The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06510, USA 1 Present address: Department of Obstetrics and Gynecology,Szent-Gyorgyi Albert Medical School, Szeged, Hungary
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: biochemical markers/creatine kinase-M/fertility/spermiogenesis/tail length
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The potential relationship between sperm immaturity, as reflected by cytoplasmic retention, and sperm morphology first occurred to us when, following CK immunocytochemistry of human spermatozoa, we found several sperm subpopulations (reflecting the sperm CK activity differences) with different degrees of cytoplasmic retention (Huszar and Vigue, 1993). Normal spermatozoa with clear heads, characteristic of mature spermatozoa that have completed cytoplasmic extrusion, showed the lowest CK content. Other spermatozoa that exhibited patterns of different degrees of CK stippling or even solid CK staining had normal or slightly abnormal morphology. Finally, spermatozoa that displayed extensive cytoplasmic retention were of abnormal morphology and/or amorphous shape. The analysis of the association between CK content and sperm morphology indicated that a relationship exists among increased cytoplasmic retention in the sperm head, a larger head area, increased sperm head roundness, and increased incidence of amorphous sperm heads. We also showed, based on 3000 spermatozoa counted in ten pairs of samples, that the incidence of immature spermatozoa was significantly higher in the supernatant versus the pellet fractions of 4080% Percoll density gradients (Huszar and Vigue, 1993
). Considering that tail sprouting also occurs during spermiogenesis, we postulated that immature spermatozoa which had not completed cytoplasmic extrusion and thus showed arrested spermiogenic development would also have shorter tails.
In the present experiments, our aim was to explore the relationship between sperm biochemical markers and sperm shape, as measured by objective morphometry, in the head, midpiece and tail regions of mature and immature human spermatozoa. This study was facilitated by the recent paper from an Edinburgh group describing an efficient method for the visualization of the midpiece in spermatozoa (Gomez et al., 1996). Using the midpiece staining method, which also highlights the contour of spermatozoa, we further studied the relationship between sperm biochemical maturity and sperm morphology. We prepared mature and diminished-maturity sperm fractions and compared the morphometric dimensions of the sperm head, midpiece and tail in order to establish which parameters of the three sperm regions are related or unrelated to sperm maturity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The sequential centrifugation is carried out as follows. Liquefied semen is diluted with pre-warmed human tubal fluid medium (Irvine Scientific, Santa Anna, CA, USA)bovine serum albumin (5 mg/ml) in a 1:2 ratio of seminal fluid:human tubal fluid medium (max: 3.0 ml per 15 ml conical tube). After mixing, the diluted semen is centrifuged at 400 g for 4 min. The supernatant is carefully removed and transferred into a second centrifuge tube. The remaining sperm pellet is fraction A. The supernatant of fraction A is centrifuged again at 400 g for 4 min. The supernatant of this step is removed and transferred into a third centrifuge tube. The remaining sperm pellet is fraction B. Finally, the third tube is centrifuged at 4000 g for 20 min. The pellet of this step is fraction C.
CK activity and CK-M isoform ratio measurements
These assays were carried out by standard procedures as described previously (Huszar and Vigue, 1990; Huszar et al., 1992
). Aliquots of semen were washed with 1015 volumes of ice-cold 0.15 M NaCl and 30 mmol/l imidazole (pH 7.0) at 5000 g to remove seminal fluid, and the sperm pellets were disrupted by vortexing in 0.1% Triton, 30 mmol/l imidazole (pH 7.0), 10% glycerol, and 5 mM DTT. The homogenate was clarified by centrifugation at 5000 g, and aliquots of the sperm extract were subjected to CK activity determinations by a spectrophotometric CK kit (Sigma Co., St Louis, MO, USA).
The isoforms of sperm CK were separated by electrophoresis on precast Agarose gels (Helena Laboratories, Beaumont, TX, USA). The separated CK isoforms were detected by overlaying the gel with a fluorescent CK substrate. The fluorescent bands corresponding to the CK-M and CK-B isoforms were quantified under long-wave ultraviolet light with a scanning fluorometer. The CK-M ratio is expressed as % [CK-M/(CK-M + CK-B)].
Image analysis of the sperm midpiece and tail
These studies were undertaken using the Image-1 analysis system (Universal Imaging Corp., West Chester, PA, USA) in concert with a histochemical stain that targets the sperm midpiece and also highlights the contour of spermatozoa. The modified technique (Gomez et al., 1996) utilizes NADH and nitroblue tetrazolium as electron donor and acceptor, respectively, to form a blue-black compound, formazan (Caldwell, 1976
). For this procedure, aliquots of A, B and C sperm fractions resuspended to ~20x106 spermatozoa/ml concentration, were dried down onto slides, overlaid with 50 µl of nitroblue tetrazolium (3 mmol/l stock in PBS) and 50 µl of NADH (3.5 mM stock in PBS), and incubated for 4 h at 37°C in a humidity chamber. As a consequence of this histochemical procedure, the entire area of the midpiece, including the residual cytoplasm, was stained blue-black (Figure 1a
). In each of the A, B and C fractions of the 20 samples (60 fractions in all), 25 cells with well-defined tails were photographed and analysed (75 spermatozoa for each man, and 1509 spermatozoa in the study).
|
Statistical analysis
Data were analysed with SigmaStat software (SPSS Inc., Chicago, IL, USA) on a Micron PC Pentium computer. Differences within and between groups were examined by analysing variance on ranks by means of the MannWhitney U-test or the unpaired t-test, as appropriate. After the analysis of variance on ranks was performed, post-hoc analysis was carried out using Dunn's test to establish the presence of significant differences in the pair-wise comparisons. Data are mean ± SEM. A value of P < 0.05 was considered significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the 20 samples, the declining sperm maturity in the A, B and C fractions, which was due to the increased cytoplasmic retention and lower density of spermatozoa remaining in the supernatant after each centrifugation step, was well reflected by both the CK activities (0.17 ± 0.06, 0.19 ± 0.06 and 0.91 ± 0.3 CK IU/108 spermatozoa, A versus C and B versus C, P < 0.001) and the CK-M ratios (54.6 ± 5.4%, 38.8 ± 5.6% and 19.8 ± 3.3%, A versus C, P < 0.001, A versus B and B versus C, P < 0.05) in the three fractions respectively. Thus, using sequential centrifugation, we were able to prepare sperm subpopulations of various maturities from the same specimens.
Sperm dimensions in the A, B and C fractions
In order to examine the relationship between sperm maturity and the sperm morphometric parameters and also to identify the sperm region(s) that differ in mature and immature sperm fractions, we divided the 20 men into two groups based on their sperm maturity in the initial semen. The first group had low CK-M ratios (LCKM: 14.6 ± 7%, n = 8, six oligozoospermic and two normozoospermic men). The second group had high CK-M ratios (HCKM: 48.0 ± 4.3%, n = 12, two oligozoospermic and 10 normozoospermic men). We examined the spermatozoa originating in the A, B and C fractions (60 fractions in all, examples in Figure 1a and b). From each slide, we took photographs of 25 spermatozoa with fully visible tails (n = 600 in the LCKM and n = 909 spermatozoa in the HCKM groups).
The data of the LCKM and HCKM groups are presented in Table I. In both groups, the CK-M ratios differed significantly in all comparisons between the A, B and C fractions. The significantly lower CK-M ratios of the respective A versus A (66.8 ± 4.8% versus 36.4 ± 8.1%), B versus B (51.3 ± 6.1% versus 17.4 ± 3.6%) and C versus C fractions (26.3 ± 4.5% versus 10.0 ± 1.7%, P < 0.001 in all three comparisons) of the LCKM versus the HCKM groups also confirmed that the overall sperm maturity was higher in the HCKM group than the LCKM group. Regarding the three sperm regions, in the midpiece seven of the 10 morphometric parameters within the LCKM and HCKM groups showed differences at the level of P < 0.001 and two at the level of P < 0.05 among the A, B and C fractions, according to the analysis of variance. In the post-hoc analysis, 15 of the 20 comparisons were significantly different. The midpiece diameter, perimeter, area and shape differed between the A and B and the A and C fractions in both the LCKM and HCKM groups. Similarly, the analysis of variance in the tail length indicated differences at the level of P < 0.001 in both the LCKM and HCKM groups. The post-hoc comparisons also indicated differences in line with the CK-M parameters of sperm maturity. The analyses showed no significant differences in eight of the 10 parameters of head dimension, which compared the A, B and C mature and diminished-maturity sperm fractions. In addition, only the head diameter and head long axis in the LCKM group differed between the A and B fractions according to the post-hoc comparison. Thus, according to the morphometric parameters, the various dimensions of the midpiece and tail reflected the differences in sperm biochemical maturity while the head dimensions were unrelated to sperm maturity.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three other laboratories have confirmed the relationship between sperm cytoplasmic marker enzymes and sperm function via the study of sperm CK, sperm lactate dehydrogenase (LHDx), and glucose-6-phosphate-dehydrogenase (Casano, 1991; Aitken et al., 1994; Orlando et al., 1994
; Sidhu et al., 1998
). More specifically, the recent midpiece studies, in accordance with previous data on sperm CK activity and the CK-M isoform ratio, LDHx activity and LDHa/LDHx ratio (Huszar et al, 1988a
; Huszar and Vigue, 1993
; Lalwani et al., 1996
), showed a close correlation between the residual cytoplasm present in the midpiece and the activities of both the CK and glucose-6-phosphate dehydrogenase enzymes (Gomez et al., 1996
).
In line with previous complementary studies by the Aitken and Huszar groups that have explored the biochemical markers of sperm function, the present work has one common set of results with Gomez et al. (1996) and there are several unique features in the two papers. The common feature is the area of the midpiece measurements in immature and mature sperm fractions, defined by CK-M ratios in our case and by CK and glucose-6-phosphate dehydrogenase activities in the Gomez paper. In spite of the differences in sperm preparation methods (differential centrifugation which separates spermatozoa strictly based on their density versus Percoll gradients which exclude the low-density/large midpiece sperm fraction) and in midpiece assessment approaches (manual tracing and measurement of individual spermatozoa versus automated area measurement combined with image enhancement), the two studies provided very comparable data. In the Edinburgh study the medians (10th90th percentile) of midpiece area in the high-density and low-density Percoll populations were 4.7 (3.76.3) µm2 and 5.7 (4.18.9) µm2, which agree very well with our respective values of 5.1 (2.86.5) µm2 and 7.1 (4.312.3) µm2 in the A fraction of HCKM and C fraction of LCKM.
This common point aside, our study differs in scope from that of Gomez et al. After establishing that the midpiece area reflects sperm maturity and immaturity, the Gomez paper focuses upon corresponding differences in the rate of lipid peroxidation and sperm function, including sperm motility, acrosome reaction and spermoocyte fusion. These data are in full agreement with previous results pertaining to the relationship between cytoplasmic retention, sperm CK-M ratios, zona-binding ability, and the occurrence of IVF pregnancies (Aitken et al., 1989a,b
, 1994
; Huszar and Vigue, 1990
; Huszar et al., 1992
, 1994
). Particularly striking is the similarity between the Gomez study's correlation between midpiece area and the reactive oxygen species generation (r = 0.48) and the correlation between CK activity and the rate of sperm lipid peroxidation (r = 0.43) reported by the Yale group (Huszar and Vigue, 1994
). Other findings that support the utility of sperm midpiece measurements are the relationship between midpiece size and rate of lipid peroxidation, the decline in motility in spermatozoa exposed to oxidative stress and the recent data demonstrating that, during spermiogenesis, a remodelling of the sperm plasma membrane occurs, which is likely to promote the formation of the sperm zona-binding sites and ion-channels necessary for the fertilization function (Rao et al., 1989
; deLamirande and Gagnon, 1992a
,b
; Huszar et al., 1997
).
Following a different line of research, in the present study we systematically examined the head, midpiece, and tail regions in sperm fractions of various maturity, as defined by the CK-M ratios, and looked for morphometric features that would reflect sperm maturity. We found that sperm tail length and midpiece dimensions, with the exception of axis length, differed among the mature and diminished maturity sperm fractions, although none of the head dimensions showed corresponding differences. The midpiece long axis length findings contain two points of interest. (i) The axis lengths were not different in the HCKM group, but the analysis of variance indicated an overall difference in the LCKM group without differences between the A, B and C fractions in the post-hoc analyses. The reason for this apparent discrepancy is that the Dunn's post-hoc comparison used is a very conservative test. Other tests indicated a difference (P < 0.05) between the A and B groups, but we choose not to mix tests of different levels of rigor in the same Table. (ii) From the physiological perspective one could argue that the length of the midpiece axis should not be subject to substantial changes because it is fixed by the underlying fibre structure, the development of which precedes cytoplasmic extrusion.
Considering the apparent relationship among tail sprouting in the elongated spermatids and the simultaneous events of cytoplasmic extrusion and commencement of CK-M isoform synthesis in the spermiogenesis, we propose a hypothesis incorporating these elements in an integrated scheme of sperm development (Figure 2). In the last phase of spermatogenesis, following the second meiotic division and formation of the round spermatid, the spermiogenic phase of sperm development occurs (Clermont, 1963
). During this period, the tail sprouts and the acrosome is formed. As the spermatid elongates further, the cytoplasm that will be left as a residual body in the adluminal area accumulates around the midpiece of the spermatozoa. This process is followed by cytoplasmic extrusion, expression of the CK-M isoform, and the developmental remodelling of the sperm plasma membrane, which facilitates the formation of sites (i.e. zona-binding sites) necessary for spermoocyte interaction (Huszar and Vigue, 1990
, 1993
; Huszar et al., 1997
). It was somewhat unexpected that all differing morphometric parameters among the A, B and C fractions were in the midpiece and the tail regions of spermatozoa, while head dimensions were similar in the fractions of varying maturity. However, once the data are considered in the light of the related events of spermiogenesis (Figure 2
), the connections between sperm immaturity, increased cytoplasmic retention in the midpiece, and shorter tail size become clear. We suggest that the morphological and biochemical parameters of spermatozoa are related because they both reflect the completion of spermiogenic development of mature spermatozoa or the arrest of maturation in diminished-maturity spermatozoa. Further, arrested or delayed spermiogenic development, which results in abnormal morphology due to the retention of the extra cytoplasm (in native or in stained spermatozoa the extra cytoplasm is not perceptible, thus the midpiecehead complex gives the appearence of larger head size), is also reflected in increased CK activity, diminished concentration of the CK-M to CK-B isoforms (lower CK-M ratios), shorter sperm tails and retarded plasma membrane remodelling, which causes diminished zona-binding ability.
|
In order to assess the relationship between biochemical markers and sperm morphology, in a blinded study, we carried out simultaneous CK determinations and strict sperm morphology determinations using the Dimensions program (IVOS; HamiltonThorne Research, Beverly, MA, USA), which is based on the strict criteria (Menkveld et al., 1990; Kruger et al., 1996
), and the CK parameters developed in our laboratory. In 81 samples, we found close correlations of r = 0.71 and r = 0.74 between the incidences of abnormal spermatozoa versus CK activity and CK-M ratios, respectively. With respect to clinical utility and the comparative predictive values, the sperm biochemical markers fared better than sperm morphology: in 14 of the 66 normozoospermic semen samples (sperm concentration: 61 ± 8 x106 cells/ml) that did not conform with the correlation, 12 men had abnormal CK activity and CK-M ratio values while only three men showed <15% spermatozoa with normal morphology (Yamada et al., 1995
).
Based on the data of the present paper, the theoretical question may arise whether sperm morphology or biochemical markers better predict male fertility. This question clearly cannot be decided without rigorous clinical studies, but two points merit consideration. (i) Measurements of CK activity and CK-M isoform ratio are objective, whereas sperm morphology determinations are known to be highly variable among laboratories and technicians. (ii) In accordance with the scheme of Figure 2, a sperm subpopulation which has substantially completed the processes of cytoplasmic extrusion and sperm tail formation during spermiogenesis may experience a late developmental failure that arrests biochemical maturation. Thus, sperm maturity and function in some spermatozoa may be diminished without appreciable changes in sperm morphology.
We can also compare the utility of strict morphology and CK-M isoform ratio in IVF studies, although not on the same population. In an excellent structured review examining the predictive value of strict sperm morphology for IVF outcome (Coetzee et al., 1998), 15 articles (of 216 considered) provided sufficient data for statistical analysis. In three key parameters there were significant differences between the groups with <4% and >4% normal forms (fertilization rates: 59.3 versus 77.6%; no transfer rates: 24.0 versus 7.4% and pregnancy rates: 15.2 versus 26.0%). As much as these data may be helpful in the management of patients, they fall short of the optimal goal of diagnostic medicine: to provide inclusion and exclusion criteria (Ombelet et al., 1997
). The CK-M ratio measurements seem to be more efficacious in predicting IVF failure. In the first blinded study, 22 of the 84 IVF husbands had CK-M ratios in the diminished maturity <10% range and none achieved pregnancy. Of the 22 men, nine were normozoospermic (Huszar et al., 1992
). A recent retrospective study confirmed these findings. No men with <10% CK-M ratio caused pregnancy, although the proportion of such men was only 15 of 194 couples, because patients with low motile sperm concentrations are now treated with intracytoplasmic sperm injection. For the same reason, however, the representation of normozoospermic men who failed to cause pregnancy has increased (eight of 15) in the <10% CK-M group (Dokras et al., 1999
).
Due to the ambiguity discussed above between sperm morphology and sperm cellular maturation (Figure 2), we believe that the predictive value of morphology alone cannot be further improved even with `stricter' criteria. However, morphology coupled with biochemical markers, as is now attempted in combination with acrosin activity, chromatin structure assessment, and, optimally, an immunoprobe for CK-M, which will allow visualization of individual mature spermatozoa along with the morphology, should be a better approach (Menkveld et al., 1996
; Duran et al., 1998
). The data of the present study, identifying the sperm regions that best reflect maturity, will likely further improve the diagnostic utility.
Another aspect of diminished sperm maturity is apparently reflected by differences in chromatin structure, detectable by the DNA-specific fluorescent dye, acridine orange (Evenson et al., 1991). It is unclear at present whether the reduced stability of DNA, signified by a shift from green (native, double-stranded DNA) to red (single-stranded DNA), is caused by early spermatogenic faults in DNA replication, defects in the histoneprotamine replacement process or by DNA nicks due to the increased rate of lipid peroxidation in immature sperm (Aitken et al., 1989a
; Huszar and Vigue, 1994
; Sakkas et al., 1995
; Lalwani et al., 1996
; Steger et al., 1998
). The value of the sperm chromatin structure assay in predicting male fertility is under investigation in several laboratories. However, a recent epidemiological study in 277 Danish men demonstrated differences with respect to age, smoking habits, and the presence of immature germ cells and leukocytes in the semen (Spano et al., 1998
).
In the context of sperm maturation, one should also address the distinction between cellular maturation which is a genomically regulated process, and `epididymal maturation', which involves modification of the spermatozoa in order to improve motility and functional integrity in both resisting premature acrosome reaction and interacting efficiently with the female reproductive tract (Amman et al., 1993). We have recently shown in men and in stallions that cellular maturation with respect to cytoplasmic extrusion and the synthesis of the CK-M isoform is completed by the time the spermatozoa arrive in the caput epididymidis. There was a high incidence of mature spermatozoa, with CK values similar to that of ejaculated spermatozoa, in all epididymal segments (Huszar et al., 1998). Similarly, in a study of prostatic carcinoma patients, there was only a slight improvement in sperm morphology among sperm fractions arising from the efferent ducts or from the corpus and cauda epididymides, whereas sperm function attributes, such as motility and accrosome response to calcium ionophore showed several-fold increases in the cauda spermatozoa (Yeung et al., 1997
). Thus, in the light of the functional but not structural sperm changes, the question arises whether the epididymal process should not be called `epididymal conditioning' rather than `maturation' in order to distinguish events that are influenced by the local environment of the epididymis.
Several groups have reported specific alterations in sperm properties due to epididymal exposure in vivo or in vitro (Turner, 1995; Boue et al., 1996
; Akhondi et al., 1997
). For instance, recent work demonstrated changes in sperm membrane anisotropy and the content of phosphatidylcholine, which is known to stimulate motility of epididymal sperm in vitro (Haidl and Opper, 1997
). In view of the spermiogenic remodelling (Huszar et al., 1997
), one may consider whether these lipid changes are homogeneous in all spermatozoa, or whether there are variations related to sperm cellular maturity and plasma membrane structure in acquiring the optimal set of lipid and protein modulators.
In conclusion, we have established the relationship between sperm morphology and the biochemical markers of sperm maturity based on principles of the cell biology of human spermiogenesis. In addition to a better understanding of this relationship, this work has a future practical application because the morphometric dimensions associated with mature sperm fractions provide objective values that will facilitate the computer-assisted determination of normal sperm morphology in semen samples. This method can be further enhanced by fluorochrome-coupled probes specific to the biochemical markers of sperm maturity, which may enhance the utility of the morphology studies in men with diminished fertility who may show disturbance of synchronicity in the spermiogenic maturation processes.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
* Part of this research was presented at the 1998 annual meeting of ESHRE, Göteborg, Sweden.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aitken, R.J., Clarkson, J.S., Hargreave, T.B. et al. (1989b) Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermic men. J. Androl., 10, 214220.
Aitken, R.J., Krausz, C. and Buckingham, D. (1994) Relationship between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions. Mol. Reprod. Dev., 39, 268279.[ISI][Medline]
Akhondi, M.A., Chaple, C. and Moore, H.D.M. (1997) Prolonged survival of human spermatozoa when co-incubated with epididymal cultures. Hum. Reprod., 12, 514522.[ISI][Medline]
Amann, R.P. Hammerstedt, R.H. and Veeramachaneni, D.N.R. (1993) The epididymis and sperm maturation: a perspective. Reprod. Fertil. Dev., 5, 361381.[ISI][Medline]
Boue, F., Blais, J. and Sullivan R. (1996) Surface localization of P34H, an epididymal protein, during maturation, capacitation and acrosome reaction of human spermatozoa. Biol. Reprod., 54, 10091017.[Abstract]
Caldwell, K. (1976) Sperm diaphorase: genetic polymorphism of a sperm specific enzyme in man. Science, 191, 11851187.[ISI][Medline]
Casano, R., Orlando, C., Serio, M. and Forti, G. (1991) LDH and LDHx activity in sperm from normospermic and oligospermic men. Int. J. Androl., 14, 257263.[ISI][Medline]
Coetzee, K., Kruger, T. and Lombard., C.J. (1998) Predictive value of normal sperm morphology: a structured literature review. Hum. Reprod. Update, 4, 7382.
Clermont, Y. (1963) The cycle of the seminiferous epithelium in man. Am. J. Anat., 112, 3551.[ISI]
David, G., Bisson, J.P., Czyglik, F. et al. (1975) Anomalies morphologiques du spermatozoide humain. 1. Prognitionen pour un système de classification. J. Gynécol. Obstet. Biol. Reprod., 4, 1736.
deLamirande, E. and Gagnon, C. (1992a) Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J. Androl., 13, 368378.[Abstract]
deLamirande, E. and Gagnon, C. (1992b) Reactive oxygen species and human spermatozoa. II. Depletion of adenosine-triphosphate (ATP) plays a role in the inhibition of sperm motility. J. Androl., 13, 379386.[Abstract]
Dokras, A., Habana, H. and Giraldo, G. (1999) Sperm cellular maturity and the treatment choice of IVF or ICSI: the contributions of the sperm creatine kinase M-isoform ratio. Abstract, American Society for Reproductive Medicine 55th Meeting, Toronto, Ontario.
Duran, E.H., Gurgan, T., Gunalp, S. et al. (1998) A logistic regression model including DNA status of spermatozoa for prediction of fertilization in vitro. Hum. Reprod., 13, 12351239.[Abstract]
Evenson, D.P., Jost, L.K., Baer, R.K. et al. (1991) Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod. Toxicol., 5, 115125.[ISI][Medline]
Gomez, E., Buckingham, D.W., Brindle, J. et al. (1996) Development of an Image-Analysis system to monitor the retention of residual cytoplasm by human spermatozoa correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J. Androl., 17, 276287.
Grow, D. and Oehninger, S. (1995) Strict criteria for the evaluation of human sperm morphology and its impact on assisted reproduction. Andrologia, 27, 325333.[ISI][Medline]
Haidl, G. and Opper, C. (1997) Changes in lipids and membrane anisotropy in human spermatozoa during epididymal maturation. Hum. Reprod., 12, 27202723.[Abstract]
Huszar, G. and Vigue, L. (1990) Spermatogenesis related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa. Mol. Reprod. Dev., 25, 258262.[ISI][Medline]
Huszar, G. and Vigue, L. (1993) Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentrations and abnormal head morphology. Mol. Reprod. Dev., 34, 292298.[ISI][Medline]
Huszar, G. and Vigue, L. (1994) Correlation between the rate of lipid peroxidation and cellular maturity as measured by creatine kinase activity in human spermatozoa. J. Androl., 15, 7177.
Huszar, G., Corrales, M. and Vigue, L. (1988a) Correlation between sperm creatine phosphokinase activity and sperm concentrations in normospermic and oligospermic men. Gamete Res., 19, 6775.[ISI][Medline]
Huszar, G., Vigue, L. and Corrales, M. (1988b) Sperm creatine phosphokinase activity as a measure of sperm quality in normospermic, variable spermic and oligospermic men. Biol. Reprod., 38, 10611066.[Abstract]
Huszar, G., Vigue, L. and Corrales, M. (1990) Sperm creatine kinase activity in fertile and infertile oligozospermic men. J. Androl., 11, 4046.
Huszar, G., Vigue, L. and Morshedi, M. (1992) Sperm creatine phosphokinase M-isoform ratios and fertilizing potential of men: A blinded study of 84 couples treated with in vitro fertilization. Fertil. Steril., 57, 882888.[ISI][Medline]
Huszar, G., Vigue, L. and Oehninger, S. (1994) Creatine kinase immunocytochemistry of human hemizonasperm complexes: Selective binding of sperm with mature creatine kinase-staining pattern. Fertil. Steril., 61, 136142.[ISI][Medline]
Huszar, G., Sbracia, M., Vigue, L. et al. (1997) Sperm plasma membrane remodelling during spermiogenetic maturation in men: Relationship among plasma membrane beta-1,4-galactosyl-transferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios. Biol. Reprod., 56, 10201024.[Abstract]
Huszar, G., Patrizio, P., Vigue, L. et al. (1998) Cytoplasmic extrusion and the switch from creatine kinase B to M isoform are completed by the commencement of epididymal transport in human and stallion spermatozoa. J. Androl., 19, 1120.
Jouannet, P., Ducot, B., Feneux, D. et al. (1988) Male factors and the likelihood of pregnancy in infertile couples. I. Study of sperm characteristics. Int. J Androl., 11, 379394.[ISI][Medline]
Kruger, T.F., Lacqut, F.A. and Ozgur, K. (1996) A prospective study on the predictive value of normal sperm morphology as evaluated by computer (IVOS). Fertil. Steril., 66, 285291.[ISI][Medline]
Kruger, T.F., Menkveld, R., Stander, F.S.H. et al. (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril., 46, 11181125.[ISI][Medline]
Lalwani, S., Sayme, N., Vigue, L. et al. (1996) Biochemical markers of early and late spermatogenesis: Relationship between the lactate dehydrogenase-X and creatine kinase-M isoform concentrations in human spermatozoa. Mol. Reprod. Dev., 43, 495502.[ISI][Medline]
Mendoza, C., Benkhalifa, M., Cohen-Bacrie, P. et al. (1996) Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridisation for evaluation of human ejaculated germ cells. Zygote, 4, 279283.[ISI][Medline]
Menkveld, R., Stander, F.S.H., Kotze, T.J.W. et al. (1990) The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum. Reprod., 5, 586593.[Abstract]
Menkveld, R., Rhemrev, J.P.T., Franken, D.R. et al. (1996) Acrosomal morphology as a novel criterion for male fertility diagnosis: relation with acrosin activity, morphology (strict criteria), and fertilization in vitro. Fertil. Steril., 6, 637644.
Ombelet, W., Bosmans, E., Janssen, M. et al. (1997) Semen parameters in a fertile versus subfertile population: a need for change in the interpretation of semen testing. Hum. Reprod., 12, 987993.[ISI][Medline]
Orlando, C., Krausz, C., Forti, G. et al. (1994) Simultaneous measurement of sperm LDH, LDHx, CPK activities and ATP content in normospermic and oligospermic men. Int. J. Androl., 17, 1318.[ISI][Medline]
Rao, B., Soufir, J.C., Martin, M. et al. (1989) Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res., 24, 127134.[ISI][Medline]
Sakkas, D., Manicardi, G., Bianchi, P.G. et al. (1995) Relationship between the presence of endogenous nicks and sperm chromatine packaging in maturing and fertilizing mouse spermatozoa. Biol. Reprod., 52, 11491155.[Abstract]
Sidhu, R.S., Sharma, R.K. and Agarwal, A. (1998) Relationship between creatine kinase activity and semen characteristics in subfertile men. Intern. J. Fertil., 43, 192197.
Spano, M., Kolstad, A.H., Larsen, S.B. et al. (1998) The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum. Reprod., 13, 24952505.[Abstract]
Steger, K., Klonisch, T., Gavenis, K. et al. (1998) Expression of mRNA and protein on nucleoproteins during human spermiogenesis. Mol. Hum. Reprod., 4, 939945.[Abstract]
Turner, T.T. (1995) On the epididymis and its role in the development of the fertile ejaculate. J. Androl., 16, 292298.
Twigg, J., Fulton, N., Gomez, E. et al. (1998) Analysis of the impact of intracellular reactive oxygen species generation on the structural functional integrity of human spermatozoa: lipid peroxidation, DNA trapmentation and effectiveness of antioxidants. Hum. Reprod., 13, 14291436.[Abstract]
Yamada, Y., Vigue, L. and Huszar, G. (1995) Sperm creatine kinase parameters and strict sperm morphology in men: Relationship between the biochemical and morphological measures of sperm maturity and fertilizing potential. American Society for Reproductive Medicine 51st Annual Meeting, Seattle, Washington.
Yeung, C.H., Perez-Sanchez, F., Soler, C. et al. (1997) Maturation of human spermatozoa (from selected epididymides of prostatic carcinoma patients) with respect to their morphology and ability to undergo the acrosome reaction. Hum. Reprod. Update, 3, 205213.
Submitted on December 17, 1998; accepted on April 9, 1999.