Characterization of human sperm populations using conventional parameters, surface ubiquitination, and apoptotic markers.

TL;DR: Sperm quality is inversely correlated with lack of viability, DNA fragmentation, and ubiquitin fluorescence intensity means, however, none of the apoptotic markers correlate with ubiquit in labeling.

About: This article is published in Fertility and Sterility.The article was published on 2007-03-01 and is currently open access. It has received 61 citations till now. The article focuses on the topics: Sperm & Semen analysis.

Summary

Biological Material

• Human sperm samples were obtained from the Fertility Clinic (University Hospitals of Coimbra) from patients undergoing routine semen analysis or fertility treatments involving both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).
• During a 2-year period, a total of 150 samples were collected and used in this study (see Table 1 for details).
• Sperm morphology was assessed using strict criteria (17) .
• Information on proven fertility of patients was not available.
• All the fluorescent tests described below were carried out blind by at least two observers.

Flow Cytometry Analysis

• Sperm population was defined after passing samples through a sorter and separating the different populations obtained in a scatterplot of complexity versus size (Fig. 1A ).
• These populations were analyzed and classified as sperm population, small cells/cellular debris population, and large cell population.

Detection of Membrane PS Exposure and Membrane Integrity Using the Annexin V Assay

• The detection of PS externalization was performed using annexin V conjugates (Molecular Probes, Eugene, OR).
• After resuspension in HEPES buffer, 5 L of allophycocyanin-annexin V and 0.1 L from an 0.005 mM stock solution of the impermeant DNA dye Green Nucleic Acid Stain (Sytox; Molecular Probes) were added for each 100 L of cellular suspension.
• Sperm was incubated in this solution for 15 minutes at room temperature, washed again, and centrifuged (800 ϫ g for 5 minutes).
• The settings included a 488-and 635-nm excitation with a 530/30-nm bandpass filter for fluorescein isothiocyanate conjugate (FITC) detection and a 661/ 16-nm bandpass filter for allophycocyanin (annexin V) detection.
• The interobserver and intraobserver variability was Ͻ5%.

DNA Fragmentation Monitored Using the TUNEL Assay

• To monitor DNA fragmentation via the TUNEL assay, the authors used the APO-BrdU TUNEL assay Kit (Molecular Probes), and the procedure was in accordance with the manufacturer's instructions.
• Briefly, after sperm fixation with 2% formaldehyde in phosphate-buffered saline (PBS; pH ϭ 7.2) for 60 minutes, and permeabilization in PBS with 1% Triton for 30 minutes, the samples were washed with the kit washing buffer.
• The cell suspension was then centrifuged and washed in 300 L of the rinse buffer.
• The stained cells were analyzed by flow cytometry and fluorescence microscopy on the day of processing.
• Percentages of TUNEL-positive cells were obtained using the FACSCalibur cytometer at a 488-nm wavelength.

Sperm-Ubiquitin Tag Immunoassay

• The sperm-ubiquitin tag immunoassay (SUTI) was essentially carried out as originally defined (11, 13) .
• The same procedure was also carried out with sperm previously attached to coverslips for fluorescence microscopy analysis.
• After fixation, samples were incubated with anti-ubiquitin antibody (clone KM 691 mouse IgM; Kamiya Biomedical, Seattle, WA), diluted 1:400 in blocking solution, at 37°C for 30 minutes; centrifuged (800 ϫ g for 5 minutes); washed in PBS with 0.1% Triton for 30 minutes, and centrifuged again.
• They were then incubated with the appropriate secondary antibody (anti-IgM 488, Molecular Probes) diluted 1:200 (37°C, 20 minutes).
• Samples were analyzed on the day of processing using a FACSCalibur flow cytometer and a fluorescence microscope.

Ubiquitin-Annexin V Assay

• For dual ubiquitin-annexin V staining, the authors first carried out the annexin V protocol, followed by formaldehyde fixation and immunostaining for ubiquitin, including the DAPI counterstain.
• All samples were analyzed immediately with a fluorescence microscope.
• The authors analyzed 200 sperm cells per coverslip to determine percentages of positive cells for surface ubiquitination, positive cells for annexin V, and positive cells for both.
• The interobserver and intraobserver variability were Ͻ5%.

Merocyanine 540 Staining

• After incubation, the sample was washed with PBS 0.1% Triton X-100 and examined by fluorescence microscopy.
• Due to quick photobleaching of the dye, it seemed often useful to omit the washing step and fix the cells.
• Samples were fixed in 2% formaldehyde, processed for ubiquitin immunostaining, and observed under fluorescence microscopy, as described above, to evaluate the presence of non-DNA-containing ubiquitin bodies.
• It should be noted that, in the current study, Merocyanine 540 was not used to assess sperm function, namely as a possible reporter for changes related to sperm capacitation (15) , but only to detect the presence of non-DNA-containing semen bodies, as discussed in previous studies (14) ,.

Statistical Analysis

• The criterion of data normality was evaluated.
• As all the variables had a normal distribution, parametric tests were performed.
• Comparisons between classes of patients were carried out by one-way ANOVA for apoptotic levels, DNA fragmentation, and ubiquitination, both for percentages of labeled cells (fluorescence microscopy) and fluorescence means (flow cytometry).
• The authors also calculated Pearson's correlation coefficients (r) (PϽ.05) to determine the correlation between apoptotic levels, DNA fragmentation, ubiquitination fluorescence means (flow cytometry), percentages of ubiquitinlabeled cells (fluorescence microscopy), and seminal param-.

FIGURE 1 CONTINUED

• The quadrants represent (a) viable sperm (AnϪ;SϪ), (b) sperm in early apoptosis (Anϩ;SϪ), (c) sperm in late apoptosis (Anϩ;Sϩ), and (d) nonviable sperm (AnϪ;Sϩ).
• (C) Typical plot of a sperm population obtained with the TUNEL assay.
• The dark dots represent unlabeled cells , the lighter dots represent cells staining in the green channel .
• FSC, forward scatter (relative size); SSC, side scatter (relative granularity or internal complexity).
• All tests were carried out using Prism 4.0 (Graph Pad Software, San Diego, CA).

Apoptotic Markers and Semen Quality

• Samples were divided into four groups: normozoospermic (N), asthenozoospermic (A), oligoasthenoteratozoospermic (OAT), and samples that presented two parameters with values lower than those indicated by the World Health Organization (P2).
• These populations formed statistically separate entities for semen parameters (PϽ.0001), and differences between them were as expected (e.g., N and A differed only in motility), except for samples with two abnormal seminal parameters, where subpopulations (i.e., oligoteratozoospermic, asthenoteratozoospermic, oligoasthenozoospermic) could not be distinguished statistically, thus prompting the creation of the P2 population.
• To assess PS exposure on the outer leaflet of the plasma membrane (plus cell integrity) and DNA fragmentation, the authors used the annexin V and the TUNEL assays, respectively.
• To ensure that the percentages of cells calculated by flow cytometry accurately characterized each sample, a simultaneous evaluation using fluorescence microscopy was carried out on the same populations (Fig. 2 ).
• The results of these distinct quantifications for both assays were statistically indistinguishable (data not shown).

FIGURE 3

• The annexin V assay carried out on samples from men with varying etiologies.
• N and A samples differed significantly from both OAT and P2 populations (PϽ.01 and PϽ.05, respectively).
• Thus, early apoptosis seemed to inversely vary with lack of cell viability but was not correlated with late apoptosis (Pϭ.2817), which, in turn, did not correlate with nonviable cells (Pϭ.804).
• When comparing results from the annexin V and TUNEL assays carried out in the same samples, the authors observed that the only significant (and positive) correlation (Pϭ.0152 and r ϭ 0.4192) was found between DNA fragmentation and lack of cell viability (AnϪ;Sϩ).

Surface Ubiquitination, Semen Parameters, and Apoptosis

• The SUTI assay was carried out by flow cytometry and fluorescence microscopy.
• In flow cytometry experiments, the authors analyzed both the percentage of ubiquitinated entities and the intensity of fluorescence.
• In the former case, no differences were found between populations (see Fig. 4B ), but in the latter case, samples with at least one abnormal WHO parameter (concentration, motility, morphology) yielded a stronger signal when compared with normal samples, as shown by the respective fluorescence means (Fig. 4A ; Pϭ.0002).
• This contradicts recent findings (14) and may suggest a lack of reproducibility for this relatively new assay.
• When surface ubiquitination was quantified by percentage of labeled sperm cells, no differences were found between populations (Pϭ.2130), confirming their flow cytometry results (Table 2 ; Fig. 4B ).

FIGURE 4

• Sperm ubiquitination (SUTI assay) monitored by flow cytometry and fluorescence microscopy on samples from men with varying etiologies: correlations with semen parameters, annexin V staining, and Merocyanine 540-positive bodies.
• (A) Fluorencence means determined by flow cytometry.
• A clear statistically significant difference was found between normozoospermic (N) samples and all other samples, with at least one abnormal semen parameter (#Pϭ.0002), but no differences were found between asthenozoospermic (A) Varum.
• All annexin V-positive sperm cells were indeed ubiquitinated under fluorescence microscopy (Fig. 4G ).
• In all cases, the three assays were performed on the same samples.

DISCUSSION

• The authors results show that seminal parameters are inversely and significantly correlated with DNA damage and percentage of nonviable cells (AnϪ;Sϩ).
• On the other hand, the percentage of viable cells (An-;S-) correlates positively and significantly with motility and morphology.
• Furthermore, in their hands TUNELϩ is positively and significantly correlated with nonviable cells but not with apoptosis, contrary to other reports showing correlations between TUNELϩ cells and late apoptosis (7) .
• When samples were divided into groups according to semen parameter criteria, results were in accordance with what has been described.
• The fact that in A samples the prevalence is for late apoptosis, while OAT and P2 samples have a higher number of nonviable cells, suggests that spermatozoa of the latter samples initiate apoptosis earlier, or that it occurs more rapidly in these groups, considering that early apoptosis, late apoptosis, and nonviability are likely to be sequential events.

FIGURE 4 CONTINUED

• Samples, samples with two abnormal semen parameters (P2), or oligoasthenoteratozoospermic (OAT) samples.
• (I) OAT sample with an ubiquitinated entity that did not contain DNA.
• Results with the SUTI assay were less clear because different conclusions can be reached, depending on whether the percentage of ubiquitinated cells or the means of ubiquitin fluorescence are considered.
• When percentages of ubiquitinated entities were considered, both by flow cytometry and fluorescence microscopy, no statistically significant difference between groups was found.
• The fact that other factors (not only morphology) seem to determine surface ubiquitination in sperm, as well as the existence of major variations between published results, indicates possible issues in assay reproducibility.

Figures

TABLE 3

TABLE 1

Frequently asked questions

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Sandra Varum, B.Sc., Carla Bento et al. this paper, Carla Sousa, M.Sc.