Population Genetics of LEBISTES (POECILIA) RETICULATUS Peters (Poeciliidae; Pisces). I. Effects of Radiation-Induced Mutations on the Segregation Ratio in Postirradiation F2

Wild-type germ cells of the viviparous fish, Lebistes (Poecilia) reticulatus, were irradiated in the stages of oogonia, spermatogonia or spermatozoa with 1000 R of X rays at a dose-rate of 200-220 R/min. The grown-up fish were mated after irradiation or sham treatment to unirradiated mutant fish homozygous for three recessive gene loci, causing albinism, lack of yellow and reddish pigmentation ("blue" constitution), and diminution of the size of melanophores ("blond" constitution). As compared to the control series, the phenotypic segregation ratios in the adult F(2) generation after irradiation was changed significantly in favour of wild-type and blue fish, whereas the proportion of albinotic, blond and "white" (= blond + blue) guppies was lowered. This unidirectional effect in postirradiation F(2) was greatest after exposure of spermatozoa with 2 x 500 R (24 hr apart), followed by single doses of 1000 R to spermatogonia and oogonia. These results were explained in terms of a synergistic interaction of recessive radiation-induced mutations in the heterozygous state with those genotypes of the guppy which affect the formation of melanophoric patterns and the synthesis of melanins.     

Effect of dose-rate on the frequency of X-linked lethal mutation in the nematode Panagrellus redivivus

A total X-ray dose of 50 Gy was applied to the nematode Panagrellus redivivus using dose-rates ranging from 0.23 Gy/min to 10.49 Gy/min, and the frequency of lethal X-chromosomes was determined. This frequency ranged from approximately 1.6% at the lower dose-rate to 4.3% at the highest dose-rate, indicating a dose-rate dependency of mutation frequency in the spermatogonia and oogonia of this organism.     

Reduction of seminal plasma concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa

This study was conducted to investigate the effect of different levels of seminal plasma (SP) and cold-shock on ram spermatozoa during 36 h storage at 5°C. In both ejaculated spermatozoa coated with egg yolk (second ejaculate; coated spermatozoa) and epididymal spermatozoa, samples were treated with 0, 50 and 100% seminal plasma. Different levels of seminal plasma were added on the basis of ram spermatocrit (32%). Then half of aliquots were suddenly put on ice water (cold-shock) and other half were gradually (0.25°C/min) chilled (non- cold shock). Sperm motility, viability and functional membrane integrity were determined in both aliquots at 0, 12, 24 and 36 h storage at 5°C. Under non- cold shock and cold-shock conditions, coated spermatozoa treated with 0% SP showed the highest motility compared to ejaculated spermatozoa (first ejaculate; uncoated spermatozoa) after 12, 24 and 36 h of storage at 5°C (P<0.05). Under non- cold shock and cold-shock conditions, viability and functional membrane integrity was higher in the coated spermatozoa treated with 0% SP than in the uncoated spermatozoa during 36 h storage (P<0.05). There was no significant difference between coated spermatozoa treated with 0 and 50% SP in the percentage of motility and viability after 24 and 36 h of storage (P>0.05). Under non- cold shock and cold-shock conditions, the percentage of motility of epididymal spermatozoa treated with 0% SP was significantly (P<0.05) higher than those treated with 100% SP after 36 h of storage at 5°C. In conclusion, removal of seminal plasma and/or reduction (up to 50%) of its concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa.     

The influence of dose rate on the lethal and mutagenic effects of X-rays in proliferating L5178Y cells differing in radiation sensitivity

The lethal and mutagenic effects of ionizing radiation delivered at high (53 Gy/h) and low (0.02 Gy/h) dose rates were measured in two closely related strains of mouse lymphoma L5178Y cells differing in radiation sensitivity (LY-R and LY-S). Strain LY-R was more resistant to the lethal effects of radiation than strain LY-S when exposed at either the high or low dose rate. The survival of strain LY-R was markedly enhanced by the reduction in dose rate. The dose-rate dependence of the survival of strain LY-S was less clear, because of the biphasic nature of its survival curve following low dose-rate radiation. However, if the initial slope of the low dose-rate survival curve is compared to the slope of the high dose-rate survival curve for strain LY-S, only a slight increase in survival at the low dose rate is apparent. Although more sensitive to the lethal effects of radiation, strain LY-S was less mutable at the hypoxanthine/guanine phosphoribosyl transferase locus by both low dose-rate and high dose-rate radiation than strain LY-R. Little dose-rate dependence was exhibited by either strain with regard to the mutagenic effects of radiation. Thus, for strain LY-R, which showed marked dose-rate dependence for survival but not for mutation, the ratio of mutational to lethal lesions was much greater following exposure to low dose-rate than to high dose-rate radiation.     

The effect of gamete co-incubation time during in vitro fertilization with frozen-thawed unsorted and sex-sorted ram spermatozoa on the development of in vitro matured adult and prepubertal ewe oocytes

In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubated with unsorted or sex-sorted frozen-thawed spermatozoa for 2 to 3 h (short) or 18 to 20 h (long) to determine the effects of reducing the gamete co-incubation time during IVF on subsequent embryonic development in vitro. For oocytes derived from adult ewes, there were no differences in oocyte fertilization and cleavage at 24 h post insemination (hpi) between types of spermatozoa or co-incubation times (P > 0.05). By 48 hpi, oocyte cleavage was higher after a short (390/602, 64.8%) compared with a long (381/617, 61.7%) co-incubation (P < 0.05), and was not significantly different for unsorted (266/372, 71.5%) and sex-sorted (505/849, 59.9%) spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (150/266, 56.4%) and sex-sorted (295/505, 58.4%) spermatozoa, but was higher after a short (240/390, 61.5%) than long (205/381, 53.8%) co-incubation (P < 0.05). Oocyte development to the blastocyst stage was not different for unsorted (150/372; 40.3%) and sex-sorted (295/847; 34.8%) spermatozoa but was significantly increased by a short (240/602, 39.9%) compared with a long (205/617, 33.2%) co-incubation. Fertilization of oocytes from prepubertal ewes was similar for types of spermatozoa and for duration of co-incubation. Oocyte cleavage (48 hpi) was similar for a short (241/377, 63.9%) and long (226/349, 64.8%) co-incubation with unsorted spermatozoa, but was increased (P < 0.05) by a long co-incubation (286/500, 57.2% versus 163/517, 31.5%) with sex-sorted spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (230/467, 49.3%) and sex-sorted (186/449, 41.4%) spermatozoa, and a short (200/404, 49.5%) or long (216/512, 42.1%) co-incubation. However, oocyte development to the blastocyst stage was higher (P < 0.05) after IVF with unsorted (230/726, 37.1%) than sex-sorted (186/1017, 18.3%) spermatozoa. Reducing the duration of gamete co-incubation did not deleteriously affect the in vitro development of adult and prepubertal ewe derived oocytes after IVF with unsorted and sex-sorted spermatozoa. In general, sex-sorting had no substantial influence on fertilization and embryo development rates.     

The minimum effective spermatozoa : egg ratio for artificial insemination and the effects of mercury on sperm motility and fertilization ability in Clarias gariepinus

The optimal ratio of spermatozoa : egg (15 000 : 1) for artificial insemination of African catfish Clarias gariepinus gave fertilization and hatching rates of 80 and 67%, respectively. Below a sperm : ova of 3000 : 1 fertilization success decreased significantly. Excessive sperm (>15 000 : 1) partly inhibited fertilization success. Sperm motility was decreased significantly by 0·001 mg 1⁻¹ Hg²⁺ as HgCl₂, but its effect on fertilization was dependent on the sperm : ova ratio, since excess sperm masked the effect of the pollutant. The most sensitive sperm : ova ratio for monitoring pollutant effects on fertilization success was 1500 : 1, which corresponds to half the minimal amount that yields a high fertilization rate in artificial insemination. There was a good correlation between fertilization and hatching rates ( r =0·83; P<0·05). Although both fertilization and hatching rates provide equally good indicators of fertilization success, the more rapid fertilization rate test is recommended since it requires only 12 h.     

Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison

This study compared variation in the quality of cryopreserved boar spermatozoa and the control and accuracy of cooling rates between three semen freezers (CryoLogic Freeze Control CL3000, Planer Products Kryo Save Compact KS1.7/Kryo 10 Control module and a controlled rate 'Watson' freezing machine developed within our laboratory). Five ejaculates were collected from each of 15 boars (five boars from each of three breeds). Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% v/v glycerol) and placed into 0.5 ml straws. Three straws per treatment, from each ejaculate were cooled to -5 degrees C at 6 degrees C/min, held at -5 degrees C for 30s while ice crystal formation was induced, then further cooled from -5 to 80 degrees C at either 40 degrees C/min (Kryo Save Compact KS1.7 and Watson) or 6 degrees C/min (Freeze Control CL3000). Precise measurements of temperature fluctuations during the programmed cooling curves were made by inserting thermocouples into the semen filled straws. Semen was assessed for %motile cells, motility characteristics using computer-assisted semen analysis (CASA), plasma membrane integrity (%SYBR-14 positive stained spermatozoa) and acrosome integrity (%FITC-PNA positive stained spermatozoa). Spermatozoa cryopreserved using the Freeze Control CL3000 system (maximum rate of 6 degrees C/min) exhibited reduced post-thaw viability (14.2+/-2.8% mean plasma membrane intact spermatozoa) when compared to both the KS1.7 and Watson freezers (optimal rate of 40 degrees C/min) (18.4+/-3.2 and 25.7+/-3.7% mean plasma membrane intact spermatozoa, respectively). Differences in motility characteristics were observed between spermatozoa cryopreserved at 40 degrees C/min with the Watson apparatus preserving a larger proportion of sperm with progressive motility. Cooling curves in the CL3000 and KS1.7 were interrupted by a pronounced increase in temperature at -5 degrees C that corresponded with the latent heat of fusion released with ice crystal formation. This temperature change was significantly reduced in the cooling curves produced by the Watson freezer. These findings suggest that preserving spermatozoa using the Watson freezer improved post-thaw semen quality, with regard to sperm motility characteristics. Furthermore, that post-thaw semen viability was enhanced by minimising temperature fluctuations resulting from the release of the latent heat of fusion at ice crystal formation.     

Is there a proportionality between the spontaneous and the X-ray induction rates of mutations?: Experiments with mutations at 13 X-chromosome loci in Drosophila melanogaster

The X-ray induction of recessive visible specific locus mutations at 14 X-chromsome loci was studied in Drosophila melanogaster using the "Maxy" technique. The X-ray exposure was 3000 R to 5-day-old males and the sampling of germ cells was restricted to mature spermatozoa. Presumptive mutant females recovered in the F1 generation were tested for transmission, allelism, fertility and viability in males. A total of 128 mutations (115 completes and 13 mosaics including those that were male viable as well as male-lethal) recovered among 38 898 female progeny were found to be transmitted. On the basis of the above frequency, the average mutation rate can be estimated as 7.8 X 10(-8)/locus/R; for mutations that were viable and fertile in males, the rate is 3.0 X 10(-5)/locus/R (49 mutations among 38 898 progeny). The frequency of mutations at the different loci encompassed a wide range: while no mutations were recovered at the raspberry and carnation loci, at others, the numbers ranged from 1 at echinus to 31 at garnet; in addition, the proportion of mutations that was male-viable was also different, depending on the locus. Schalet's extensive data on spontaneous mutations at 13 (of the 14 loci employed in the present study) loci permit an estimate of the spontaneous rate which is 6.1 X 10(-6)/locus (a total of39 mutations among 490 000 progeny); for mutations that were viable and fertile in males, the rate is 3.0 X 10(-6)/locus (19 mutations among 490 000 progeny). The mutability of the different loci varied over a 9-fold range. When the different loci are ranked depending on their relative mutability (for spontaneous and induced mutations) it is found that in general, loci that mutate spontaneously relatively more frequently are also those at which more mutations have been recovered in the radiation experiments and likewise, those that are less mutable spontaneously are also those that mutate less after irradiation. Since the data are limited, it is concluded that the above finding is not inconsistent with the assumption of proportionality between spontaneous and induction rates of mutations. On the basis of the above results, a doubling dose of 100 R can be calculated for the X-ray induction of specific-locus mutations in Drosophila spermatozoa.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. VII. Effects of repair deficiency in males on X-ray-induced sex-linked recessive lethals in spermatozoa

The response of mature spermatozoa to the X-ray induction (500 R and 3000 R) of sex-linked recessive lethals was studied in Drosophila melanogaster males known to be deficient in excision- or post-replication repair of UV damage in somatic cells. The results show that the induced frequencies of recessive lethals in the excision-repair-deficient males (mei-9a and mei-9L1) are similar to those in the appropriate repair-proficient males (mei+ and Berlin-K). However, in the post-replication-repair-deficient males (w mus(1)101D1), these frequencies are significantly lower than in the comparable repair-proficient males (w) after 500 R, but not after 3000 R.     

Specific-locus experiments show that female mice exposed near the time of birth to low-LET ionizing radiation exhibit both a low mutational response and a dose-rate effect

Female mice were exposed to 300 R of 73-93 R/min X-radiation either as fetuses at 18.5 d post conception (p.c.) or within 9 h after birth. Combining the similar results from these two groups yielded a specific-locus mutation frequency of 9.4 X 10(-8) mutation/locus/R, which is statistically significantly higher than the historical-control mutation frequency, but much lower than the rate obtained by irradiating mature and maturing oocytes in adults. Other females, exposed at 18.5 days p.c. to 300 R of 0.79 R/min gamma-radiation, yielded a mutation frequency that was statistically significantly lower than the frequency at high dose rates. The low-dose-rate group also had markedly higher fertility. It appears that the dose-rate effect for mutations induced near the time of birth may be more pronounced than that reported for mature and maturing oocytes of adults. A hypothesis sometimes advanced to explain low mutation frequencies recovered from cell populations that experience considerable radiation-induced cell killing is that there is selection against mutant cells. The reason for the relatively low mutational response following acute irradiation in our experiments is unknown; however, the finding of a dose-rate effect in these oocytes in the presence of only minor radiation-induced cell killing (as judged from fertility) makes it seem unlikely that selection was responsible for the low mutational response following acute exposure. Had selection been an important factor, the mutation frequency should have increased when oocyte killing was markedly reduced.     

Change in the radiation responses of oogonia in the embryos and fry of the fish Oryzias latipes.

Embryos of the fish Oryzias latipes were irradiated with 1000 R of X-rays 1 day before hatching,and the post-irradiation change in the female germ-cell population was observed. Scarcely any reduction in the number of oogonia was observed, but their proliferation was inhibited. Repopulation occurred between 12 and 20 days after hatching. These responses were quite different from those of germ cells in the irradiated fry (Hamaguchi and Egami 1975). Embryos and/or fry were also exposed to 1000 R of X-rays 1 day before hatching and 0, 1, 2, and 3 days after hatching. A comparison of their responses suggested that the change in the radiation responses of oogonia is correlated with the initiation of meiosis.     

Inverse dose-rate effect of DNA breaks and inactivation of transforming activity induced by tritiated water and60COγ-rays

Summary When an aqueous solution of plasmid DNA at a constant low concentration of 5 µg/cm³ was irradiated with⁶⁰Co-rays, D₃₇ dose of single-strand breaks was decreased from 18 Gy at a dose-rate of 6.77 Gy/h of acute irradiation to 2.3 Gy at a dose-rate of 0.00212 Gy/h. OrG value was increased from 0.0010 to 0.0081. Similar dose-rate dependency of D₃₇ dose andG value were also found when the plasmid DNA solution was treated with various concentrations of tritiated water at various dose-rates, ranging from 5.13 Gy/h to 0.000118 Gy/h. RBE of tritiumß-rays for single-strand breaks was ranged from 0.3 to 0.5 in a wide range of dose-rates. When the DNA solution was saturated with argon to remove oxygen, the dose-rate dependency of-rays was abolished and that of tritiumß-rays was significantly supressed. When the DNA solution in air was kept at 4° C for 50 h or 25 days after acute irradiation, theG value of DNA breaks was the same as that kept at —20° C for the same period, but much lower than that of the solution irradiated for the same period at a lower dose-rate to give the same total doses. This shows that the inverse dose-rate effect could not be induced from the different exposure periods but from continuous irradiation of different dose-rates. The inverse dose-rate effect for inactivation of transforming activity of DNA irradiated with tritiated water was also observed in the range from 0.0588 Gy/h to 0.00118 Gy/h.     

Relative biological effectiveness of 103Pd and 125I photons for continuous low-dose-rate irradiation of Chinese hamster cells

Monolayers of Chinese hamster lung cells (CCL-16) in a polystyrene phantom were irradiated in vitro by 103Pd and 125I sources at dose rates of 6 to 72 cGy/h. Cell survival curves for acute high-dose-rate irradiation (over 30 Gy/h) were also measured using nearly monoenergetic X-ray beams which were designed to simulate the mean energies of photons emitted by 125I and 103Pd and also using a clinical 250 kVp X-ray beam. A profound dose-rate effect is observed over the dose-rate range of 6 to 20 cGy/h. An inverse dose-rate effect was observed for both radionuclides, with its onset occurring at a dose rate of about 20-30 cGy/h. The average RBE of 103Pd relative to 125I was determined to be 1.45 +/- 0.07, 1.41 +/- 0.07, 0.70 +/- 0.07 and 1.49 +/- 0.07 at dose rates of 6.9, 12.6, 19.0 and 26.7 cGy/h, respectively. Because 103Pd implants are generally prescribed at a higher initial dose rate (21 cGy/h) than the corresponding 125I implants (7 cGy/h), the effects of both dose rate and photon energy on biological response must be considered together. For the CCL-16 cells, the RBE of 103Pd at 19.0 cGy/h relative to that of 125I at 6.9 cGy/h was estimated to be 2.3 +/- 0.5.     

The effect of caffeine on repair systems in oocytes of Drosophila melanogaster. I

Young Drosophila females were treated with caffeine, then mated for 24 h to males that had been irradiated with 2000 R X-irradiation, so that only mature spermatozoa were sampled. The radiation-induced frequency of dominant lethals and sex chromosome loss in the paternal genome was determined. The results show that treatment of females with caffeine leads to an increase in the frequencies of radiation-induced dominant lethals and to sex-chromosome loss.When young virgin females of the radioresistant stock RöI₂ were treated with caffeine and then irradiated with 3000 R X-irradiation, a striking increasein dominant lethals (in the maternal genome) was observed; caffeine treatment increased the X-ray response of the radioresistant stock to the level of the normal (+60) stock. It is suggested that caffeine reduces the efficiency of a system in Drosophila oocytes that repairs X-ray-produced chromosome breaks in both the paternal and maternal genomes.     

Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson

This paper compares some morphological and endocrinological characteristics of diploid and triploid rainbow trout.
Significant differences were found between diploid and triploid females in GSI, condition factor, gut weight, liver weight and percentage dress-out, and between diploid and triploid males in GSI, condition factor and gut weight.
Diploid females had large, well-developed ovaries containing yolk-filled secondary oocytes whereas the triploids had only string-like ovaries containing nests of oogonia. No primary oocytes were present.
All the diploid males produced copious quantities of milt but it was possible to express a thin, watery milt containing motile spermatozoa from only two of the 12 triploid males. Testes weights in triploids were similar to those of diploids but, while the diploid testes were packed with spermatozoa, those of the triploids consisted mainly of spermatocytes and spermatids with few spermatozoa present. Measurements of the heads of spermatozoa revealed that those from triploids were larger and had a wider size range than those from diploids.
Levels of testosterone and 11-ketotestosterone in triploid and diploid males were not significantly different. However, levels of testosterone and 17β-oestradiol in diploid females were considerably higher than those of triploid females.     

Species variation in osmotic, cryoprotectant, and cooling rate tolerance in poultry, eagle, and peregrine falcon spermatozoa

Potential factors influencing spermatozoa survival to cryopreservation and thawing were analyzed across a range of the following avian species: domestic chicken (Gallus domesticus), domestic turkey (Meleagris gallopavo), golden eagle (Aquila chrysaetos), Bonelli's eagle (Hieraaetus fasciatus), imperial eagle (Aquila adalberti), and peregrine falcon (Falco peregrinus). Studies focused on spermatozoa tolerance to the following: 1) osmotic stress, 2) different extracellular concentrations of the cryoprotectant dimethylacetamide (DMA), 3) equilibration times of 1 versus 4 h, 4) equilibration temperature of 4 versus 21 degrees C, and 5) rapid versus slow cooling before cryopreservation and standard thawing. Sperm viability was assessed with the live/dead stain (SYBR-14/propidium iodine). Sperm viability at osmolalities >/=800 mOsm was higher (P: < 0.05) in raptor than poultry semen. Return to isotonicity after exposure to hypertonicity (3000 mOsm) decreased (P: < 0.05) number of viable spermatozoa in chicken, turkey, and golden and Bonelli's eagle spermatozoa but not in imperial eagle or peregrine falcon spermatozoa. Differences were found in spermatozoa resistance to hypotonic conditions, with eagle species demonstrating the most tolerance. Semen, equilibrated for 1 h (4 degrees C) in diluent containing DMA (> or =2.06 M), experienced decreased (P: < 0. 05) spermatozoa survival in all species, except the golden eagle and peregrine falcon. Number of surviving spermatozoa diminished progressively with increasing DMA concentrations in all species. Increased equilibration temperature (from 4 to 21 degrees C) markedly reduced (P: < 0.05) spermatozoa survival in all species except the Bonelli's eagle and turkey. Rapid cooling was detrimental (P: < 0.05) to spermatozoa from all species except the imperial eagle and the chicken. These results demonstrate that avian spermatozoa differ remarkably in response to osmotic changes, DMA concentrations, equilibration time, temperature, and survival after fast or slow freezing. These differences emphasize the need for species-specific studies in the development and enhancement of assisted breeding for poultry and endangered species.     

The Effect on Intracytoplasmic Sperm Injection Outcome of Genotype,Male Germ Cell Stage and Freeze-Thawing in Mice

Background

Intracytoplasmic sperm injection (ICSI) has been widely used to study the mechanisms of mammalian fertilization and to rescue male-factor infertility in humans and animals. However, very few systematic analyses have been conducted to define factors affecting the efficiency of ICSI. In this study, we undertook a large-scale series of ICSI experiments in mice to define the factors that might affect outcomes.

Methodology/Principal Findings

We used a 5×3×2 factorial design with the following factors: mouse genotype (ICR, C57BL/6, DBA/2, C3H/He, and 129/Sv strains), type of male germ cells (epididymal sperm, elongated or round spermatids), and their freeze–thawing treatment. The efficiencies (parameters) of each developmental step were analyzed by three-way ANOVA (significance level P<0.01). The type of male germ cells affected all the four parameters observed: oocyte survival after injection, cleavage of oocytes, implantation, and birth of offspring. Genotype affected the oocyte survival, cleavage and birth rates, whereas freeze–thawing had no effects on any of the parameters. There were significant genotype/cell type interactions for oocyte survival and cleavage, indicating that they were determined by a combination of strain and germ cell maturity. Multiple comparisons revealed that spermatozoa and elongated spermatids gave better implantation and birth rates than did round spermatids, while spermatozoa and elongated spermatozoa were indistinguishable in their ability to support embryonic development. The best overall efficiency (birth rate per oocytes injected) was obtained with frozen–thawed DBA/2 strain elongated spermatids (23.2±4.2%).

Conclusions/Significance

The present study provides the first comprehensive information on ICSI using the mouse as a model and will contribute to the efficient use of materials, time, and efforts in biomedical research and clinics involving ICSI.     

On-Chip Cryopreservation: A Novel Method for Ultra-Rapid Cryoprotectant-Free Cryopreservation of Small Amounts of Human Spermatozoa

Cryopreservation of human spermatozoa free from cryoprotectant can avoid toxicity caused by highly concentrated cryoprotectant and a series of specific carriers have been previously explored, except for PDMS chip. Our study is aimed at exploring a novel device for ultra-rapid cryopreservation of small numbers of spermatozoa without cryoprotectant based on polydimethylsiloxane (PDMS) chips. Spermatozoa from 25 healthy men were involved in this study, comparing on-chip cryopreservation with different micro-channel height (group A: 10 µm height, group B: 50 µm height, group C: 100 µm height) and conventional freezing (group D) in liquid nitrogen for 72 h. The viability, motility, DNA integrity by comet assay and acrosome integrity by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining of frozen-thawed spermatozoa of each group were compared. The motility and viability of post-thawed spermatozoa was significantly decreased than that of pre-freezing spermatozoa. There was no difference of viability and motility of frozen-thawed spermatozoa between group A and D, while viability and motility of group B and C decreased compared to group A. Comet assay showed that no matter for group A or D, there was no difference of CR, TL, TD and OTM between pre-frozen and post-thawed spermatozoa. There was no difference of CR, TL, TD and OTM of post-thawed spermatozoa between group A and group D neither, while spermatozoa DNA damage was more serious in group B and group C with increasing height of micro-channel compared with group A. The proportion of intact acrosome of post-thawed spermatozoa in group A was the highest when compared with group B and group C, though similar to that of group D. In conclusion, PDMS chip with 10 µm height micro-channel is ideal for ultra-rapid cryopreservation of small quantity of spermatozoa without cryoprotectant.     

Stimulation by n6-cyclopentyladenosine of A1 adenosine receptors, coupled to galphai2 protein subunit, has a capacitative effect on human spermatozoa

The effects of selective A(1) receptor agonist on human spermatozoa were examined to verify physiological responses and to investigate the signal transduction pathway. N6-Cyclopentyladenosine on uncapacitated spermatozoa did not induce spontaneous acrosome reaction after 5 h capacitation, whereas the number of capacitated spermatozoa, assessed by lysophosphatidylcholine-induced acrosome reaction with Pisum sativum agglutinin staining, was significantly increased. N6-Cyclopentyladenosine was also added to capacitated human spermatozoa to find out whether the agonist could induce the acrosome reaction. Results, although statistically significant, could not be considered biologically significant. A1-Mediated capacitation was followed by the increase of tyrosine phosphorylation of a protein subset ranging between M(r) = 200 000 and 30 000. Stimulation of A1 receptor with the selective agonist elicited an agonist-induced inositol phospholipid hydrolysis leading to a transient rise of inositol triphosphate (IP3). This increase was not induced by A(1) receptor antagonist and was blocked by phospholipase C inhibitor. Coimmunoprecipitation experiments showed that the A(1) receptor is coupled to Galphai2 subunit suggesting that the activation of phospholipase C is mediated by betagamma subunits. In conclusion, the A(1) adenosine receptor in human spermatozoa is coupled to Galphai2, signals via IP3, and affects the capacitative status of ejaculated spermatozoa.