| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gamete Biology |
The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology3
Department of Genetics,4 Yale School of Medicine, New Haven, Connecticut 06510
Department of Histology and Embryology,5 School of Medicine, Akdeniz University, 07070, Antalya, Turkey
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
sperm
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One of these biochemical parameters is the concentration of the enzyme creatine phosphokinase (CK) in sperm, which reflects cytoplasmic retention, the hallmark of diminished maturity [3–5]. The CK immunocytochemistry studies of individual spermatozoa have also demonstrated a relationship between cytoplasmic retention and aspects of abnormal sperm morphology, including larger head size, roundness of the head, and increased proportion of amorphous heads. Furthermore, in CK-immunostained sperm-hemizona complexes, we have shown that sperm which bind to the zona are exclusively those of the normal and clear-headed type, without cytoplasmic retention [6].
Another biochemical marker of sperm maturation is the testis-specific HspA2 chaperone protein, a member of the highly conserved HSP70 chaperone family, which has been thoroughly investigated in the mouse [7, 8]. The developmental and clinical significance of the human homologue HspA2 has been established in our laboratory [9]. Using an HspA2 polyclonal antibody, we have identified two waves of expression of HspA2 protein family in human spermatogenesis and spermiogenesis [9]. The HspA2 first appears in the primary and secondary spermatocytes as a component of the synaptonemal complex, the structure formed between homologous chromosomes during meiosis. The second wave of chaperone expression occurs during terminal spermiogenesis, simultaneously with cytoplasmic extrusion, plasma membrane remodeling, and formation of zona-binding sites. We have suggested that the underlying factor in both sperm immaturity and chromosomal aneuploidies is diminished HspA2 expression, which may cause both the higher incidences of aneuploidies and, perhaps, the retention of cytoplasm and consequential abnormal shape that characterizes immature sperm. Numerical chromosomal abnormalities (i.e., aneuploidies or diploidies) occur when a sperm cell possesses less or more than one copy of an autosomal or sex chromosome or two copies of the entire genome. In line with our hypothesis, significant correlations (r = 0.7–0.78) were found between proportions of immature sperm with cytoplasmic retention and frequencies of disomies, indicating that disomies are primarily found in immature spermatozoa with cytoplasmic retention and diminished HspA2 levels [10].
Because diminished expression of HspA2 is related to increased frequencies of disomies/diploidies, and because cytoplasmic retention and maturity are directly related to sperm shape, we hypothesized that a relationship may exist between sperm shape and disomies or diploidies within the same sperm. To our knowledge, such studies aimed at identification of abnormal morphology and aneuploidy in sperm have not been carried out previously. To perform fluorescent in situ hybridization (FISH), sperm DNA must be decondensed and denatured, and these processes may alter sperm shape. Thus, any potential relationship between shape and aneuploidy cannot be assessed unless it has been established that the shape attributes of sperm remain conserved after decondensation.
Earlier studies demonstrated an association between sperm shape properties and various chromosomal abnormalities. Men with oligoasthenoteratozoospermia have increased frequencies of numerical chromosomal aberrations, suggesting that these patients produce higher proportions of aneuploid gametes [11–14]. Infertile men with normal karyotypes and low sperm concentrations or higher levels of morphologically abnormal sperm have significantly increased risks of producing aneuploid spermatozoa, particularly for the sex chromosomes [15]. Semen samples with certain forms of morphological anomalies, such as megalopinhead spermatozoa, were associated with increased rates of chromosomal aneuploidies [16–18].
Further research data suggest that the association between sperm morphology and incidences of sperm with aneuploidies and diploidies is not consistent. Lee et al. [19] found that incidences of structural chromosomal aberrations were approximately 4-fold higher in semen samples with high frequencies of sperm having amorphous, round, or elongated heads than in those with greater frequencies of morphologically normal sperm. However, incidences of aneuploidies were not significantly different between the two groups. When human spermatozoa with abnormally large or small heads were injected into mouse oocytes, no increases in sperm chromosomal aberrations were found [19]. In another study, disomy frequencies in infertile males were directly correlated with the severity of oligospermia, but no relationship was established between aneuploidy rates and morphology [20]. Furthermore, in a case study of men with increased levels of globozoospermia, shortened flagella syndrome, or sperm with abnormal acrosomes, no association was found between sperm shape and chromosomal status [21]. Additionally, Ryu et al. [22] studied approximately 100–150 morphologically normal sperm (according to the strict criteria of Kruger) from both normal and infertile couples and found that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Moreover, in a man with globozoospermia, the frequencies of aneuploidy and diploidy were within the range of normozoospermic men [23].
To resolve these discrepancies, and to better assess the relationship between sperm shape and chromosomal aberrations, it is necessary to study the same spermatozoon for both parameters. However, the important question of whether the steps of decondensation and denaturation, which are necessary for performing FISH analysis, would alter the original shape of a sperm cell has not been adequately ascertained. In the present study, we approached this question by objective, computer-assisted morphometry of individual spermatozoa in the native shape and after the decondensation and denaturation processes with consideration of sperm of various morphological properties.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The study population was composed of men who presented for semen analysis at the Sperm Physiology Laboratory of the Department of Obstetrics and Gynecology at Yale University School of Medicine. All studies were approved by the Human Investigation Committee of Yale School of Medicine.
Preparation of Slides
Aliquots of liquefied semen (100–200 µl) from eight patients (sperm concentration: 16.9 ± 3.1 million sperm/ml [median: 19.5 million sperm/ml]; motility: 43.2% ± 2.7%; all data are presented as the mean ± SEM) were diluted with physiological saline containing 0.3% BSA and 30 mM imidazole (SAIM) up to a final volume of 5–8 ml. We selected low-sperm-concentration samples as subjects for the shape-decondensation studies, because our earlier work had demonstrated that these ejaculates are likely to contain a higher proportion of immature sperm with cytoplasmic retention, abnormal morphology, and chromosomal aneuploidies [3, 10]. The semen samples were centrifuged at 400 x g for 18 min at room temperature. After the supernatant was discarded, each sperm pellet was resuspended in the SAIM solution to a concentration of 10–25 million sperm/ml. Sperm slides were prepared by smearing 10 µl of sperm onto clean glass slides and allowed to air-dry. Slides were subsequently fixed in a 3:1 methanol-acetic acid solution for 15 min; dehydrated in 70%, 85%, and 100% ethanol for 5 min at each step; and then air-dried for 20 min at room temperature.
Decondensation and Pre- and Postdecondensation Imaging of Sperm
Our basic experimental design, including preparation of slides, pre- and postdecondensation imaging of sperm, and morphometric analysis, is summarized in Figure 1.
|
The fixed slides were stained with one drop of antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA), and images were captured via a black-and-white digital camera using the 40x phase-contrast objective of an Olympus BX51 microscope (Olympus, Melville, NJ). Although the antifade-mounting medium is not a dye, it facilitated the recording of consistently dark sperm contours via the digital camera. Such contours are necessary to establish a threshold level for the separation of sperm from the background for the Metamorph assessments.
The phase-contrast images of individual sperm were digitized using a Sanyo VCB-3524 B/W CCD camera (Sanyo, Richmond, IN) and the Metamorph program (Version 4.6; Universal Imaging Corporation, Downingtown, PA) and were saved. A magnification of 40x was used in visualizing not only the individual sperm but also the sperm fields to facilitate relocalization of sperm after decondensation. In addition to recording the x and y coordinates of each digitized microscopic field, we also sketched the configuration of the sperm cells in the notebook. After the decondensation step, the fields that had been studied initially were recaptured, and the decondensed sperm were digitized and saved. Further morphometric assessments were carried out with Metamorph.
For the DNA decondensation step, the cover slips that were placed for the initial microscopic observation were carefully removed by rinsing with distilled water. Slides were placed in a humidity chamber and decondensed by flooding with a 10 mM solution of dithiothreitol (DTT; Sigma, St. Louis, MO) in 0.1 M Tris-HCl (pH 8.0) for 25 min at room temperature. Subsequently, the DTT was replaced with a 10 mM solution of LIS (lithium 3,5-diiodosalicylicacid; Sigma) in 0.1 M Tris-HCl (pH 8.0), and slides were incubated in the dark for 2.5 h at room temperature. The LIS solution was then discarded, and the slides were rinsed gently in distilled water.
In control experiments (after we had established that decondensation does not further alter sperm head size or shape), the decondensed sperm were treated with denaturation solution (28 ml of 70% formamide and 4 ml of 20x SSC [1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate]) and then heated to 75°C for 8–10 min. The denatured slides were immediately cooled to -20°C in 70% ethyl alcohol for 2 min and then in 100% ethyl alcohol for 2 min at the same temperature. Slides were allowed to air-dry.
The decondensed sperm were stained, as performed initially, with Vectashield antifade medium. The same microscopic fields that were studied initially were recaptured, and the now-decondensed sperm were digitized and saved. Further morphometric assessments were carried out with Metamorph.
Morphological Classification of the Sperm Head
To better evaluate changes in sperm head shape because of decondensation or to detect differences between normal and abnormal sperm forms, which also reflect cytoplasmic retention and sperm maturity, we classified the sperm cells, before Metamorph assessment, into four groups: symmetrical, asymmetrical, irregular, and amorphous (Fig. 2). This classification serves only to distinguish among the various types of mature and immature spermatozoa; it has no relationship to the andrological or clinical assessments of normal or abnormal sperm morphology.
|
FISH Methods
All steps, including the preparation of sperm nuclei and the processes of FISH, were carried out essentially as described by Kovanci et al. [10]. Briefly, three-color FISH was performed using centrometric probes for the X, Y, and 17 chromosomes (Vysis, Downers Grove, IL). A 12-µl sample of hybridization mixture (50% formamide and 10% dextran sulfate in 2x SSC) containing the probes was denatured at 75–80°C for 8 min and applied to the slide specimens previously denatured in 70% formamide and 2x SSC for 8 min at 70°C. The hybridization was carried out at 37°C in a moist chamber for 12–14 h. Posthybridization washes were performed with 50% formamide and 2x SSC three times at 42°C and another three times with 0.1x SSC at 60°C to remove the excess probe reagents. The slides were then washed with 4x SSC and 0.1% Tween at 42°C three times, and after staining with 4',6'-diamino-2-phenylindole (DAPI; Sigma), they were mounted with an antifade solution (Vectashield).
The overall hybridization efficiency in these experiments was greater than 98%. Sperm nuclei were scored according to published criteria [24]. Nuclei were not scored if they overlapped or if they displayed no signal because of hybridization failure. A spermatozoon was considered to be disomic when it showed two fluorescent domains of the same color, comparable in size and brightness in the approximately same focal plane and clearly positioned inside the edge of the sperm head and at least one domain apart. Diploidy was recognized by the presence of two double-fluorescence domains with the above criteria. Scoring was performed on an Olympus AX70 epifluorescence microscope, primarily with the triple-pass filter for DAPI, fluorescein isothiocyanate, and rhodamine (Chroma Technologies Co., Brattleboro, VT). All aneuploid or diploid spermatozoa were also examined with a phase-contrast objective to verify the presence of the tail and to exclude apparent diploidy in two closely positioned spermatozoa.
Calibration of the Morphometry Program
Calibration was performed by viewing an objective micrometer scale (OB-M 1/100) at 40x magnification and digitizing the image with the Metamorph program. The automated, computerized conversion of pixels to micrometers was 0.29 µm/pixel.
Computerized Morphometry Measurements
After digitizing the images, Metamorph overlay tools were used to delineate the head versus tail regions of individual spermatozoa to measure the head and the tail parameters separately. In the assessment of head parameters, Metamorph recognizes the following elements (Fig. 3): area (area of entire object), perimeter (distance around edge of object, measuring from the midpoints of each pixel that defines its border), long head axis (length of longest diameter through the object), short head axis (width measured perpendicular to the longest diameter), and shape factor (a value from 0–1, representing how closely the object represents a circle, with 1 being a perfect circle). For the sperm tail measurements, Metamorph distinguishes the fiber length (the length of an object, assuming that it is a fiber). In addition, in our laboratory, we have developed two sperm parameters that are not standard to the Metamorph program but that well reflect sperm cellular maturity: roundness ratio (short head axis:long head axis) and tail length:long head axis ratio [2]. These additional parameters were calculated using Microsoft Excel (Microsoft, Redmond, WA).
|
Validation of Methods
Because of the multistep study design for the comparison of sperm in the native and decondensed states, we invested substantial effort in demonstrating the validity and reproducibility of our methods. We determined 1) the potential effects of the antifade mounting medium, 2) the increase in sperm size caused by DTT decondensation versus LIS swelling, 3) the overall percentage of sperm undergoing the decondensation process, 4) the potential effects of denaturation coupled with the decondensation and hybridization steps, and 5) the intraassay variation regarding the accuracy of the various Metamorph measurements.
To ensure that antifade mounting medium has no effect on sperm size and shape, and to verify that its use does not block DNA decondensation with DTT, we substituted one drop of distilled water for mounting medium. The results were compared to those found with the same sperm subsequently mounted with antifade (20 sperm each from the symmetrical, asymmetrical, and irregular groups and 10 sperm from the amorphous group; n = 70 sperm). All Metamorph parameters were measured and evaluated with the paired Student t-tests. The dimensions in any parameter studied were similar between sperm before and after addition of antifade. Furthermore, the antifade application did not interfere with decondensation.
To demonstrate that changes in sperm size are directly related to the DTT-induced decondensation and not to the swelling effect of LIS, three slides from each semen sample were incubated with DTT alone, with LIS alone, or with DTT followed by LIS. The sperm head dimensions were determined before and after treatment. Sperm treated with DTT showed less than 10% increases (P < 0.05), with no significant changes in shape factor. Sperm treated with LIS alone showed less than 20% increases in head parameters and head area (P < 0.001 and P = 0.005, respectively). The combined effects of DTT and LIS, when LIS is applied first and DTT second, resulted in a head size increase of only 25%. However, sperm treated with DTT and then LIS (as carried out in the present study) showed increases comparable to those indicated by the data in Table 2 (area: +40%; perimeter: +22%; long axis: +22%; short axis: +15%; all P < 0.001, with no significant changes in shape factor; n = 250).
|
To assure that the majority of sperm undergo decondensation in response to DTT-LIS treatment and that sperm selected for measurements are typical in this regard, we reviewed 100 sperm from each of the eight men (800 sperm in all). We determined the percentage that appeared to be decondensed, overdecondensed, and completely undecondensed. Overall, 82.2% ± 2.2% of sperm were decondensed, 12.2% ± 2.0% were overdecondensed, and 5.0% ± 1.2% were not decondensed. Sperm that were not decondensed or that were overdecondensed were not further evaluated.
Because the FISH protocol calls for decondensation followed by denaturation to make DNA fully accessible to probes, we tested the effects of denaturation on decondensed sperm. One hundred sperm from three men (n = 300) were studied. When decondensed sperm were subjected to the denaturation step, no additional dimensional changes occurred. Moreover, hybridization of the sperm with the FISH chromosome probes caused no further dimensional shape changes.
We determined the intraassay variations of the Metamorph assessment by measuring the various parameters five different times in the same sperm (n = 70 sperm; five parameters measured in the native state and five parameters in the decondensed state; 3500 measurements in all). The data were developed independently by two different investigators. In this respect, the present study may be viewed as blinded, because it was impossible to predict the outcome until all data were available and compiled for analysis. The intraassay variation was 3.4% ± 0.6%. The highest variation was in the head area assessment (5.9%) and the lowest in the short axis assessment (2.4%), whereas the perimeter, long axis, and tail length measurements all showed a 2.9% variation.
Statistical Analysis
To compare the various sperm shape parameters before and after the decondensation process, we used the paired Student t-test analysis using the computer-based SigmaStat program (Version 2.0; Jandel Scientific Corporation, San Rafael, CA). We compared each sperm parameter (area, perimeter, long axis, etc.) in each sperm group. Subsequently, the percentage changes (proportion of increase before vs. after decondensation) were also calculated. The differences before and after decondensation among the sperm shape groups of symmetrical, asymmetrical, irregular, and amorphous, with respect to head and tail parameters, were separately tested utilizing the one-way ANOVA and post hoc Dunn tests. Level of significance was selected as P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Before the Metamorph studies, we performed morphological classification of the sperm head attributes by conventional microscopic assessment. The conceptual basis for this visual assessment is the relationship between sperm maturity and shape. Because of cytoplasmic retention in sperm of diminished maturity, abnormal sperm head shape is, in most cases, also a reflection of arrested maturation [4].
We assigned the sperm studied to the following four groups (Fig. 2): symmetrical (n = 115), or sperm that were normally shaped with oval, symmetrical heads and axial tail insertion; asymmetrical (n = 115), or sperm that did not satisfy the descriptions of symmetrical or irregular sperm because of somewhat tapered and/or elongated or slightly rounded heads, moderately enlarged midpieces, slightly asymmetrical postacrosomal regions, etc.; irregular (n = 115), or sperm with large, round, or asymmetrical heads, bulging midpieces, and/or abaxial tail insertions; and amorphous (n = 50), or sperm with grossly asymmetric and distorted heads.
In evaluating the effects of decondensation on sperm shape in the eight men studied, we digitized 307 fields of the native, predecondensed sperm representing all four categories of spermatozoa. Of these 307 fields, we were able to relocalize 277 fields (90%) after the decondensation step, with 395 sperm in all. Because it was easier to find sperm in the symmetrical and asymmetrical classes in any man but the irregular and amorphous types were less frequent in some men, we captured more fields in some men than in others.
Conservation of Sperm Shape after Decondensation
The Metamorph data indicate that the sperm dimensions of area, perimeter, long axis, and short axis were significantly increased after the decondensation process (Table 1 and Fig. 2). After performing paired Student t-tests, significant differences were seen in the comparisons of sperm dimensions in the native and decondensed states. This was true for all sperm groups, whether they were symmetrical, asymmetrical, irregular, or amorphous. However, in spite of the significant increases in dimensions, the sperm shape was conserved, as indicated by the lack of change in the shape factor and roundness ratios, although both the long and short axis lengths were longer in the decondensed sperm. This means that the increases in the dimensions contributing to the shape factor and roundness ratio were proportional. From the perspective of andrology, it is of note that no differences were observed in the dimensions of sperm within the symmetrical, asymmetrical, or irregular sperm categories when sperm from different men were compared. The dimensions of the amorphous sperm were random and showed no resemblance to other amorphous sperm, either within the same man or in the other study subjects.
|
Because the tail is a one-dimensional object, its length was not affected by the decondensation, and it is not considered in the tables. However, the ratio of tail length to long head axis, which is a measure of sperm maturity, is of interest (Table 1). First, as in our previous work [2], we observed that the ratio is higher in mature sperm with symmetrical head shape than in the other less mature, asymmetrical, irregular, and amorphous sperm shape categories. Also, on decondensation, the tail length:long head axis ratios were lower, because the tail length did not change but the long head axis became longer (Table 1). However, the relative tail length:long head axis ratio differences between the symmetrical and the other less mature sperm groups were maintained. This is further confirmation of the notion that sperm head dimensions are related to sperm maturity.
The visual observation of sperm before and after the decondensation process has also demonstrated very convincingly that shape was precisely maintained whether the sperm were in the symmetrical, asymmetrical, irregular, or amorphous groups (Fig. 2). It is of note that the visual and morphometric assessments of sperm were carried out under phase-contrast microscopy, because the fluorescence illumination of sperm cells obliterates the definition of sperm contours (comparisons of phase-contrast and fluorescence images are shown in Fig. 4).
|
Consistency of Decondensation-Related Changes
To examine potential differences in decondensation-related changes of sperm with symmetrical or irregular shapes, the sperm dimensions in the native and decondensed states were compared between the symmetrical, asymmetrical, irregular, and amorphous sperm categories (Table 2). Increases were observed in all four categories of the head parameters, including area, perimeter, and long and short axes. The degrees of increase were similar within a narrow range in the four groups. For instance, the four values were very close in perimeter (mean and range: 23.5% and 5 µm, respectively), in long axis (18.5% and 6 µm, respectively), and in short axis (21.8% and 2 µm, respectively). The area parameter did not quite follow this pattern, because the irregular amorphous sperm group showed a higher percentage change. However, the areas of the symmetrical, asymmetrical, and irregular groups showed the similarity pattern of the other parameters (52.3% and 7 µm, respectively). Thus, the sperm heads underwent a proportional increase in dimensions yet conserved their shapes after decondensation. The shape factor and roundness ratio parameters further supported this conclusion. Whereas the sperm head dimension increased in the 15%–87% range, the shape factor and roundness ratio remained the same or showed very minor changes (1.7% ± 0.6%).
We analyzed the sperm dimensions before and after decondensation and compared their morphometrical attributes. After performing ANOVA and post hoc Dunn tests, significant differences were seen in nearly all pairwise comparisons between the mean initial and decondensed values of symmetrical, asymmetrical, irregular, and amorphous sperm with respect to total area, perimeter, shape factor, ratio of tail length to long axis, and roundness ratio (P < 0.001). Differences in mean long axis were not significant only between asymmetrical and amorphous sperm groups. Mean values for short axis were not significantly different only between symmetrical and asymmetrical categories. No values were significantly different between the irregular and amorphous categories, except for long axis and ratio of tail length to long axis (P < 0.001). Comparisons among the four sperm categories showed significant differences (P < 0.001) in mean postdecondensation values for all head attributes except the perimeter and short axis of symmetrical and asymmetrical groups, which were not significantly different. Also, the irregular and amorphous categories did not show significant differences for the head parameters in pairwise comparisons.
Studies of Sperm Shape and FISH
In ongoing studies, we are exploring both shape properties and chromosomal complements of individual decondensed sperm. In one experiment [25], we are focusing on the potential relationships between sperm shape and chromosomal disomies and diploidies (Fig. 4). As Figure 4 indicates, relationships between numerical chromosomal aberrations and sperm shapes are inconsistent. Sperm with haploid or aberrant nuclei are in the heads of sperm with both normal and abnormal shapes. However, an association is found with respect to abnormal sperm head shape and incidences of disomies and diploidies. We sorted 600 haploid spermatozoa according to the parameters of head area, long head axis, and short head axis and established the size ranges for the upper, middle, and lower third. Subsequently, we established the dimensions of 150 sperm with disomic nuclei and 60 sperm with diploid nuclei. Finally, we compared the dimensions of sperm with aberrant chromosomes with the size ranges of the sorted, normal haploid sperm. Sperm having disomic or diploid nuclei were approximately 2- to 4-fold as likely to have sizes corresponding to the upper tertile of haploid sperm than to the middle or lower tertiles. These results indicate that aneuploid or diploid sperm may occur with abnormal or normal shapes, but aberrant nuclei are increased in sperm with abnormal shape.
In another ongoing study [26], we reexamined the long-debated question of whether there are size or shape differences between X chromosome-bearing and Y chromosome-bearing spermatozoa [27, 28]. Our combined method of FISH identification of sperm and objective morphometry appears to represent an ideal approach to this question. Based on various measurements of 150 X chromosome-bearing sperm and 150 Y chromosome-bearing sperm, we found no size or shape differences. For instance, the parameters of head area (X chromosome-bearing sperm: 31.4 ± 0.6 µm2; Y chromosome-bearing sperm: 31.3 ± 0.6 µm2) or long head axis (X chromosome-bearing sperm: 7.8 ± 0.08 µm; Y chromosome-bearing sperm: 7.8 + 0.08 µm) were identical in the two groups. In addition to the lack of differences in the mean values, the distributions of dimensions (e.g., the short axis:long axis ratios) also showed identical patterns across the dimension ranges from the elongated to the round sperm heads (Fig. 5).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We have been studying various objective biochemical markers of sperm maturity and function, such as CK content and HspA2 expression, that have been used for the assessment and evaluation of male fertility [4, 30, 31]. These measures are more reliable indicators of fertility than are sperm concentration and motility. The sperm CK level is a measure of cytoplasmic retention in diminished maturity sperm, whereas levels of the 70-kDa HspA2 chaperone protein provide an independent measure of sperm development and maturation. Indeed, because HspA2 is part of the meiotic synaptonemal complex, diminished expression of the HspA2 causes meiotic defects, such as aneuploidies [10]. A major expression of the testis-specific chaperone proteins also occurs during terminal spermiogenesis simultaneously with cytoplasmic extrusion [7, 9]. Thus, these HspA2-related chaperone proteins may also facilitate cytoplasmic extrusion, a process that, if incomplete, substantially affects sperm morphology by virtue of retained surplus cytoplasm. These concepts are the theoretical bases for relationships among diminished sperm maturity, abnormal sperm morphology, and increased frequencies of chromosomal aneuploidies.
To study the potential relationships between sperm morphology and numerical chromosomal aberrations in the same sperm, we examined whether original sperm shape would be conserved after the decondensation/denaturation steps, which are prerequisites for performing FISH on sperm. These experiments were carried out using two methodologies. First, we utilized the morphometry software, Metamorph, for objective measurements of sperm both before and after the decondensation process. Second, we divided the sperm into four groups based on sperm head shape (symmetrical, asymmetrical, abnormal, and amorphous), and we considered the maintenance of shape in the individual spermatozoa of various morphological properties both before and after decondensation. The data of all studies have been confirmatory: The sperm heads become larger after decondensation/denaturation, but the head shape remains conserved.
We have carefully validated our methods by testing the technical aspects of our protocol. We established that the mounting medium does not affect decondensation or perceived sperm size after digitization. The overall efficiency of decondensation reached 82.0%, with 5.0% of the sperm remaining nondecondensed and another 13% of the sperm being overdecondensed. The intraassay variation of the Metamorph measurements was in the range of less than 4.0%. When we examined the effects of DTT and LIS on the decondensation process, we found that these reagents, when used as in the FISH studies, cause well-reproducible effects. Finally, we also investigated the effects of denaturation and FISH hybridization steps on sperm morphology. Decondensation of sperm DNA is a prerequisite for performing FISH to reveal the DNA double helix and to make it accessible for hybridization by the FISH probes. The data have shown that after the decondensation process, neither the denaturation nor the FISH hybridization steps caused any further appreciable increase in sperm head dimensions, and the shape of sperm was still preserved.
Regarding the decondensation-related enlargement of the sperm head, it is of interest that the degree of enlargement (% increase) was uniform among the four shape groups, although differences were found among the various dimensional parameters (Tables 1 and 2). The exception to this finding is the area parameter of the amorphous group, which showed an 86% increase in contrast to the approximately 52% increase in the symmetrical, asymmetrical, and irregular groups. This difference likely relates to the fact that the amorphous group is the most immature and their membrane structure the least developed in light of the arrested maturation and sperm membrane remodeling [32].
The data further show that we were successful in our classification of sperm into the symmetrical, asymmetrical, irregular, and amorphous shape groups. The sperm categories represented well the spectrum of sperm, and we confirmed that their shape remained unchanged before and after the decondensation/FISH process (Fig. 2). The assessment of sperm dimensions also supports the conclusions (Tables 1 and 2). The percentage increases for each of the categories, such as head area, perimeter, long axis, and short axis, were quite similar, except for a larger percentage increase in the mean head area in the amorphous category. The data also indicate that sizes of sperm heads in the symmetrical, asymmetrical, and irregular groups increase within a 20%–50% range in a manner proportional to their original sizes and that, in all categories, sperm maintained their shape properties.
A recent preliminary communication reported that FISH studies and preservation of head morphology may be carried out without a decondensation step [33]. This work seems to have multiple deficiencies: First, the study is without controls, both because the authors did not also perform FISH with a decondensation protocol to compare the aneuploidy frequencies and because sperm shape was not recorded before and after a 90°C incubation. Thus, the preservation of sperm shape cannot be confirmed. Second, the study is based on two-color FISH, either with probes for the X and Y or the 18 and 21 chromosomes. This makes the distinguishing of disomies from potential diploidies problematic. Third, very few sperm were evaluated (
1000 with either the X/Y or 18/21 probe pairs per sample). Fourth, the very high rate of nuclei with disomies and diploidies with the four probes (means of 2.8% in 14 men with sperm concentrations of 19 million/ml semen and of 6.5% and 9% in two men with sperm concentrations of 19 and 37 million/ml, respectively) likely indicates nonspecific probe binding.
In summary, the present data clearly reveal that sperm shape is preserved, as determined not only visually but also by objective morphometry, after the decondensation/denaturation protocols. Moreover, it is now well substantiated that sperm which undergo FISH maintain their original shapes with high fidelity. Thus, post-FISH shape can be further used to evaluate relationships between sperm shape and the nuclear chromosomal complement. We are presently pursuing this line of research.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Gabor Huszar, Sperm Physiology Laboratory, Yale School of Medicine, 333 Cedar Street, New Haven, 06510, Connecticut. FAX: 203 737 1200; gabor.huszar{at}yale.edu
Received: 6 March 2003.
First decision: 24 March 2003.
Accepted: 21 May 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
si J, Ward D, Huszar G. The dimensions of X- and Y-bearing human sperm are not different: a study of objective morphometry and FISH. In: Program of the 28th Annual Meeting of the American Society of Andrology and XVIIth Testis Workshop; Phoenix, Arizona. Abstract 78 This article has been cited by other articles:
|
|
 |
|
  C. Celik-Ozenci, A. Jakab, T. Kovacs, J. Catalanotti, R. Demir, P. Bray-Ward, D. Ward, and G. Huszar Sperm selection for ICSI: shape properties do notpredict the absence or presence of numericalchromosomal aberrations Hum. Reprod., September 1, 2004; 19(9): 2052 - 2059. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Huszar, C. Celik-Ozenci, S. Cayli, T. Kovacs, L. Vigue, and E. Kovanci Semen Characteristics After Overnight Shipping: Preservation of Sperm Concentrations, HspA2 Ratios, CK Activity, Cytoplasmic Retention, Chromatin Maturity, DNA Integrity, and Sperm Shape J Androl, July 1, 2004; 25(4): 593 - 604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Cayli, D. Sakkas, L. Vigue, R. Demir, and G. Huszar Cellular maturity and apoptosis in human sperm: creatine kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm Mol. Hum. Reprod., May 1, 2004; 10(5): 365 - 372. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
x1500
