Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.

ABSTRACT:We hypothesized that cryopreservation and incubation in conditions that mimic the female genital tract following insemination increases the susceptibility of ram sperm DNA to denaturation. Ram sperm samples (n ϭ 12) underwent the sperm chromatin structure assay (SCSA) and semen quality tests, including motility parameters, viability, and chlortetracycline fluorescence (CTC) patterns. We also assessed correlations between SCSA variables and semen quality parameters. Analyses were performed for both fresh and cryopreserved samples at 0, 3, and 20 hours of incubation in synthetic oviductal fluid (SOF; 39ЊC, 5% CO 2 ). The SCSA variables, mean alpha t (X␣ t ) and standard deviation of alpha t (SD␣ t ), were higher because of cryopreservation (P Ͻ .05, P Ͻ .001, respectively) after 20 hours in SOF. For both fresh and frozen spermatozoa, SCSA values (X␣ t , SD␣ t , and the percentage of cells outside the main population of ␣ t [%COMP␣ t ]) increased during incubation in SOF. Motility was negatively correlated with both SD␣ t and %COM-P␣ t , ranging from Ϫ0.39 (P Ͻ .01) to Ϫ0.59 (P Ͻ .001) for both fresh and cryopreserved semen; viability also was negatively correlated with X␣ t , SD␣ t , or %COMP␣ t (Ϫ0.36; P Ͻ .05, Ϫ.40 and -.46; P Ͻ .01, respectively) in fresh semen. The %COMP␣ t was positively correlated to the percentage of CTC pattern AR (P Ͻ .001) and negatively correlated to the percentages of patterns F and B (Ϫ0.33 to Ϫ0.60, P Ͻ .05 to P Ͻ .001). Variation among ejaculates within ram was observed (P Ͻ .01). Cryopreservation clearly facilitates DNA damage in physiological conditions. The low to moderate correlations between SCSA variables and classical semen quality parameters indicate that the SCSA provides additional information to standard tests for evaluating ram sperm quality.

This study was performed in order to validate flow cytometry as an acceptable method for analyzing follicular atresia in bovine granulosa cells by comparing it to two other techniques, histology and ELISA. Ovaries from 35 nontreated cows, all at different times of their estrous cycle, and 12 superovulated cows were collected. Superovulation treatments began between Days 9 and 12 (Day 0 = estrous), and animals were administered 8 doses of FSH-P (32 or 20 mg) at 12-h intervals over 4 days with or without the addition of 1 mg of prostaglandin s.c. on the third day. Animals were slaughtered after the last FSH-P injection. Granulosa cells from 133 follicles from non-treated cows and 85 follicles from superovulated cows were analyzed. Follicular diameters ranged from 2 to 20 and 2 to 16 mm, respectively. Because of the ample amounts of cells collected, it was possible to perform more than one technique for each follicle. Flow cytometry detected in most follicles a subpopulation of cells that possessed less DNA than normal, viable cells (referred to as -G1 cells). Histological classes used (established in previous work) were nonatretic (< or = 5% picnotic nuclei), slightly atretic (> 5 to < 15% picnotic nuclei), and atretic (> or = 15% picnotic nuclei). A strong linear correlation existed between the percentage of picnotic nuclei and the percentage of -G1 cells (R = 0.86; p < 0.001) with granulosa cells from follicles from nontreated cows. In some cases, flow cytometry detected a certain percentage of cells with reduced DNA content while histology revealed very few picnotic nuclei, indicating a higher sensitivity of flow cytometry. Superovulation decreased considerably the percentage of atretic cells seen with both techniques. The linear correlation was not as strong because follicles from superovulated animals represent a very homogenous population (R = 0.54; p < 0.001). The ELISA technique coincided with flow cytometry as seen in the strong correlation between the two techniques (R = 0.91; p < 0.001). Flow cytometry appeared to be very effective and rapid in evaluating the atretic states of follicles from nontreated and superovulated cows. Strong correlations existed between this method and histology and ELISA.

The addition of the peroxovanadium (pV) derivatives potassium bisperoxo(1,10-phenanthroline)oxovanadate(v) (bpV[phen]) or potassium bisperoxo(pyridine-2-carboxylato) oxovanadate(v) (bpV[pic]), both of which are potent inhibitors of protein tyrosine phosphatases (PTPs) [Posner et al. (1994): J Biol Chem 269:4596-4604], to the culture medium of neuroblastoma NB 41 and glioma C6 cells resulted in a marked decrease in their proliferation rates and a progressive accumulation at the G2/M transition of the cell cycle. The effect was dependent on dose, cell type, and a pV compound employed. Mean values of the RNA-to-DNA and RNA-to-protein ratios in NB cells treated for 48 h with increased doses of bpV[phen] showed that general synthetic functions were not altered, nor did we observe oxidative damage to DNA using a sensitive DNA-nick detection assay. No changes in the expression and localization of vimentin, a component of the intermediate filament cytoskeleton, were observed by indirect immunofluorescence, showing that treatment did not disturb the cytoskeleton network. Measurements of BrdU incorporation into newly synthesized DNA showed that cells treated were not totally arrested. Furthermore, cells arrested G2/M were able to reenter the cycle rapidly after the release of inhibition. This progressive accumulation of G2/M coincided with the detection of tyrosine-phosphorylated p34cdc2 and a dramatic reduction in its kinase activity toward histone H1 by 48 h of culture. Both compounds were equally potent in inhibiting the catalytic activity of a yeast and the structurally distant mouse cdc25B in vitro, suggesting that augmented tyrosine phosphorylation of p34cdc2 derived from the in vivo inhibition of cdc25. Their equal in vitro potency contrasted with the considerably greater potency of bpV[phen] in vivo, in vivo suggesting that factors regulating the intracellular access of these compounds to cdc25 might be critical in determining in vivo specificity. In conclusion the final consequence of long-term exposure to potent and structurally defined PTP inhibitors on two highly proliferative nerve cell lines is to restrict cell growth. The corresponding hyperphosphorylation and reduced activity of p34cdc2 likely reflects the unusual sensitivity of cdc25 as an in vivo target for peroxovanadium compounds.

SummaryCoelomocytes were extruded from three earthworm species: Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum. Featuring a simple low-vacuum holding device, the proposed methodology allows the recovery of cells with minimum risk of contamination by faecal material. The viability of O. tyrtaeum coelomocytes was highly reproducible (average 93%), with an average yield of 0.92 x 10 6 viable cells per earthworm. Cell viability for 1. terrestris and E. fetida averaged -68% but the cell yields were higher (respectively 1.67 x 10 6 and 1.28 x 10 6 ). Large inter-individual differences in cell yields were observed with 1. terrestris. Flow cytometric analyses indicated species to species differences in cell populations.Coelomocytes from E. fetida were the smallest with -57% of the total viable cells recovered being monitored between 2 and 10 tim. Large granulated cells I~20 tim) were detected in fairly large proportions in 1. terrestris and O. tyrtaeum [-52 and -96%, respectively I while they were less abundant in E. fetida (-9%). Using the vital dye neutral red to assess functional integrity, average cellular uptakes were significantly higher for 1. terrestris and O. tyrtaeum than for E. fetida (2.94, 2.66 and 0.64 tig/2 x 10 5 cells, respectively). In summary, the extrusion methodology herein described is applicable for the recovery of coelomocytes from a wide range of earthworm sizes and species. Moreover, this study strengthens the fact that extruded coelomocytes could be used for the evaluation of cell dysfunction and/or cell death following an in vitro and/or in vivo treatment. Keywords