventral massages after cloacal relaxation and directly from genital papilla inside the cloaca. A total of 28 ejaculates from 39 animals were obtained, representing collection efficiency of 71.80%. Semen volume and concentration in Brazilian rattlesnakes ranged from 3–70 µl and from 0.94–2.23 × 10⁹ spermatozoa/ml, respectively. Zoo Biol 0:1–6, 2007. © 2007 Wiley‐Liss, Inc.     

Sexual selection drives weak positive selection in protamine genes and high promoter divergence,enhancing sperm competitiveness

Phenotypic adaptations may be the result of changes in gene structure or gene regulation, but little is known about the evolution of gene expression. In addition, it is unclear whether the same selective forces may operate at both levels simultaneously. Reproductive proteins evolve rapidly, but the underlying selective forces promoting such rapid changes are still a matter of debate. In particular, the role of sexual selection in driving positive selection among reproductive proteins remains controversial, whereas its potential influence on changes in promoter regions has not been explored. Protamines are responsible for maintaining DNA in a compacted form in chromosomes in sperm and the available evidence suggests that they evolve rapidly. Because protamines condense DNA within the sperm nucleus, they influence sperm head shape. Here, we examine the influence of sperm competition upon protamine 1 and protamine 2 genes and their promoters, by comparing closely related species of Mus that differ in relative testes size, a reliable indicator of levels of sperm competition. We find evidence of positive selection in the protamine 2 gene in the species with the highest inferred levels of sperm competition. In addition, sperm competition levels across all species are strongly associated with high divergence in protamine 2 promoters that, in turn, are associated with sperm swimming speed. We suggest that changes in protamine 2 promoters are likely to enhance sperm swimming speed by making sperm heads more hydrodynamic. Such phenotypic changes are adaptive because sperm swimming speed may be a major determinant of fertilization success under sperm competition. Thus, when species have diverged recently, few changes in gene-coding sequences are found, while high divergence in promoters seems to be associated with the intensity of sexual selection.     

Inhibition of adenosine-metabolizing enzymes modulates mouse sperm fertilizing ability: A changing role for endogenously generated adenosine during capacitation

The effect of inhibiting adenosine-metabolizing enzymes on sperm fertilizing ability was studied to investigate a possible role for endogenously generated adenosine in the regulation of capacitation. The compounds used have been shown to be effective inhibitors of the relevant enzymes in similarly incubated mouse sperm suspensions. Inhibition of 5′-nucleotidase activity with α,β-methylene adenosine 5′-diphosphate (AMPCP), to reduce available endogenous adenosine, caused a dose-dependent inhibition of the fertilizing ability of partially capacitated spermatozoa, which was significant with 100 and 250 μM AMPCP. Conversely, inhibition of adenosine deaminase with 100 nM coformycin, to increase available endogenous adenosine, promoted the fertilizing ability of partially capacitated spermatozoa when the fertilization rate of control suspensions was low. However, coformycin had no effect on sperm suspensions with moderate fertilizing ability, and it inhibited fertilizing ability when added to capacitated spermatozoa. These data are consistent with a promotion of the early stages of capacitation by endogenously generated adenosine and suggest that sensitivity to adenosine changes as capacitation proceeds. Because the majority of adenosine-metabolizing enzyme activity resides in or is directed toward the extracellular compartment in such suspensions, these effects of adenosine may be mediated at the outer surface of the cell. By interacting with receptors on adenylate cyclase, externally produced adenosine could modulate intracellular levels of cyclic adenosine monophosphate (cAMP), thereby influencing fertilizing ability.     

In vitro embryo production in camel (Camelus dromedarius) from in vitro matured oocytes fertilized with epididymal spermatozoa stored at 4 degrees C

Experiments were conducted to study the effect of storing epididymal spermatozoa, in tris-tes- and tris-lactose egg yolk extenders, on their fertilizing ability and subsequent in vitro embryo development. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 degrees C and on ice (0-1 degrees C), respectively. Cumulus oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25COCs/well) containing 500 microL of maturation medium and cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in air for 36 h. Spermatozoa were collected from the cauda epididymides in syringes containing 2-3 mL of either tris-tes- or tris-lactose egg yolk extender. They were cooled down slowly and stored at refrigeration (4 degrees C) temperature. The spermatozoa were evaluated for motility and used for IVF of IVM oocytes on the day of collection and after 2, 4, 6 and 8 days of storage. On the day of IVF, spermatozoa were prepared by the swim up technique and both spermatozoa and oocytes were co-incubated at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air for 15-16 h. Presumptive zygotes were either fixed and stained with Hoechst 33342 for evaluation of fertilization or were cultured in 500 microL of the culture medium at 38.5 degrees C in an atmosphere of 5% CO(2), 5% O(2) and 90% N(2) in air. There was no significant difference (P>0.05) in the proportion of oocytes fertilized with spermatozoa stored in either of the two extenders for up to 8 days. The proportion of oocytes that cleaved (43-60%) and those that developed to blastocysts (14-21%) did not show any difference (P>0.05) either, when spermatozoa from different days of storage were used. First cleavage was observed as early as 16 h after IVF, early blastocysts had developed by day 4, expanded blastocysts after day 5 and hatching of blastocysts started after day 6 of culture. It may be concluded that dromedary epididymal spermatozoa survive in storage for at least 8 days in tris-lactose- and tris-tes egg yolk diluents at 4 degrees C. These spermatozoa maintain fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures.     

Magnetic-activated Cell Sorting before Cryopreservation Preserves Mitochondrial Integrity in Human Spermatozoa

Superparamagnetic annexin-V conjugated microbeads are able to eliminate spermatozoa with externalized phosphatidylserine, a membrane feature of apoptotic cells as well as spermatozoa with deteriorated plasma membrane. Our objective was to evaluate the effects of annexin-V Magnetic-Activated Cell Sorting (MACS) in cryopreservation–thawing protocols and on integrity of sperm mitochondrial transmembrane potential and mitochondrial integrity survival rate (MSR). Mature spermatozoa of 10 healthy donors were prepared by density gradient centrifugation and divided into 2 aliquots afterwards. The first one was subjected to annexin-V MACS followed by cryopreservation and thawing, while the second was cryopreserved–thawed without MACS to serve as control. Annexin-negative sperm separated by MACS showed significantly higher levels of intact mitochondria following cryopreservation–thawing (45.4±8.6%) compared to sperm that were not separated (15.8±4.6%, p<0.01). Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize cryopreservation–thawing outcome. MACS using annexin-V microbeads enhances the percentage of spermatozoa with intact transmembrane mitochondrial potential and mitochondrial integrity survival rates following cryopreservation.     

Hyperactivation is the mode conversion from constant-curvature beating to constant-frequency beating under a constant rate of microtubule sliding

Flagellar beating of hyperactivated golden hamster spermatozoa was analyzed in detail using digital image analysis and was compared to that of nonhyperactivated (activated) spermatozoa in order to understand the change in flagellar beating during hyperactivation and the active microtubule sliding that brought about the change in flagellar beating. Hyperactivated flagellar beating, which was characterized by a sharp bend in the proximal midpiece and low beat frequency, was able to alter the waveform with little change in beat frequency (constant-frequency beating), whereas activated flagellar beating, which was characterized by a slight bend in the proximal midpiece and high beat frequency, was able to alter beat frequency with little change in the waveform (constant-curvature beating). These results demonstrate that flagellar beating of hyperactivated and activated spermatozoa were essentially different modes and that hyperactivation was the mode conversion from constant-curvature beating to constant-frequency beating. Detailed analysis of flagellar bends revealed that the increase in curvature in the proximal midpiece during hyperactivation was due to the increase in total length of microtubule sliding in a nearly straight region between bends, while the rate of microtubule sliding remained almost constant.