Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.     

Oriented spermatozoa in the spermatophore of the palaemonid shrimp, Macrobrachium rosenbergii

Estimates of the degree of orientation of spermatozoa within spermatophores of the freshwater shrimp, Macrobrachium rosenbergii, were made using electron micrographs of ultrathin sections cut transverse and parallel to the longitudinal axis of the spermatophores. Counts of the total number of longitudinal and non-longitudinal profiles of sperm spikes in transverse sections of spermatophores indicated that 67% of the spermatozoa were oriented with their spikes approximately perpendicular to the long axis of the spermatophore. In longitudinal sections, ~30% of the sperm spike profiles were oriented parallel to the longitudinal axis of the spermatophore, leaving ~70% of the profiles oriented in planes not parallel to the longitudinal spermatophore axis. Stereologic analyses based on the number of intersections of longitudinally sectioned sperm spike profiles per unit length of test lines placed over photographic images of ultrathin sections cut in both transverse and longitudinal planes of spermatophores gave a Saltykov approximation of 0.607, favoring the view that the spermatozoa were oriented with their spikes perpendicular to the long axis of the spermatophore. This orientation brings many of the sperm bases approximately parallel to the surface of the spermatophore, an orientation which may facilitate normal sperm-egg interaction during fertilization in this species.     

Fertilization by sperm injection in the rabbit

Whole rabbit spermatozoa and isolated sperm nuclei were microinjected directly into the ooplasm of hamster and rabbit ova. These injected sperm decondensed and formed male pronuclei during subsequent in-vitro culture. Injection of whole spermatozoa and sperm nuclei prepared by a protocol known to allow in-vitro capacitation of ejaculated spermatozoa yielded a significantly higher (P < 0.01) number of activated rabbit ova containing male pronuclei than did injection of uncapacitated epididymal sperm nuclei or ejaculated sperm nuclei. Rabbit ova fertilized by sperm injection were capable of undergoing normal-appearing cleavage division during 22 h of culture.     

Scoring mitotic activity in longitudinal sections of crypts of the small intestine

Various counts have been made of the number of mitotic figures in whole crypts and sections of crypts of the small intestine of the mouse. Samples were analysed from animals killed at different times of the day and at different times after administration of vincristine. Measurements have been made of the size of mitotic and interphase nuclei and of the radial position of mitotic figures. The correction factor, f, which is required to take into account the enhancement of mitotic counts in sections as a consequence of their centripetal position has been investigated. The results indicate the following: (1) transverse sections of the crypt differ from longitudinal sections if they involve cutting the intestine before fixation which may result in a relaxation of the crypt and its widening by 25%; (2) columnar cell nuclei have a shape that resembles a sphere flattened so that the average diameter is 20% greater in crypt transverse sections; (3) mitotic nuclei tend to be about half-way between the crypt edge and the central axis of the crypt; (4) between about four and seven times more mitotic figures have their mitotic axis parallel to the long axis of the crypt; (5) about one-third of all mitotic figures in a crypt are seen in a longitudinal section of the crypt. If this is related to the number of cells in the crypt as a whole and in a section, a correction factor fD for the mitotic index of 0.59 is obtained; (6) the correction factor fT derived from the shape and position of the mitotic figures measured in 3 microns longitudinal sections is 0.53; (7) relating cell cycle and mitotic accumulation data using a computer-based model of the crypt also permits a correction factor fmod to be estimated. This gives a value of 0.66. When sectioned material is used to calculate a mitotic index the most appropriate correction factor is fD; for mouse small intestine it is 0.59.     

Feulgen Staining of Rabbit Spermatozoa: Hydrolysis Time and Distribution of Chromatin

A period of 12-16 min was found to be optimum for hydrolysis, in 1 N HCl at 60 C, of rabbit spermatozoa for quantitative Feulgen staining. The gradient of optical density along the longitudinal axis of Feulgen-stained rabbit sperm cell nuclei followed a typical pattern, deviating from a linear regression. A 2 μ diameter spot centered at the midpoint of the nucleus with uniform density approximating the mean density was present in most of the cells measured. This central spot can be used with reliance in DNA assays using light beam core sampling methods of microspectrophotometry.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

Role of guinea-pig sperm autoantigens in sperm binding to the zona pellucida and oocyte penetration

In vitro, binding of acrosome-reacted spermatozoa to the zona pellucida of mature guinea-pig oocytes was inhibited by guinea-pig sperm anti-T IgG and antibodies. Anti-P IgG antibodies prevented oocyte penetration without interfering with sperm-zona binding. The fusion of acrosome-reacted spermatozoa with zona-free oocytes was prevented by anti-T IgG and it was diminished by anti-P IgG. In the same conditions anti-S antibodies had no effect in these in-vitro fertilization events. Immunization of female guinea-pigs with P antigen resulted in a significant decrease of the number of tubal cleaved eggs. T antigens were less clearly implicated in fertilization in vivo. This study provides evidence that well characterized autoantigenic molecules of guinea-pig spermatozoa are involved in fertilization events.     

两种石龙子精子超微结构的比较

利用透射电镜研究多线南蜥和印度蜓蜥附睾精子的超微结构。两种卵胎生石龙子的精子具有一些有鳞类精子的共同特征,即具有顶体囊、顶体下锥、单个核前穿孔器和核喙,无核内管,纤维鞘伸入中段,与双联微管3和8相邻的外周致密纤维具双份纤维结构。多线南蜥和印度蜓蜥精子超微结构的种间差异主要表现在:多线南蜥精子核前方的顶体下锥电子密度较小,顶体囊具单侧嵴,横切面上可见非连续的致密体环或11个线粒体;印度蜓蜥无单侧嵴,横切面上可见连续的致密体环或12个线粒体。迄今未发现石龙子科精子的独征,但该科不同类群的顶体囊、顶体下腔、核前方的顶体下锥电子致密程度、核肩、纵切面线粒体与致密体的排列方式、横切面致密体环形状和线粒体等精子超微结构特征有一定程度的差异。这些差异可为研究石龙子科系统发生提供辅助信息。     

DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes.

DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 +/- 0.16) and testicular spermatozoa (4.03 +/- 0.34), and then decreased sharply (p < 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 +/- 0.03). However, the ratio was not different among corpus (0.69 +/- 0.01), cauda epididymis (0.68 +/- 0.11) and ejaculated spermatozoa (0.63 +/- 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p < 0.01) during passage of spermatozoa from testis (4.74 +/- 0.16) through epididymis (caput, 2.72 +/- 0.08; corpus, 1.07 +/- 0.03; cauda, -0.05 +/- 0.05; ejaculated, 0.08 +/- 0.03). The acridine orange red/green fluorescence ratio increased (p < 0.01) during zona penetration (binding sperm, 0.52 +/- 0.09; perivitelline sperm, 0.64 +/- 0.16) and sperm decondensation (decondensed sperm, 0.69 +/- 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.     

Effects of peristaltic and longitudinal wave motion of the channel wall on movement of micro-organisms: application to spermatozoa transport

A mathematical model is presented to study the motion of the spermatozoa in the cervical canal by considering the transverse waves along its tail and the transverse and longitudinal motions of the cervical wall. In an attempt to control fertility by reducing the speed of sperm, the transverse waves have been considered in the direction opposite to the motion of the spermatozoa. It has been shown that by having appropriate transverse wave motion and longitudinal velocity, the sperm may not be able to move towards the oviduct even if it could continue to have its own propelling velocity. A particular case of the motion of a thin plane sheet in a channel under peristaltic motion of its walls has also been obtained and studied.     

In vitro metabolism of androgens by mammalian cauda epididymal spermatozoa.

The in vitro metabolism of [3H] testosterone (17beta-hydroxy-4-androsten-3-one), [3H] androstenedione (4-androstene-3,17-dione) and [3H] dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) by cauda epididymal spermatozoa from the rat, rabbit, hamster, guinea-pig and ram, varied between species. There were differences in the androgens utilized, the extent of their conversion and the identities of the metabolites formed. Of the steroid substrates tested rat spermatozoa metabolized testosterone preferentially while spermatozoa from guinea-pig transformed [3H] dehydroepiandrosterone (DHEA) almost exclusively. Rabbit spermatozoa converted all three [3H] androgens while hamster sperm utilized [3H] testosterone and [3H] DHEA. Spermatozoa collected from rams killed at the abattoir metabolized both [3H] androstenedione and [3H] DHEA but this capacity was dramatically reduced in spermatozoa collected from rams subjected to short-term anaesthesea. The results are discussed in relation to the possible direct roles of androgens in sperm physiology.     

Preferential attachment of cock spermatozoa to the perivitelline layer directly over the germinal disc of the hen's ovum.

In vitro incubation of cock spermatozoa with perivitelline layer (PL) from recently ovulated ova of the hen resulted in binding of spermatozoa to the PL and activation of the acrosome reaction. A simple quantitative technique was developed for assessing these events. Following incubation of the PL (0.5 cm2 sections) with spermatozoa, the PL section was rinsed and stained with Schiff's reagent. Microscopic examination revealed holes in the PL that were assumed to be sites of spermatozoa penetration. Utilizing this technique, a correlation was demonstrated between sperm concentration and the number of spermatozoa attaching to the PL and undergoing an acrosome reaction. Pre-treatment of spermatozoa with solubilized PL inhibited spermatozoa binding to pieces of intact PL. The PL overlying the germinal disc and a similarly sized section of PL from another area of the ovum were removed and incubated separately with spermatozoa (1 x 10(5) sperm/100 microliters). Spermatozoa showed preferential attachment and digestion of the PL from the germinal disc area (809 sperm/mm2) as compared to PL from other areas of the ovum (608 sperm/mm2). Spermatozoa attached to the PL in a circular, doughnut-shaped fashion in the area directly over the germinal disc.     

The effect of the trypsin inhibitor aprotinin (Trasylol) and TLCK on the gelatinolytic activity of acrosin and the motility of rabbit sperm in vitro

In a series of experiments the influence of the trypsin inhibitors aprotinin (Trasylol) and TLCK (N-p-tosyl-L-lysin chloromethyl ketone) on the gelatinolytic activity of acrosin and motility of rabbit spermatozoa was tested. Ejaculated, highly motile spermatozoa were washed in Brackett-Medium. 12.5 to 1000 microns Aprotinin and 50 to 1000 micrograms TLCK, respectively, were added to samples of 1 ml sperm suspension: the specimens were incubated at 37 degrees C. Increasing aprotinin concentrations reduced the gelatinolytic activity of acrosin and the sperm incubation at a concentration of 1000 micrograms Aprotinin/ml sperm. Spermatozoa in all TLCK specimens were entirely immotile 1.5 hours after incubation. The gelatinolytic activity of acrosin was obviously not inhibited at any TLCK concentration. These results suggest that, under these experimental conditions, aprotinin and TLCK may impair primarily the motility spermatozoa.     

COMPARATIVE ANALYSIS OF MAMMALIAN SPERM MOTILITY

Spermatozoa of several mammalian species were studied by means of high-speed cinematography and electron microscopy. Three types of motile patterns were observed in mouse spermatozoa. The first type involved an asymmetrical beat which seemed to propel the sperm in circular paths. The second type involved rotation of the sperm and appeared to allow them to maintain straight paths. In the third type of pattern, the sperm appeared to move by crawling on surfaces in a snakelike manner. Spermatozoa of rabbit and Chinese hamster also had an asymmetrical beat which sometimes caused them to swim in circles. In spite of the asymmetry of the beat, these spermatozoa were also able to swim in straight paths by rotating around a central axis as they swam. Spermatozoa of some species appeared very flexible; their flagella formed arcs with a very small radius of curvature as they beat. Spermatozoa of other species appeared very stiff, and their flagella formed arcs with a very large radius of curvature. The stiffness of the spermatozoan appeared to correlate positively with the cross-sectional area of the dense fibers. This suggests that the dense fibers may be stiff elastic elements. Opossum sperm become paired as they pass through the epididymis. Pairs of opossum spermatozoa beat in a coordinated, alternating manner.     

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.     

Relationship between human sperm motility characteristics and sperm penetration into human cervical mucus in vitro

A series of 100 modified Kremer tests of human sperm penetration into human cervical mucus was carried out as part of the routine investigation of couples presenting with infertility. The outcome of these tests was significantly correlated with the concentration and progressive motility of the spermatozoa in the semen sample used for the test. Other semen characteristics significantly correlated with the test result were the mean velocity of progression (VP) and the amplitude of lateral head displacement about the axis of progression (AH) of the progressive spermatozoa. Normal sperm morphology was also correlated with the outcome. Using these semen characteristics as the independent variables to predict the test outcome in a discriminant analysis (normal vs abnormal tests), 34.2% of the variance was accounted for. From the discriminant function equation 75.0% of the test results could be predicted correctly. In the 30 cases in which the semen samples used for the tests showed greater than or equal to 25 X 10(6) progressively motile spermatozoa per ml, mean VP of greater than or equal to 25 microns/sec and mean AH of greater than or equal to 7.5 microns, 83.3% had normal test results. Conversely, all 13 cases for which the semen characteristics were below these limits had abnormal test results. Therefore, both the concentration of progressively motile spermatozoa and their movement characteristics are significant factors determining the outcome of homologous tests of human sperm-cervical mucus interaction.     

Monoclonal antibodies to rabbit sperm autoantigens. I. Inhibition of in vitro fertilization and localization on the egg

Biochemical and antigenic similarities exist among members of what can be considered a family of low molecular weight rabbit sperm autoantigens. These autoantigens are intrinsic plasma membrane glycoproteins specific to spermatogenic cells and spermatozoa. The amino acid and carbohydrate compositions of rabbit sperm autoantigen-1 (RSA-1) and RSA-2 were compared and monoclonal antibodies (mAb) were raised in mice against rabbit sperm autoantigens. The epitopes recognized by the antibodies were present on RSA-1, 2 and 3. A monoclonal anti-RSA-1, 2 and 3 (designated A.F. 1) was used to localize the antigen on spermatozoa and testis cells and investigate the epitope's tissue specificity. This mAb inhibited in vitro fertilization but did not block the sperm from dispersing the cumulus cells surrounding the egg. The mAb also demonstrated the presence of RSA-1, 2 and 3 on the plasma membrane of the egg after fertilization. It is concluded that the RSA family plays a central role in zona penetration.     

Correlation between the spermatozoal characteristics and sperm penetration distance in polyacrylamide gel and bovine cervical mucus

Correlation between the spermatozoal characteristics and the sperm penetration distance in polyacrylamide gel was assessed, utilizing frozen thawed semen samples obtained from 6 bulls, and it was compared with the correlation between sperm penetration in bovine cervical mucus and spermatozoal characteristics. In vitro sperm penetration tests were performed with mucus and gel. The sperm penetration in gel and mucus was significantly and positively correlated with post-thaw motility (r=0.81; r=0.89:P<0.01) and acrosome integrity (r=0.88; r=0.94:P<0.01). A significant negative correlation with abnormal spermatozoa (r=-0.84;r=0.83:P<0.01) was observed. Both sperm concentration and post-thaw live spermatozoa were not significantly correlated. A significant multiple regression between sperm penetration and the spermatozoal characteristics both in gel (R2=0.87; F=40.27; P<0.01) and mucus (R2=0.91; F=60.48; P<0.01) was observed. The major spermatozoal characteristics determining the capacity of spermatozoa to penetrate gel were post-thaw motility, percentage of abnormal spermatozoa and acrosome integrity. The acrosome integrity has a more significant contribution. The correlation established with sperm penetration in gel was very similar to that of sperm penetration in mucus. The utility of gel as a mucus substitute in in vitro sperm penetration tests was discussed.     

Decrease of internal free calcium and human sperm movement

In order to elucidate the effects of calcium on the movement of human spermatozoa, studies were conducted using motile cells selected by swim-up migration at 37 degrees C in 5% CO2 in air in a synthetic BWW medium containing 1.7 x 10(-3) M CaCl2 or BWW without added calcium (BWW-Ca). Preliminary experiments have confirmed that the addition of EGTA (5 x 10(-3); 10(-2) M) to BWW medium decreased the intracellular calcium concentration ((Ca++)i) of spermatozoa, as measured in cells loaded with a fluorescent Ca++ indicator, Quin-2. Concomitant measurements of (Ca++)i and sperm movement (analysed by videomicrography at 200 f/s at room temperature) were carried out on Quin-2 loaded cells incubated in BWW-Ca medium plus EGTA (10(-5) M; 10(-4) M; 10(-3) M). Under these conditions a decrease in (Ca++)i was observed and associated with a decrease in mean amplitude of lateral head displacement (ALH). Analysis using an automatic analyser (Hamilton Thorn at 37 degrees C) confirmed these results: the percentage of spermatozoa swimming with ALH greater than or equal to 6 microns is decreased when the external free calcium in BWW-Ca is decreased by the addition of 10(-5) M, 10(-4) M, or 10(-3) M EGTA. Flagellar analysis of the sperm population characterized by ALH greater than or equal to 6 microns showed a large proximal curvature of the tail associated with a low propagation wave velocity and a low beat frequency as compared to the spermatozoa with ALH less than 6 microns with similar progressive velocities. These characteristics result in a high flagellar beat efficiency (in terms of head displacement per beat). The disappearance of this pattern of movement when intracellular calcium is lowered indicates that calcium plays a complex role in the relationship between curvature and wave propagation. The ability of spermatozoa to modulate their movement in response to an alteration in the intracellular calcium level confirms the role of calcium in controlling flagellar movement in intact cells.     

Sources of variation in the morphological characteristics of sperm subpopulations assessed objectively by a novel automated sperm morphology analysis system

There is evidence that the mammalian ejaculate contains distinct subpopulations of spermatozoa and that the variability among these subpopulations may have adaptive and functional significance. This study investigated the precision, reproducibility and operating characteristics of a novel automated sperm morphology analysis system, the Hobson Morphology package, establishing protocols to investigate boar sperm characteristics. Five ejaculates were collected from each of three boars from different genetic lines: Landrace-Meishan introgression, Sireline Large White and Damline Large White. Five semen smears per ejaculate were stained with haematoxylin and eosin. Two hundred spermatozoa per slide were analysed. No significant differences among slides within an ejaculate were detected for sperm tail length (P = 0.770), head width (P = 0.736) and head length (P = 0.615), indicating that both staining and morphology analysis were precise and reproducible. Among the boars, variability in tail length was detected (P = 0.001), but head width (P = 0.114) and length (P = 0.069) did not differ significantly. Multivariate pattern analysis (PATN computer package) highlighted three sub-populations of spermatozoa objectively on the basis of tail length (10.0-22.0 microns, 22.1-73.0 microns and 73.1-130.0 microns). The Landrace-Meishan introgression boar possessed more spermatozoa (P < 0.0001) with tails 73.1-130 microns long. Subsequent analysis of morphology parameters in a pure-bred Meishan boar showed similar measurements for tail length (mean +/- SD; 66.36 +/- 24.70 microns) to the Landrace-Meishan introgression boar (mean +/- SD; 67.09 +/- 21.80 microns). Sperm subpopulations originate during spermatogenesis, when heterogeneous genotypic effects determine the structural features of spermatozoa. The findings of this study confirm that tail length differs between boars and that subpopulations of spermatozoa can be detected within a single ejaculate.