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Biology of Reproduction 66, 354-360 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Sperm Apoptosis in Fresh and Cryopreserved Bull Semen Detected by Flow Cytometry and Its Relationship with Fertility1

Muhammad Anzara, Liwei Heb, Mary M. Buhra, Thomas G. Kroetschc, and Karl P. Pauls2,,b

a Department of Animal and Poultry Science and b Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 2W1 c Gencor, Guelph, Ontario, Canada N1H 6J2


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was conducted to detect sperm apoptosis in fresh and frozen semen and to determine its relationship with bull fertility. Three ejaculates were collected from five breeding bulls with different fertility levels and were cryopreserved using standard methods. Two flow cytometric methods were employed to measure apoptosis: an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labeled Annexin V and propidium iodide (PI), and an assay for nicked DNA using bromodeoxyuridine (BrdU), terminal deoxynucleotidyl transferase, and fluorescein-labeled anti-BrdU monoclonal antibody. Both assays showed that fresh sperm contained 10%–20% apoptotic sperm. Significant differences in the percentage of apoptotic sperm were observed among the bulls. Cryopreservation induced translocation of PS to the outer leaflet of the plasma membrane and caused most of the necrotic cells in fresh sperm to disintegrate. Bull fertility was significantly related to the percentage of necrotic or viable sperm in fresh semen as detected by the Annexin V/PI assay, to the number of apoptotic sperm in fresh semen as detected by the TUNEL assay, and to the level of chromatin or DNA condensation as detected by PI staining. The present study suggests that the presence of apoptotic spermatozoa in fresh semen could be one of the reasons for poor fertility in breeding bulls.

apoptosis, gamete biology, sperm, sperm maturation, stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Necrosis and apoptosis are two forms of cell death. Necrosis results from injury and affects large numbers of cells, causing cell swelling and membrane rupture. In contrast, apoptosis (meaning "the dropping of leaves" from tree) is physiologically programmed cell death that affects single cells without any associated inflammation in the surrounding tissues [1]. Apoptosis is characterized by distinct ultrastructural [1, 2] and biochemical changes [1, 3] in cells, including chromatin aggregation, cytoplasmic condensation, and indentation of nuclear and cytoplasmic membranes. At the end of this process, the nucleus undergoes fragmentation, and the whole cell blebs and fragments into apoptotic bodies. These bodies disperse in the intercellular spaces, are extruded from the tissue, or are phagocytosed by neighboring tissue cells. Apoptotic cells that are not removed by phagocytosis undergo secondary necrosis characterized by increased membrane permeability and distension of cytoplasmic membrane structure. These classical morphological events have been reported for a wide variety of cells [1, 4].

In addition, apoptosis-associated biochemical changes occur on the cell surface and in the DNA. During early apoptosis, a cell looses its membrane asymmetry. Phosphatidylserine (PS), normally present on the inner cytoplasmic leaflet of the plasma membrane of healthy cells, is translocated and exposed on the outer leaflet [5]. The externalization of PS tags apoptotic cells to promote their phagocytosis by neighboring healthy cells. In apoptotic cells, internucleosomal cleavage of DNA by specific endonucleases produces ~180-base pair DNA fragments, which can be identified as distinct bands of DNA using agarose gel electrophoresis [1].

Annexin V is a Ca2+-dependent, phospholipid-binding protein (35–36 kDa) that has a high affinity for PS (Kd, ~5 x 10-2) and binds to cells with exposed PS [6, 7]. Annexin V conjugated to fluorescein isothiocyanate (FITC) fluorochrome retains its high affinity for PS and, therefore, serves as a sensitive probe that can be used for flow cytometric detection of cell death (apoptotic or necrotic) characterized by the loss of membrane asymmetry. Use of Annexin V in combination with propidium iodide (PI; a vital dye) allows the detection of apoptotic and necrotic cells distinct from viable cells. The 3'-OH end of DNA fragments can be labeled in situ by the enzyme terminal deoxynucleotidyl transferase (TdT) in the presence of fluorescently labeled nucleotide (e.g., bromolated deoxyuridine triphosphate nucleotide [BrdU]). This method is known as TUNEL (TdT-mediated deoxyuridine triphosphate nick end-labeling) [8, 9], and it allows the detection of DNA fragments by flow cytometry.

In adult males, germ cell death during normal spermatogenesis plays an important role in sperm production. Approximately 25%–75% of potential spermatozoa degenerate and die in mammalian (adult) testis [10, 11]. Spontaneous apoptosis has been observed in the seminiferous epithelium of rat testis [12], spermatogonia [13, 14], and spermatocytes and spermatids [12, 15]. That sperm apoptosis is under hormonal (FSH and LH) control is generally accepted [16–18]. The withdrawal of gonadotropins and testosterone promotes germ cell death in the testis [11]. Apoptosis-specific proteinases, called caspases, are also involved in Fas-associated germ cell apoptosis [19]. A tumor suppressor protein, p53, mediates spontaneous apoptosis in testicular germ cells. Rat Sertoli cells phagocytose apoptotic spermatogenic cells in vitro by recognizing PS exposed on the outer leaflet of the plasma membrane of degenerating spermatogenic cells [20]. Failure to remove apoptotic (defective) germ cells results in high numbers of abnormal sperm in semen and low fertility [21]. A high incidence of sperm apoptosis is found in the ejaculates of sterile and infertile men [22]. In humans, morphologically abnormal spermatozoa possess DNA strand breaks [10].

Mammalian systems undergo apoptosis in response to environmental stress via sphingomyelin and c-Jun kinase/stress-activated protein kinase pathways [23]. Short-term exposure of rat testis to heat causes apoptosis of germ cells in stage- and cell-specific fashions [24]. Scrotal insulation also causes changes in the chromatin structure of bull and stallion spermatozoa [25, 26]. Extensive DNA fragmentation with the loss of sperm motility and ability to fuse with the egg has been observed in conditions of high oxidative stress [27]. During cryopreservation, the spermatozoa undergo tremendous chemical and physical stresses. Cryopreservation changes the lipid composition of boar sperm plasma membrane [28], reduces the head size of bull sperm [29], and translocates PS from the inner to the outer leaflet of ram [30] and human [31] spermatozoa.

Three kinds of bull sperm populations—green (live), red (dead), and dual-stained (partially damaged)—have been detected by flow cytometry [32] and by fluorescence microscopy [33] in fresh and frozen bull semen. Recently, it has been found that sorted, dual-stained spermatozoa die rapidly after incubation at 37°C (unpublished results), suggesting that bull semen contains an apoptotic sperm population. However, to our knowledge, no report of apoptosis in bull sperm has appeared to date. Therefore, the present study was conducted to detect apoptotic sperm in fresh bull ejaculates by flow cytometry, to assess if cryopreservation induces apoptosis in bull sperm, and to test relationships between apoptotic sperm and fertility potential of semen processed for artificial insemination.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bull Semen

Five Semex-Alliance (Guelph, ON, Canada) Holstein breeding bulls (age, 5 yr and 3 mo) maintained at Gencor (Guelph, ON, Canada) under uniform feeding and housing conditions were used in this study. Two ejaculates were collected from each bull, on the day of experiment, with prewarmed artificial vagina (37°C). After an initial microscopic evaluation, the acceptable ejaculates (possessing >65% motile sperm) from one bull were pooled. Three (pooled) ejaculates were collected from each of the five bulls over 3 wk (one pooled ejaculate per bull per week) for flow cytometric analyses. One milliliter of fresh semen per bull was taken for the Annexin V/PI assay, and another 1 ml was used (separately) for the TUNEL assay, as detailed below. The remainder of the semen was cryopreserved and stored in liquid nitrogen (N2) for at least 48 h until evaluation by both assays. The spermatozoa were diluted in Tris-citric acid extender (pH 6.8; osmotic pressure, ~300 mOsm/kg) containing egg yolk (20% v/v) and glycerol (6% v/v). Semen was cooled to 4°C over 2.5 h and frozen in liquid N2 vapor as follows: from 4°C to -12°C at -4°C/min, from -12°C to -40°C at -40°C/min, and from -40°C to -140°C at -50°C/min before plunging into liquid N2. Thawing was performed at 37°C for 50 sec.

Annexin V/PI Assay

An Annexin V-FITC Apoptosis Detection Kit (catalog no. 6710KK; Pharmingen Canada, Mississauga, ON) was used to detect the translocation of PS from the inner to the outer leaflet of the plasma membrane of fresh and frozen-thawed spermatozoa as recommended by the manufacturer with slight modifications. Both kinds of spermatozoa were washed (500 x g, 10 min, 25°C) twice in Hepes-buffered saline (0.15 M NaCl and 0.01 M Hepes/NaOH [pH 7.0]). The sperm pellet was resuspended in Annexin V Binding Buffer (0.01 M Hepes/NaOH [pH 7.2], 0.15 M NaCl, and 2.5 mM CaCl2) at room temperature to a concentration of 2 x 106 sperm/ml. Aliquots (100 µl each, 2 x 105 cells) of fresh and frozen sperm were transferred into culture tubes. Five microliters of Annexin V-FITC + 1 µl of PI (50 µg/ml), or nothing was added to the samples. The tubes were gently mixed and incubated for 15 min at room temperature in the dark, and additional Binding Buffer (500 µl) was added to each tube. Flow cytometric evaluation was conducted within 5 min.

TUNEL Assay

An APO-BRDU Kit (catalog no. APT115; Chemicon International, Inc., Temecula, CA) was used for the detection of nicked DNA in fresh and frozen spermatozoa as recommended by the manufacturer with slight modifications. Frozen spermatozoa were first centrifuged (800 x g, 10 min, 25°C) to remove egg yolk extender. In all subsequent washings, the sperm suspensions were centrifuged at 500 x g for 10 min at 25°C. Fresh and frozen-thawed spermatozoa were diluted to 2 x 106 cells/ml in PBS (0.01 M NaH2PO4 [pH 7.2] and 1.5 M NaCl). Spermatozoa of both kinds were fixed by adding 10 ml of paraformaldehyde (1% w/v) in PBS per milliliter of sperm suspension on ice for 15 min. After fixation, sperm were washed twice in PBS, and the pellet was resuspended in 0.5 ml of PBS followed by 5.0 ml of ice-cold ethanol (70% v/v). The fixed sperm suspensions were stored at -20°C until analyzed for nicked DNA. On the day of analysis, the fixed sperm were centrifuged to remove ethanol. Then, spermatozoa were washed twice in PBS. Five milliliters of proteinase K (20 µg/ml in 10 mM Tris-HCl [pH 7.6]) were added to each sperm pellet, and the sample was mixed by swirling and then incubated for 20 min at room temperature. The sperm suspensions were washed twice in 1 ml of Wash Buffer (APO-BRDU Kit). Then, the pellet was resuspended in 51 µl of freshly prepared DNA-labeling solution (APO-BRDU Kit) containing TdT Enzyme (0.75 µl), BrdU (8.0 µl), TdT Reaction Buffer (10.0 µl), and distilled H2O (32.25 µl). The sperm were incubated in the DNA-labeling solution at 37°C for 4 h. These suspensions were gently shaken every 30 min. At the end of the incubation period, the sperm were washed twice in 1 ml of Rinse Buffer (APO-BRDU Kit), and the pellet was resuspended in 200 µl of freshly prepared antibody solution (5 µl of fluorescein-labeled anti-BrdU [called fluorescein PRB-1] monoclonal antibody and 195 µl of Rinse Buffer). The sperm suspensions were incubated in the dark for 30 min at room temperature. Spermatozoa were analyzed in 300 µl of PI/RNase A solution for counterstaining the total DNA by flow cytometry.

Flow Cytometric Analysis

An EPICS Elite ESP flow cytometer (Coulter Corporation, Inc., Hialeah, FL), equipped with an argon-ion laser (488 nm) was used to analyze sperm labeled with Annexin V and PI. The green fluorescence (FITC) was detected with PMT2 (behind 550 DL and 525 BP filters) and the red fluorescence (PI) with PMT4 (behind 600 DL and 575 BP filters). For the TUNEL assay, green fluorescence due to fluorescein-labeled anti-BrdU monoclonal antibody was collected with PMT2 (behind 525 DL and 550 BP filters). The integrated and peak red fluorescence (PI) were collected through PMT3 (behind 640 DL and 610 BP filters) to measure total DNA per cell. In both assays, the sperm population was identified by a combination of side-scatter (PMT1) and forward-scatter (FS) information. Acquisition gates on the FS x PMT1, two-dimensional histogram were used to eliminate small particles and cell debris from subsequent analyses. Ten thousand spermatozoa were analyzed per sample.

After completion of the experiment, the fertility data of the bulls (based on 56-day nonreturn rate out of 1769 total first-service inseminations) was obtained from Canadian Dairy Network (Guelph, ON, Canada). The individual bull fertility was expressed as the deviation from the average breed fertility of 69% [34, 35]. Fertility, based on the 56-day nonreturn rate, ranged from 61% to 74% for the group of bulls that were used.

Data Analysis

All data expressed as percentages were normalized with an arcsine transformation. A 5 (bulls) x 2 (fresh/frozen semen) factorial analysis was used to evaluate the main effects and their interactions. If the analysis of variance revealed significant differences among means (P < 0.05), the differences between fresh and frozen semen and among the five bulls were tested for significance with multiple-range tests. Pearson correlation coefficients were calculated to test the relationships between fertility and different kinds of spermatozoa in fresh and frozen samples. All analyses were conducted using the statistical software SYSTAT (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A typical two-dimensional flow cytometric profile of fresh sperm labeled with Annexin V and PI is shown in Figure 1. Using the Annexin V/PI assay, three kinds of cells were detected in human spermatozoa [31]. In the present study, four different kinds of sperm were observed. Apoptotic sperm were labeled with Annexin V-FITC, but not with PI, and fell into the green-only quadrant (Fig. 1D). Early necrotic sperm, in the top right quadrant (Fig. 1B), had a significant green fluorescence on the sperm head and midpiece regions and a little red fluorescence confined to the postacrosomal region of sperm head. Necrotic sperm, labeled with PI, but not with Annexin V-FITC, were found in the top left quadrant (Fig. 1A). The majority of the cells, which fell in the lower left quadrant (Fig. 1A), were nonfluorescent, fully viable sperm. These various patterns of staining were confirmed by fluorescence microscopy of sperm samples, and representative images of stained spermatozoa are shown as insets in Figure 1.



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  		FIG. 1. Flow cytometric profile of green versus red fluorescence of fresh sperm labeled with Annexin V and PI. A) Necrotic cells labeled with PI but not with Annexin V-FITC. B) Early necrotic cells with significant green and red fluorescence. C) Nonfluorescent, fully viable cells. D) Apoptotic cells labeled with Annexin V-FITC but not with PI. Images of cells with the appropriate fluorescence characteristics were identified in a stained sample by fluorescence microscopy. Bar = 10 µm

The most consistent effect of freezing and thawing was the increase in the fraction of apoptotic sperm (Annexin V+, PI-) in the frozen semen (Fig. 2A). All sperm parameters varied significantly among bulls and between semen types, but the interaction between bulls and semen type was nonsignificant and, therefore, removed from subsequent analyses. In fresh semen, the apoptotic sperm made up less than 17% of the total number of sperm. However, after freezing and thawing, the apoptotic sperm accounted for more than 31% in the sperm samples from all the bulls. Bull 2 had the highest number of apoptotic sperm (40%) in the frozen-thawed samples.



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  		FIG. 2. Apoptotic (A), early necrotic (B), necrotic (C), and viable (D) sperm in fresh and frozen semen of 5 dairy bulls (mean ± SEM, n = 3 per bull) in Annexin V/PI assay. Fresh (upper case) and frozen (lower case) sperm labeled with different letters indicate significant differences (P < 0.05) among bulls. Asterisks indicate significant differences (P < 0.05) between fresh and frozen samples. The relationships between fertility and the number of necrotic sperm (C, r = -0.93) and the number of viable sperm (D, r = 0.87) were statistically significant (P < 0.05)

The fraction of early necrotic, dual-stained sperm (Annexin V+, PI+) was significantly higher in fresh semen from bull 1 than in bull 4 or 5 (Fig. 2B). Although the freezing of semen significantly reduced (P < 0.05) the fraction of these sperm in samples from bulls 2 and 3, its overall effect averaged over all five bulls was negligible (17.0 vs. 17.1, P > 0.05).

The percentage of necrotic sperm (Annexin V-, PI+) was significantly different among fresh semen from the five bulls (Fig. 2C). In particular, bull 1, possessing the lowest fertility, had the highest percentage of necrotic sperm (24%) in fresh semen. Bulls 2 and 3 had intermediate values (14%), and bulls 4 and 5, possessing high fertility, had the minimum number of necrotic sperm (8% and 9%, respectively). Freezing and thawing significantly reduced the number of necrotic sperm in all bulls except bull 5.

The percentage of viable sperm (Annexin V-, PI-) in fresh semen was significantly different among the five bulls tested (Fig. 2D). In general, bull 1, with low fertility, had the lowest number of viable sperm (46%). Bulls 2 and 3, possessing average fertility, had intermediate viability (56% and 51%, respectively), and bulls 4 and 5, with high fertility, had the highest (64% and 67%, respectively) number of viable sperm. Freezing and thawing significantly decreased the percentage of viable sperm in all bulls except bull 3.

The green fluorescence histograms of sperm samples stained with the TUNEL assay kit had a distinct population of apoptotic sperm with intense green fluorescence (Fig. 3) at least 10-fold higher than that of the negative cells. A fluorescent microscopic examination of these samples identified a subpopulation with distinct green staining in the head region. All sperm parameters varied significantly among bulls and between semen types, but the interaction between bulls and semen type was nonsignificant and, therefore, removed from subsequent analyses. Significant differences in sperm with nicked DNA were found among the five bulls that were tested (P < 0.05) (Fig. 4). Bulls 1 and 2, with low fertility, had the highest percentages of sperm with nicked DNA (25% and 20%, respectively), whereas bulls 3, 4, and 5, possessing average or high fertility, had less than 15% apoptotic sperm identified with the TUNEL assay. For all bulls, freezing and thawing decreased the percentage of apoptotic (DNA-nicked) sperm.



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  		FIG. 3. Flow cytometric profiles of fresh sperm labeled with the APO-BRDU Kit. A) Integrated versus peak red fluorescence assay of the fixed sperm used to gate the analysis of cells for green fluorescence measurements. B) Green fluorescence of the APO-BRDU-stained cells. C) Green and red fluorescent cells in a sample stained with the APO-BRDU kit obtained from a stained sample by microscopy. Bar = 10 µm



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  		FIG. 4. Apoptotic sperm in fresh and frozen semen of five dairy bulls (mean ± SEM, n = 3 per bull) using TUNEL assay. Fresh (upper case) and frozen (lower case) sperm samples labeled with different letters indicate statistically significant differences (P < 0.05) among bulls. Asterisks indicate significant differences (P < 0.05) between fresh and frozen samples. The relationship between fertility and the number of apoptotic sperm, as characterized by nicked DNA, was significant (r = -0.90, P < 0.05)

The red fluorescence peak positions of PI-stained fresh and frozen sperm samples are shown in Figure 5. All the fluorescence profiles consisted of two distinct peaks that were measured separately (left and right peaks; Fig. 5). The mean fluorescence peak values in the cryopreserved samples were consistently lower than those in the fresh samples.



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  		FIG. 5. Red fluorescence peak positions for PI-stained sperm in fresh and frozen semen of five dairy bulls. The red fluorescence flow cytometric profiles had two distinct peaks, identified as the left (A) and right (B) peak, respectively

To explore a possible reason for the decreased number of sperm with nicked DNA after freezing and thawing, it was hypothesized that necrotic sperm undergo fragmentation during cryopreservation. To test this hypothesis, the necrotic sperm stained with PI were sorted with an EPICS Elite ESP flow cytometer (Coulter Corporation, Inc.). These cells were mixed with fluorescent beads (Beckman Coulter, Fullerton, CA) at a ratio of approximately 1:1, and the mixed samples (n = 2) were analyzed by flow cytometry to determine the actual ratio between beads and sperm. The mixed samples were frozen, thawed, and then reanalyzed by flow cytometry to quantify any change in sperm number induced by the freezing process. The results of these experiments indicated that the freeze-thaw process produced an 18% decrease in the number of PI-labeled sperm (data not shown).

Pearson correlation coefficients between bull fertility and different sperm populations (apoptotic, early necrotic, necrotic, and viable) in fresh and frozen semen using Annexin V/PI, TUNEL, and PI assays are presented in Table 1. Bull fertility was significantly (P < 0.05), but negatively, correlated with the apoptotic sperm possessing nicked DNA as detected by the TUNEL assay, necrotic sperm as detected by the Annexin V/PI assay and mean fluorescence values for the left and right peaks after staining with PI. Fertility was positively correlated with viable sperm as detected by the Annexin V/PI assay. All significant relationships were found in fresh spermatozoa.


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  		TABLE 1. Relationships of apoptosis in fresh and frozen spermatozoa using Annexin V/PI assay, TUNEL assay, and PI staining with bull fertility (n = 5)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Both flow cytometric methods indicated that fresh bull sperm contained significant numbers of apoptotic cells. To our knowledge, this is the first report of apoptosis in bull sperm using flow cytometry, and the results are consistent with previous findings that apoptosis plays an important role in normal spermatogenesis [2, 10, 11]. This technique can be used to analyze thousands of spermatozoa in a few minutes, and the laser illumination has very little adverse effect on bovine sperm DNA [36]. Moreover, during preliminary trials on several ejaculates, the flow cytometric data were confirmed by counting the same sample under a fluorescent microscope (data not shown). The flow cytometric patterns of the sperm samples stained with fluorescent Annexin V were similar to those observed for human spermatozoa [31].

Sperm apoptosis was observed in fresh ejaculates, and the number of apoptotic cells varied among different bulls. The origin of these apoptotic sperm is an interesting question. Because spermatogenesis is a continuous process, a mechanism is needed to control sperm production. Apoptosis has been observed in male germ cells [12, 37, 38], which are phagocytosed, after the recognition of PS on the outer leaflet of the plasma membrane [20] by Sertoli cells in the testis [37]. This process is mediated by a cell-surface protein, Fas [19]; a tumor-suppressor gene, p53 [27]; and a death-promoting gene, Bclw of the Bcl2 family [38]. The failure of Sertoli cells to remove apoptotic cells results in their release into the lumen of seminiferous tubules and, consequently, in an increased number of abnormal sperm in semen and reduced fertility [21]. Print and Loveland [39] suggested that selective apoptosis of genetically damaged germ cells prevents the transmission of genetic abnormalities to offspring. Therefore, the apoptotic sperm represent abnormal sperm present in the semen. For example, the percentage of human apoptotic sperm ranges from 0.1% in fertile men to 50% in carriers of testicular seminoma [22].

The current study found that the number of apoptotic sperm in fresh semen differed among bulls, suggesting that the bulls differed with respect to their ability to remove apoptotic germ cells from the semen. Because pituitary gonadotrophins (FSH and LH) and androgen (testosterone) play key roles in preventing apoptosis in male germ cells [11, 16, 17], and because Fas and p53 mediate phagocytosis of apoptotic sperm by Sertoli cells [19, 21], it would be very interesting to monitor hormone profiles as well as Fas and p53 levels in bulls yielding higher numbers of apoptotic sperm in their semen for diagnostic and treatment purposes.

The number of apoptotic sperm characterized by translocation of PS on the outer leaflet of the plasma membrane, as measured by the Annexin V/PI assay, increased up to 40% during cryopreservation in all bulls. To our knowledge, the subpopulation of apoptotic sperm in frozen semen has been totally ignored to date. There could be several reasons for the induction of apoptosis in frozen semen. During cryopreservation, the sperm plasma membrane is destabilized due to low temperatures and high salt concentrations [40], leading to marked changes in the lipid components of sperm plasma membranes in ram [41], boar [28], and bull [42]. In addition, chemical toxicity due to glycerol [43, 44] and immune responses due to egg yolk in semen extender [45] cause alterations in the plasma membrane of spermatozoa. Under normal physiological conditions, cell membrane asymmetry is maintained by the phospholipid translocators (known as flippases). Freezing and thawing might lead to damage of the flippase present in the plasma membrane, causing translocation of PS from the inner to the outer leaflet, an early sign of apoptosis. Like somatic cells, spermatozoa live in an aerobic environment and face reactive oxygen species that cause fatty acid peroxidation of membrane phospholipids and, thus, affect sperm function [46]. Lipid peroxidation of plasma membrane increases after freezing and thawing of bull [47] and human [48] sperm, which may cause apoptosis in these cells.

In apoptotic sperm, the plasma membranes are intact, whereas in necrotic cells, the plasma membranes lose their integrity and become leaky [7]. In this study, 10%–22% early necrotic sperm, characterized by red staining over the postacrosomal region of the head due to infiltration of PI, were found in fresh semen. The number of early necrotic sperm did not change due to cryopreservation, but it fluctuated in frozen-thawed semen among different bulls. Perhaps the sensitivity of the postacrosomal region of sperm to cooling and freezing varies from bull to bull. Red fluorescence at the postacrosomal region is observed in bull spermatozoa stained with SYBR-14/PI [32] or 6-carboxy-fluorescein-di-acetate/PI [33]. These findings indicate that the plasma membrane over the postacrosomal region of the sperm head may be particularly sensitive to freezing and thawing.

The number of completely necrotic sperm decreased significantly after freezing and thawing. It is speculated that completely necrotic sperm in fresh semen (8%–24%) have weak or leaky plasma membranes and undergo fragmentation during the freezing and thawing processes, resulting in a decrease in their number. Approximately 18% loss of necrotic sperm from semen during cryopreservation was experimentally confirmed in this study by sorting them with flow cytometry, determining their number, freezing and thawing them, and then counting them again (data not shown).

The viability of bull spermatozoa in the Annexin V/PI assay was lower in fresh semen than the viability as assessed with the SYBR-14/PI assay. The Annexin V/PI assay is a more precise and reliable assay than the common live/dead SYBR-14/PI assay. The former is sensitive to alterations in sperm plasma membrane at the molecular level. SYBR-14 stains the DNA green, and intact plasma membrane is a prerequisite for its penetration into the cell. Thus, SYBR-14 stains apoptotic as well as live sperm in a population, because in both cases, the plasma membrane is intact. In contrast, the Annexin V/PI assay is able to distinguish between apoptotic and live cells. This may be why a significant relationship between bull fertility and viable (fresh) sperm was found in this study. After cryopreservation, the number of viable sperm decreased in most of the bulls by 10%–20%. The sensitivity of spermatozoa to ultralow temperature varies from bull to bull. During freezing, ice crystal formation and an increase in solute concentration are mainly responsible for the reduction in sperm viability [49].

This is the first report, to our knowledge, regarding apoptosis in fresh and frozen bull spermatozoa as detected by flow cytometry using an APO-BRDU kit [50, 51] to perform a TUNEL assay. Using this assay, apoptotic sperm were detected in fresh semen, and their number was higher than that detected by the Annexin V/PI assay. The lack of a direct correspondence between the Annexin V/PI and TUNEL assay numbers indicate that both are independent assays, but both assays indicated that sperm apoptosis is occurring and that the number of apoptotic cells varied among samples from different bulls. The Annexin V/PI assay identifies the loss of plasma membrane asymmetry (especially externalization of PS on the outer leaflet), whereas the TUNEL assay detects the changes in DNA. In the Annexin V/PI assay, necrotic cells can be easily detected because their damaged membranes allow infiltration of PI to stain DNA red. In contrast, in the APO-BRDU assay, necrotic cells cannot be distinguished from other cells, because the plasma membranes of all the cells are compromised by the addition of ice-cold alcohol, which facilitates the movement of BrdU into the cell and its binding to the 3'-OH end of nicked DNA. Reduced fertilization rate in frozen semen has been mainly attributed to altered membrane structure and function during cooling, freezing, and thawing [52]. This study shows that sperm apoptosis, characterized by nicked DNA, may also contribute to low fertility in the field.

In the TUNEL assay, the number of apoptotic sperm decreased markedly after freezing and thawing. Overcondensation of human and porcine [53] and of bull [29] sperm chromatin has been observed during cryopreservation. In agreement with these results, we detected a decrease in the fluorescence of PI staining in the semen samples after cryopreservation. These structural changes may make the 3'-OH end of DNA fragments inaccessible to BrdU in the frozen spermatozoa, thus accounting for the decrease in the TUNEL assay value after cryopreservation. However, it is possible that these overcondensed sperm might, or might not, decondense during their transit in the female reproductive tract or after fertilization of the oocyte and, thus, have a negative effect on fertility, because they carry nicked DNA. Experiments with sorted apoptotic spermatozoa and intracytoplasmic sperm injection would be of great interest to study the precise effects of spermatozoa with nicked DNA on fertilization and early embryonic development.

An intriguing conclusion of the present study was that the correlation between apoptotic cells (with nicked DNA) in fresh sperm and reduced post-freeze/thaw fertility was significant, but that the correlation for cells with nicked DNA and fertility in the frozen-thawed samples was not significant (although the trend was the same). Because we found that necrotic (PI-stained) cells were prone to fragmentation, we believe that proteases or other cyototoxic materials released by the lysing cells may affect the fertility of freshly thawed samples. In this model, the apoptotic cells in cryopreserved and thawed samples are symptomatic, but not the cause, of the poor fertility.

Maintaining high fertility in semen samples is the ultimate goal of andrologists. Cryopreservation of spermatozoa increases the availability of male germplasm for artificial insemination of females. Unfortunately, the fertility of frozen semen is not as high as that of fresh semen. The present study revealed that cryopreservation causes fragmentation of spermatozoa, overcondensation of spermatozoal DNA, and sperm apoptosis (translocation of PS from the inner to the outer leaflet of the sperm membrane). All these changes likely contribute to the general decline in fertility that is observed in semen samples after cryopreservation.

No single physical or biochemical measurement of semen quality can represent the essential sperm characteristics that are required to achieve fertilization, including assays for sperm movement, cervical mucus, penetration, and structural integrity. The present study provides another method for evaluating fresh semen quality that is highly correlated with fertility. Therefore, assays for apoptotic sperm can be used with other assays of semen quality to reduce the risk of using poor-fertility bulls in artificial breeding or assisted-reproduction programs.


   ACKNOWLEDGMENTS
 
Assistance with preparation of the manuscript by A. Hill is gratefully acknowledged.


   FOOTNOTES
 
First decision: 14 May 2001.

1 Supported by Natural Sciences and Engineering Research Council of Canada grant to K.P.P. and M.M.B and by Semex-Alliance, Canada. Back

2 Correspondence. FAX: 519 763 8933;ppauls{at}uoguelph.ca Back

Accepted: September 13, 2001.

Received: April 20, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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