Protective effects of iodixanol during bovine sperm cryopreservation

The aim of cryopreservation is to maintain cellular integrity, thereby enabling resumption of proper biological functioning after thawing. Here we propose OptiPrep™ (60% iodixanol in water) as a protectant during sperm cryopreservation using pooled bull semen as the model. We evaluated OptiPrep concentration effect and its relation to cryopreservation by comparing frozen-thawed and chilled samples. Semen, extended in Andromed^® with 0 (control), 1.25%, 2.5%, and 5% OptiPrep™, was compared after either chilling or freezing in large volume by directional freezing. Sample evaluation included sperm motility upon thawing and after 3 h incubation at 37 °C for frozen-thawed samples and after 3 h and 6 h of chilling for chilled samples; viability, acrosomal integrity, and hypoosmotic swelling were also tested for frozen-thawed and chilled samples. Chilled samples with 5% OptiPrep™ showed inferior viability (P = 0.047) and 3 h motility (P = 0.017) relative to that for chilled samples with 2.5% OptiPrep and inferior viability (P = 0.042), acrosomal integrity (P = 0.045), and 0 h motility (P = 0.024) relative to that for chilled samples with 1.25% OptiPrep. The 1.25%, 2.5%, and control samples did not differ. In frozen-thawed samples, 2.5% OptiPrep was superior to all other concentrations for 3 h motility (control, P = 0.007; 5% OptiPrep, P = 0.005; 1.25% OptiPrep, P = 0.004) and to 1.25% OptiPrep for acrosomal integrity (P = 0.001). In a search for a protection mechanism, we measured glass transition temperature (T_g) of Andromed^® and of Andromed^® with 1.25%, 2.5%, and 5% OptiPrep™. Andromed^® (-58.78 °C) and 1.25% OptiPrep™ (-58.75 °C) groups had lower mean T_g than that of the 2.5% (-57.67 °C) and the 5% (-57.10 °C) groups. Directional cryomicroscopy revealed that the presence of iodixanol alters ice crystal formation into an intricate net of dendrites. Thus, iodixanol appears to possess cryoprotective properties by helping spermatozoa maintain motility and membrane integrity, possibly through altering ice crystals formation into a more hospitable environment and increasing the glass transition temperature.     

Ability of chicken spermatozoa to undergo acrosome reaction after liquid storage or cryopreservation

The effects of in vitro storage on the sperm's ability to undergo the acrosome reaction (AR) have never been studied in avian species despite its major importance for reproduction management.The ability of chicken sperm to undergo the AR was measured after liquid storage at 4 °C and after cryopreservation, and its relationship with other semen quality parameters, including viability, mass motility and objective motility parameters measured by computer semen analyser (CASA) was analysed in two different flocks. The percentage of intact acrosome-reacting spermatozoa (IAR) was dramatically decreased by 48 h liquid storage (loss of 2/3 among the spermatozoa initially able to undergo the AR) whereas motility, viability and morphological integrity were reduced by 10-15%. By contrast, cryopreservation did not affect the induction of AR in flock 1 (29% IAR) whereas it was strongly affected in flock 2 (7% IAR). Motility parameters, viability and morphology were considerably altered by freezing in every case (more that 50% loss). Positive correlations were found between the percentage of intact acrosome-reacting spermatozoa and viability, mass motility and many objective motility parameters.Our results showed that the sperm's ability to undergo the AR was much more affected than other sperm functions after storage at 4 °C, while cryopreservation only had an effect in semen with the lowest initial quality. These results raise questions regarding the specific features of chicken sperm biology that must be taken into account in the treatment of semen.     

Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation

The objective was to evaluate apoptotic markers in ejaculated equine spermatozoa after separation by density-gradient centrifugation and after cryopreservation. Subpopulations of percoll-separated equine spermatozoa differed (P < 0.05) in the percentage of live, caspase-activated spermatozoa (2.9 ± 0.7% vs 14.2 ± 6.4%; mean ± S.E.M.), low mitochondrial membrane potential (MMP; 6.8 ± 1.1 vs 23.8 ± 3.7), altered plasma membrane permeability (1.3 ± 0.2 vs 3.0 ± 0.5), DNA fragmentation (2.0 ± 1.3 vs 14.3 ± 3.6), total motility (81.8 ± 3.3 vs 35.1 ± 5.4), and progressive motility (66.3 ± 4.3 vs 24.1 ± 4.5) for high-density versus low-density subpopulations, respectively. Phosphatidylserine externalization did not differ (P = 0.67) between the high- and low-density subpopulations (2.6 ± 0.7 vs 3.1 ± 0.9). After cryopreservation, equine spermatozoa differed (P < 0.01) in the percentage of active caspases (19.1 ± 1.6 vs 52.1 ± 2.8), low MMP (18.2 ± 2.5 vs 48.7 ± 2.6), altered plasma membrane permeability (6.8 ± 1.7 vs 17.6 ± 2.0), total motility (75.5 ± 2.4 vs 45.2 ± 5.6), and progressive motility (53.9 ± 3.1 vs 28.3 ± 4.5) for pre-freeze versus cryopreserved spermatozoa. There was no difference (P = 0.21) in percentage of DNA fragmented cells before (5.5 ± 1.2) versus after cryopreservation (6.6 ± 1.1). We concluded that apoptotic-like changes were detectable in ejaculated equine spermatozoa and were more prevalent after cryopreservation.     

Effect of quercetin on the motility of cryopreserved canine spermatozoa

The addition of an antioxidant to cryopreservation solutions for preventing oxidative stress to sperm from several species, including that from humans, has been studied previously. Quercetin is a flavonoid contained in subarctic trees with freeze resistance and is known to be a strong antioxidant. Therefore, the effect of quercetin on the cryopreservation of dog spermatozoa was examined in this study. The proportions of total motile spermatozoa were significantly higher at 30, 60, 90, 120, and 150 min and at 60, 120, and 150 min after thawing in groups treated with 5 μg/ml and 10 μg/ml of quercetin dissolved in 0.1% DMSO added to the second extender based on skim milk compared to that in the control group, respectively. There were no differences between the experimental groups in the proportion of total motile spermatozoa during the observation periods. The proportion of total motile spermatozoa among those treated with 5 μg/ml of quercetin in 0.1% DMSO was improved by approximately 10–20% at 30–180 min after thawing compared to that in the control group. To evaluate the fertility of cryopreserved spermatozoa treated with quercetin, 2 × 10⁸ spermatozoa were transcervically inseminated into bitches, and a total of 18 puppies were delivered in three bitches. These results indicated that supplementation of quercetin as a cryoprotectant to the skim milk-based extender improved the motility of cryopreserved spermatozoa from dogs compared to those of the control group. And fertility of cryopreserved spermatozoa with quercetin supplementation was proven with higher efficiency.     

A comparison between freezing methods for the cryopreservation of stallion spermatozoa

The effects of sperm freezing concentration (40 × 10⁶ mL⁻¹ vs. 400 × 10⁶ mL⁻¹), straw size (0.25 mL vs. 0.5 mL) and freezing method (liquid nitrogen vapour in a Styrofoam^® box vs. programmable freezing machine) were evaluated in a 2 × 2 × 2 factorial experimental design using 3 split ejaculates from each of 4 stallions. Immediately after thawing, the total motility and forward progressive motility of spermatozoa frozen at a concentration of 40 × 10⁶ mL⁻¹ was higher than for spermatozoa frozen at 400 × 10⁶ mL⁻¹. No significant differences were observed in the semen parameters assessed after cryopreservation in either 0.25 or 0.5 mL straws. However, the programmable freezer provided a more consistent and reliable freezing rate than liquid nitrogen vapour. We conclude that an effective protocol for the cryopreservation of stallion spermatozoa at low concentrations would include concentrations of 40 × 10⁶ mL⁻¹ in 0.25 mL straws using a programmable freezer. This freezing protocol would be suitable for emerging sperm technologies such as sex-preselection of stallion spermatozoa as the sorting process yields only low numbers of spermatozoa in a small volume available for either immediate insemination or cryopreservation.     

The influence of cryopreservation and seminal plasma on the chromatin structure of dog spermatozoa

It was the aim of the current study to investigate effects of seminal plasma on the chromatin structure of frozen-thawed canine (Canis lupus familiaris) spermatozoa. A total of 20 ejaculates were collected. Ejaculates were divided, and one half was centrifuged for removal of seminal plasma (c) while the other was left uncentrifuged (nc) before cryopreservation. This was performed according to the Uppsala system in a computerized freezing machine. Before freezing (bf) and after thawing (at), samples were investigated for motility (M), viability (CASA), and chromatin status (sperm chromatin structure assay; SCSA). Before freezing, the average DFI% and the SD-DFI from 20 nc ejaculates were 1.7 ± 4.0% and 18.6 ± 1.2, respectively. After thawing, all motility parameters decreased and were significantly lower in centrifuged than in noncentrifuged samples, whereas the percentage of morphologically abnormal spermatozoa (Morph) was significantly higher (nc: M bf, 84.1 ± 20.6%; M at, 51.9 ± 15%; c: M bf, 84.1 ± 20.6%; M at, 43.3 ± 22.2%; Morph nc: 28.3 ± 7.8% vs. c: 31.0 ± 9.8%). Furthermore, only in c samples did the DFI increase within 6 h after thawing (DFI c: bf, 41.8 ± 1.5%; 6 h at, 45.4 ± 6.6%; P < 0.01). The SD-DFI as well as the DFI% increased within 3 h of storage in both groups (SD-DFI nc: bf, 18.6 ± 1.2%; 3 h at, 25.8 ± 5.4%; DFI% nc: bf, 1.1 ± 4.0%; 3 h at, 6.1 ± 12.9%; P < 0.05). For both parameters, there was no significant difference between c and nc samples at any time investigated. In conclusion, centrifugation of semen samples before freezing decreased postthaw motility and increased the percentage of morphologically abnormal spermatozoa as well as the degree of sperm chromatin denaturation over time. Centrifugation of canine ejaculates before cryopreservation can therefore no longer be recommended.     

Effects of glycine and alanine on short-term storage and cryopreservation of striped bass (Morone saxatilis) spermatozoa

Three experiments were designed to examine the effects of the amino acids glycine and alanine on short-term storage and cryopreservation of striped bass spermatozoa. In the first experiment, the effect of glycine on post-equilibration motility was evaluated. In the presence of 2.5 or 5.0% Me(2)SO, glycine treatments (25, 50, and 75 mM) yielded higher (P<0.05) post-equilibration motility at all equilibration times examined compared to the control. There was no difference (P>0.05) among these three glycine treatments. In the second experiment, glycine and alanine at concentrations of 25, 50, 75, or 100mM were evaluated for post-thaw motility in the presence of 2.5 or 5% Me(2)SO. When compared to the control, both the glycine and alanine treatments showed positive effects on post-thaw motility at all concentrations tested. The highest (P<0.05) post-thaw motility was achieved with 50mM glycine or 75 mM alanine using 5% Me(2)SO. No interaction (P>0.05) between Me(2)SO and glycine or alanine was observed, indicating that the effect of glycine or alanine was independent of the concentrations of Me(2)SO. In the third experiment, glycine was evaluated for sperm motility, after short-term refrigerated storage and after cryopreservation of the same refrigerated semen. Sperm motility decreased after 24h of refrigerated storage in 50mM glycine treatment and the control, when compared to fresh sperm motility. However, 50mM glycine treatment yielded higher (P<0.01) sperm motility after both 24 and 48 h of storage as well as higher (P<0.01) post-thaw motility when compared to the control. An average of 30+/-2.9% and 16+/-2.4% post-thaw motility was achieved with the 50mM glycine treatment after 24 and 48 h of refrigerated semen, respectively.     

Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri)

Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 °C over 1.5 h (−0.16 °C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration–equilibration, after freezing and thawing, and 2 h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.     

The spermatozoa of ascidians: Acrosome and nuclear envelope

Summary The spermatozoon of Ascidia callosa has a head with a wedge-shaped tip. Between the nuclear envelope and the plasmalemma, at the tip of the head, there are one or two previously undescribed vesicles, 45 to 55 nm in diameter. These vesicles have the characteristics of an acrosome. Their role in the process of fertilization has not been determined. Ultrastructural studies of sperm activation are needed, but claims that the spermatozoa of ascidians do not have an acrosome should be reconsidered.Behind the tip of the sperm there are pores in the nuclear envelope. This part of the envelope also contains a dense band of amorphous material that may have a supportive function. A nearly identical structure, associated with pores has been found in the spermatozoon of Boltenia villosa. An analysis of the nuclear envelope of Ascidia callosa indicates that the same structure has previously been misinterpreted as an acrosome in the spermatozoon of Ascidia nigra.     

Effect of lactoferrin on ram sperm motility after cryopreservation

ObjectiveThe objective of this study was to analyse the differentially abundant proteins caused by freeze–thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro.MethodsSperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 μg/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS).ResultsCryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 μg/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 μg/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 μg/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05).ConclusionThe LTF is an important protein during cryopreservation, and the addition of 10 μg/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.     

Single layer colloid centrifugation technique improves motility,viability and chromatin integrity of ram spermatozoa after thawing

The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe's cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (%DFI) of the samples was assessed 0 h after thawing, by SCSA™. Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing.The proportion of motile spermatozoa was significantly higher in SLC – selected samples in comparison to control (not SLC – selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC – selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower %DFI values in SLC – selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC – selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.     

Effects of post-thaw incubation on motility,acrosomal integrity and in vitro fertilizing capacity of boar spermatozoa cryopreserved in 0.5 and 5 ml straws

The effect of post-thaw incubation (0 vs. 5 h at 15 °C) and straw size (5 vs. 0.5 ml) on motility, acrosomal integrity and in vitro fertilizing (IVF) capacity of cryopreserved boar spermatozoa was studied. In samples assessed immediately after thawing, no differences were found between the two straw sizes. After 5 h post-thaw incubation, all parameters, except polyspermy, decreased and, spermatozoa packaged in 5 ml straws showed better functional and IVF parameters than these in 0.5 ml straws.     

Influence of cryoprotectants on abnormality and motility of baung (Mystus nemurus) spermatozoa after long-term cryopreservation

Study on the effect of cryoprotectants on abnormality and motility of baung, Mystus nemurus spermatozoa were evaluated using transmission and scanning electron microscopy. Four cryoprotectants, dmso, ethanol, methanol and glycerol at concentration of 10% were tested in triplicates. Three ml of fresh sperm which was diluted with 60 ml of ringer solution was added to each of twelve 5-ml vials containing of 0.50-ml of the cryoprotectants. The vials were placed in an icebox containing dry ice 5 min and then storage into container containing liquid nitrogen for 13 months. The effect of cryoprotectants on the spermatozoa abnormality and motility were significant (P < 0.05). The spermatozoa abnormality was significantly lower in methanol (62.65%) compared with the other cryoprotectants. The spermatozoa motility was higher in methanol, but not significantly different with ethanol (P > 0.05). It is a negative correlation between sperm motility and abnormality. Generally, higher abnormalities of spermatozoa resulted low motility.     

Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs

The acrosome of the stallion spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody (18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 +/- 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up to 12 hours in TALP medium maintained motility and exhibited a significant progressive loss of acrosomes as detected by immunofluorescence. Alternatively, a similar loss of acrosomes could be induced with calcium ionophore A23187 over a 90 minute incubation. Ultrastructural observations and incubation with zona-free hamster eggs indicated that only with ionophore treatment was immunofluorescent acrosome loss correlated with a physiological acrosome reaction, while prolonged sperm incubation led to degenerative membrane changes. It was concluded that, if carefully validated, immunofluorescent localization of the acrosome of stallion sperm with monoclonal antibody could be used to monitor the acrosome reaction. Furthermore, definitive acrosome visualization would be valuable in assessing semen quality.     

Osmotic tolerance and membrane permeability characteristics of rhesus monkey (Macaca mulatta) spermatozoa

Biophysical characteristics of the plasma membrane, such as osmotic sensitivity and water and cryoprotectant permeability are important determinants of the function of spermatozoa after cryopreservation. A series of experiments was conducted with rhesus macaque spermatozoa at 23 degrees C to determine their: (1) cell volume and osmotically inactive fraction of the cell volume; (2) permeability coefficients for water and the cryoprotectants dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; (3) tolerance to anisosmotic conditions; and (4) motility after a one step addition and removal of the four cryoprotectants. An electronic particle counter and computer aided semen analysis were used to determine the cell volume and permeability coefficients, and motility, respectively. Rhesus spermatozoa isosmotic cell volume was 27.7+/-3.0 microm3 (mean+/-SEM) with an osmotically inactive cell fraction of 51%. Hydraulic conductivity in the presence of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol was 1.09+/-0.30, 0.912+/-0.27, 1.53+/-0.53, and 1.94+/-0.47 microm/min/atm, respectively. Cryoprotectant permeability was 1.39+/-0.31, 2.21+/-0.32, 3.38+/-0.63, and 6.07+/-1.1 (x10(-3)cm/min), respectively. Rhesus sperm tolerated all hyposmotic exposures. However, greater than 70% motility loss was observed after exposure to solutions of 600 mOsm and higher. A one step addition and removal of all four cryoprotectants did not cause significant motility loss. These data suggest that rhesus sperm are tolerant to hyposmotic conditions, and ethylene glycol may be the most appropriate cryoprotectant for rhesus sperm cryopreservation, as it has the highest permeability coefficient of the tested cryoprotectants.     

The cryopreservation of spermatozoa of the burbot,Lota lota (Gadidae,Teleostei)

The cryopreservation of spermatozoa of a teleost fish, the burbot, Lota lota (Gadidae) was investigated. Cryopreserved semen had the highest motility rate (46.6+/-8.0%, fresh semen control 86.5+/-8.2%) and fertility (78.1+/-2.7% embryo survival in hatching stage, fresh semen control 82.2+/-2.9%) when 10% methanol, 1.5% glucose and 7% hen egg yolk were used as cryoprotectants. Freezing was performed in 0.5-ml straws in the vapour of liquid nitrogen at 1cm above the level of liquid nitrogen and thawing in water at 25 degrees C for 20s. For optimal fertilization cryopreserved semen was first mixed with the eggs and then 25 or 50 mmol/L NaCl solution (pH 8.5) was added at a ratio of 1:24 (semen:saline solution). Under these conditions fertilization ratios in the range of fresh semen control were obtained at minimal sperm to egg ratios of 1.7 x 10(6):1. Fertilization with cryopreserved semen had no influence on the embryonic development, as the ratio of embryos which stopped development and the ratio of embryonic malformations were similar to fresh semen.     

Characterization and potential roles of calretinin in rodent spermatozoa

Calretinin has been detected in various excitable cells but the presence and putative roles of such a calcium-binding protein has never been characterized in sperm.Epididymal spermatozoa were collected from C57Bl6 (wild-type, WT) or calretinin knockout (CR^−/−) mice and Wistar rats. A specific staining for calretinin was detected by immunofluorescence in the principal piece of the flagellum, both in WT mouse and rat spermatozoa. Western blots confirmed the expression of calretinin in rat and WT spermatozoa as well as its absence in CR^−/− mice.No significant difference was observed in the spontaneous acrosome reaction between WT and CR^−/− sperm. The addition of the calcium-ionophore A-23187, Thapsigargin or Progesterone to WT or CR^−/− incubated spermatozoa induced increases in the acrosome reaction but the stimulatory effects were identical in both genotypes. Motility measurements assessed by computer-assisted sperm analysis indicated that, under basal non-stimulatory conditions, CR^-/- sperm exhibited a lower curvilinear velocity and a smaller lateral head movement amplitude, although no difference was observed for the beat cross frequency. After incubation with 25 mM NH₄Cl, the curvilinear velocity, the amplitude of the lateral head movement and the hyperactivation were increased, while the beat cross frequency was decreased, in both genotypes.Evaluation of the in vivo fertility potential indicated that the CR^−/− litter sizes were clearly reduced compared to the WT litter sizes.Our study describes, for the first time, the expression of calretinin in sperm. These data extend the potential implication of calcium-binding proteins in the sperm calcium-signaling cascade and bring new insights into the understanding of sperm physiology.     

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatoza by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization. © Wiley-Liss, Inc.     

Changes in rat spermatozoa function after cooling,cryopreservation and centrifugation processes

Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing–thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (P < 0.05). Basal (without ROS inducer, tert-butyl hydroperoxide [TBHP] treatment) and stimulated ROS (with TBHP treatment) were increased in viable cooled spermatozoa compared to viable fresh spermatozoa (P < 0.01), with equal susceptibility to TBHP among fresh, cooled, and frozen–thawed spermatozoa. Centrifugation decreased motility and PMI of frozen–thawed spermatozoa (P < 0.05). Centrifugation decreased basal ROS of all spermatozoa (P < 0.01), while it led to higher susceptibility to TBHP in viable cooled spermatozoa, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled spermatozoa (P < 0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, PMI, and MMP. Centrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa.