Flow cytometry of mammalian sperm: progress in DNA and morphology measurement.

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.     

The spermatozoon of Eurasian murine rodents: Its morphological diversity and evolution

The murine rodents are the most speciose subfamily of mammals. Here the morphology of the spermatozoon, as determined by scanning and transmission electron microscopy of representative species from four Eurasian clades, is described. Much interspecific variability in all components of the spermatozoon was found to occur, although most species have a bilaterally flattened sperm head with a single apical hook of variable length and orientation. Ultrastructural observations indicate that this apical hook invariably contains a nuclear projection as well as a large extension of the subacrosomal cytoskeleton, as a perforatorium rostrally, and a complex asymmetrical acrosomal extension. These spermatozoa also have relatively long tails that are attached to the lower concave surface of the sperm head. Uniquely, in species in the Apodemus clade, the apical hook is orientated caudally. In a few species a highly derived sperm head morphotype that does not contain an apical hook is present. These sperm heads vary in morphology from being globular in two species of Bandicota, to bilaterally flattened and paddle-shaped in Tokudaia and Micromys. In spermatozoa of the latter two genera the subacrosomal cytoskeleton, which is less extensive than in species with a hooked sperm head, forms an apical extension, but that is not the case in Bandicota. In all species where the sperm head lacks an apical hook the acrosome is more symmetrical. The sperm tail is much shorter in these species, with attachment to the head occurring on the ventral surface in Tokudaia and basal in Micromys and the two species of Bandicota. As the sperm head morphotype with a complex apical hook is present in all the major clades of murine rodents, it is likely to be a plesiomorphic character within each of these clades, with the nonhooked sperm heads, which vary greatly in structure between species of the different lineages, probably being independently derived. The ultrastructural organization of the sperm head of Bandicota, but not those of Micromys or Tokudaia, suggest divergence in some of the morphological events associated with sperm-egg interaction at the time of fertilization.     

Uniform lateral orientation, caused by flow forces, of flat particles in flow-through systems.

Recently, it was shown that the lateral orientation of sperm cells disturbs the deoxyribonucleic acid distribution measured by fluorescence in a laterally laser-illuminated flow system. The present results show how flat particles may be influenced to assume a uniform lateral orientation. This was achieved by choosing the geometrical dimensions of the hydrodynamic focusing flow path. High speed photographs of fixed chicken erythrocytes oriented in experimental chambers are presented.     

Utility of light scatter in the morphological analysis of sperm.

We were able to differentiate the morphologically diverse sperm nuclei of four animal species by using an Ortho flow cytometer to detect the forward light scatter from a red (helium-neon) laser. Cytograms depicting the axial light loss and forward red scatter signals revealed unique, but reproducible, sigmoid distributions that reflected not only interspecies differences in shape and size, but variations in particle refractive index and orientation within the flow cell at the time of analysis. Consequently, we were able to use regional gating of the light scatter cytogram to minimize the influence of orientation on the resolution of the fluorescence signal. We also observed that sperm enlarging as a result of chemically induced decondensation exhibit over time a biphasic shift (increase, then decrease) in light scatter at a species-dependent rate. These results suggest that, without any special adaptations to the flow cytometer, light-scatter parameters can be used to discriminate morphologically different sperm, to enhance the resolution of fluorescence measurements that may otherwise be confounded by variability in radial orientation, and to detect alterations in the rate of a biochemical/biophysical process such as decondensation.     

Hydrodynamic orientation of sperm heads for flow cytometry.

Two specially shaped sample injection tubes orient flat-shaped mature sperm heads in a flow cytometer. Orientation allows control of a photometric difficulty experienced with some flow cytometers in the measurement of fluorescent and scattered light from sperm and other flat cells. Both relative DNA-stain content and a measure of cell flatness can now be determined simultaneously for each cell at the high rates possible with flow instruments.     

A means for orienting flat cells in flow systems.

Flattened cells, such as red blood cells, epithelial cells, and sperm of many species, cause problems for fluorescence-activated cell analysis and sorting machines because the flow systems of such devices are unable to control the orientation of these cells as they flow past the detectors. For this reason, the fluorescence or scattered light measurements for identical cells may vary greatly. A flow geometry is here described that orients flat cells in a coaxial flow system so that each cell presents the same aspect to the observation device. A wedge-shaped exit on the sample injection tube in a coaxial flow system is sufficient to produce the desired orientation effect when used with low sample flow rates. Data is presented showing the effect of orientation of fixed chicken erythrocytes on histograms of small forward-angle light-scattering measurements.     

The Fluorescent Dye 3, 3' Dihexyloxacarbocyanine Iodide Selectively Stains the Midpiece and Apical Region of the Heads of Murid Rodent Spermatozoa

Fluorescence microscopy of caudal epididymal spermatozoa stained with 3, 3' dihexyloxacarbocyanine iodide (DiOC₆(3)) showed intense fluorescence along the concave surface of the apical hook of spermatozoa of Rattus species and along the upper concave margin of the sperm head in Mus musculus In the spermatozoa of Hydromys chrysogaster, Melomys cervinipes, and Pseudomys australis, the two ventral processes also fluoresced brightly. In P. australis, fluorescence in the apical hook of sperm heads was largely localized to its upper and lower surfaces. The sperm of N. alexis did not show consistent positive fluorescence. The localization of fluorescence in these spermatozoa after staining with DiOC₆(3) was mainly restricted to regions where a large accumulation of perinuclear theca material lies beneath the plasmalemma. The reason for this remains to be determined, but DiOC₆(3) may be useful for quickly demonstrating areas of abundant perinuclear thecal material in sperm heads of eutherian mammals by light microscopy.     

Measurement of mammalian sperm deoxyribonucleic acid by flow cytometry. Problems and approaches.

Measurement of mammalian sperm deoxyribonucleic acid content is of importance in several areas of biomedical research. When measured in flow systems with orthogonal axes of illumination, flow and detection, an unexpected, distorted distribution consisting of a narrow peak with a lateral extension to the right is observed. Several lines of evidence lead to the conclusion that this effect is an optical-geometric artifact attributable to the flat shape and high index of refraction of mammalian sperm heads. This artifact disappears when an epiillumination flow system is used in which the optic axes for illumination and detection and the flow axis are all coincident. Other approaches also eliminate the artifact. The resulting coefficients of variation observed after acriflavine-Feulgen staining are 4-5%, short of the goal of 1.5% required to distinguish between human sperm bearing X and Y chromosomes and to develop a mutagen test system using mice.     

Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads

We investigated the development to the blastocyst and subsequent live-offspring stages of in vitro-matured bovine oocytes intracytoplasmically injected with flow cytometrically sorted bull sperm heads. Bull sperm heads, prepared by ultrasound sonication, were distinguished and sorted on the basis of their relative DNA contents using a flow cytometer/cell sorter modified for sorting sperm. By fluorescence in situ hybridization, the proportion of sperm confirmed as having Y specific DNA in the fraction sorted for the Y sperm was 82%. Injection with single sorted sperm heads of in vitro-matured oocytes (cultured for 24 h) resulted in 46.6% cleavage and 6.9% blastocyst development rates. Embryo transfer of 48 blastocysts (Days 7-8) to recipients (one per recipient) resulted in 20.8% pregnancy and 20.8% normal live offspring production rates. The birth of 8 male and 2 female calves represents an 80% sex preselection accuracy rate.     

A collagen-binding protein on the surface of ejaculated rabbit spermatozoa

A rabbit antibody to mouse 3T3 cell fibronectin was used in conjunction with a fluorescein-tagged second antibody to detect fibronectin-like activity on the surface of rabbit spermatozoa. Only ejaculated sperm displayed an intense and highly localized fluorescence over the acrosomal region. Cauda epididymal sperm of the rabbit as well as several other species did not exhibit any reaction. The fluorescent activity could be eliminated by trypsin treatment but was re- established by incubation in cell-free seminal fluid. Sperm recovered from females 10-12 h after mating showed a reduction or absence of antifibronectin fluorescence, suggesting that this component's loss could be a factor in sperm capacitation. Because fibronectins show strong binding to collagen, mixtures of ejaculated sperm and collagen were examined in the light and electron microscope. Living sperm appear to have a strong affinity for collagen and quickly adhere to the filaments by their heads, while continuing vigorous flagellations. Surface labeling of sperm with the galactose-oxidase-NaB[3H]4 technique, extraction with urea-detergent mixtures and affinity chromatography of extracts on gelatin-Sepharose revealed a single radioactive band of mot wt approximately 40,000 after SDS polyacrylamide gel electrophoresis and fluorography.     

Estimation of the frequency of malformed sperm by slit scan flow cytometry

We have investigated the utility of Slit Scan Flow Cytometry (SSFCM) for measuring the frequencies of malformed sperm heads in control and mutagen treated B6C3F1/CRL mice. In SSFCM, fluorescence profiles of sperm heads stained with the DNA-specific fluorescent dye acriflavine were recorded for sperm flowing lengthwise through a 2.5-microns-thick laser beam. Malformed sperm were detected as having fluorescence profiles that differed substantially from an average fluorescence profile for sperm from untreated mice. Specifically, a sum of squared difference (SSD) value was calculated for the fluorescence profile of each sperm according to the equation (Formula: see text) where c(i) and t(i) are the ith values for the fluorescence profiles from control and test sperm, respectively. Profiles whose SSD exceeded a threshold value of 20 were considered to be from malformed sperm. We measured fluorescence profiles for 500 sperm per mouse from five control mice, five mice injected intraperitoneally daily for 5 days with a total of 375 mg/kg of body weight methyl methane sulfonate (MMS), and for 30 mice injected intraperitoneally daily for 5 days with total doses of procarbazine ranging from 125 mg/kg to 1,250 mg/kg. Sperm were collected from the caudae epididymides 35 days after the last injection. Frequencies of malformed sperm in these samples were also estimated by visual analysis. All samples were analyzed in double blind fashion. The visual and SSFCM malformed sperm frequencies for the samples from control, MMS-treated, and procarbazine-treated mice were correlated (r = 0.83). A dose effect was seen with both the visual and SSFCM estimates for the sperm from the procarbazine-treated mice.     

Visualization of GM1 with cholera toxin B in live epididymal versus ejaculated bull, mouse, and human spermatozoa

The organization of membrane subdomains in mammalian sperm has recently generated controversy, with several reports describing widely differing localization patterns for the ganglioside GM1. Using the pentameric B subunit of cholera toxin (CTB), we found GM1 to be restricted to the plasma membrane overlying the acrosome in the heads of live murine sperm. Interestingly, CTB had minimal binding to live bovine and human sperm. To investigate whether this difference in GM1 localization was because of species differences or differences between collection from the epididymis (mouse) or an ejaculate (bull, human), we examined epididymal bovine and human sperm. We found that GM1 localized to the plasma membrane overlying the acrosome in sperm from these species. To determine whether some component of seminal plasma was interfering with the ability of CTB to access GM1, we incubated epididymal mouse sperm with fluid from murine seminal vesicles and epididymal bull sperm with bovine seminal plasma. This treatment largely abolished the ability of the CTB to bind to GM1, producing a fluorescence pattern similar to that reported for the human. The most abundant seminal plasma protein, PDC-109, was not responsible for this loss. As demonstration that the seminal plasma was not removing GM1, sperm exposed to seminal plasma were fixed before CTB addition, and again displayed fluorescence over the acrosome. These observations reconcile inconsistencies reported for the localization of GM1 in sperm of different species, and provide evidence for the segregation of GM1 to a stable subdomain in the plasma membrane overlying the acrosome.     

Estimation of sperm numbers in insects by fluorometry

Summary To facilitate the study of mating biology in the desert leaf-cutter antAcromyrmex versicolor, methods were developed that allowed storage and easy quantification of sperm samples collected from both male and female reproductive tracts. Sperm samples stored frozen were sonicated, stained with a fluorescent DNA stain, and the fluorescence emitted by the stained sperm heads was measured. The intensity of fluorescence was shown to be a linear function of the number of sperm in the sample as determined by counting.     

Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy

To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments.     

Sperm morphology in four species of African platypleurine cicadas (Hemiptera: Cicadomorpha: Cicadidae)

Mature spermatozoa from four species of platypleurine cicadas (Albanycada albigera, Azanicada zuluensis, Platypleura capensis and P. hirtipennis) were examined by light and electron microscopy. The filiform sperm have a similar ultrastructure in all species but notable variations were found in sperm dimensions. All species produce more than one discrete length of nucleated, motile sperm, a form of polymorphism termed polymegaly. Polymegaly is expressed in two ways: sperm have bi- or trimodal head and tail lengths. The anterior parts of sperm heads are embedded in an elongate homogenous matrix forming a spermatodesm. The conical acrosome is deeply invaginated posteriorly, and sits on top of the nucleus. The acrosomal contents are differentiated internally with an electron-lucent central medulla and a denser cortex. The homogenously electron-dense nucleus is pointed anteriorly and is generally cylindrical, although posteriorly there is a lateral invagination that extends part-way along the nucleus. This invagination houses fine granular material of the centriolar adjunct. Vesicle-like elements that are associated with both the posterior nucleus and the centriolar adjunct are also found within the invagination. Immediately posterior of and adjoining the centriolar adjunct is a pair of mitochondrial derivatives that are elongated and extend for almost the entire length of the tail. The absence of accessory bodies in cicada sperm suggests that within the Cicadomorpha, the families Cicadidae and Cercopidae are closely affiliated.     

Evolutionary Modification of Echinoid Sperm Correlates with Developmental Mode

A significant fraction of living sea urchin species have completely or partially eliminated the pluteus larval stage and instead develop directly from embryo to adult. Direct developing sea urchins develop from large buoyant eggs. We present data to show that evolution of these large eggs is accompanied by the evolution of spermatozoa with elogate heads, in contrast with the conical sperm heads typical of most echinoids. Two congeneric Australian species, Heliocidaris tuberculata , which develops via a pluteus, and H. erythogramma , a direct developer, were investigated in detail. The sperm of H. erythrogramma have an elongate head (11 μm in length) as compared to the conical sperm head (5.6 μm) of H. tuberculata . Electrophoretic analysis of the sperm histones indicates that no unusual histones or protamines are associated with modified head morphology. Genome sizes were determined by flow cytometry. H. erythrogramma has a haploid genome size of 1.3 pg as compared to a haploid genome size of 0.95 pg for H. tuberculata . Other direct developing echinoids have elongate sperm heads, and co-evolution of gametes is indicated as a common feature of evolution of direct development in echinoids. The most extreme case, the direct developing cidaroid sea urchin, Phyllacanthus parvispinus , possesses the longest and narrowest sperm head (20 μm × 1 μm) ever observed in an echinoid.     

The Fluorescent Dye 3, 3′ Dihexyloxacarbocyanine Iodide Selectively Stains the Midpiece and Apical Region of the Heads of Murid Rodent Spermatozoa

Fluorescence microscopy of caudal epididymal spermatozoa stained with 3, 3′ dihexyloxacarbocyanine iodide (DiOC₆(3)) showed intense fluorescence along the concave surface of the apical hook of spermatozoa of Rattus species and along the upper concave margin of the sperm head in Mus musculus In the spermatozoa of Hydromys chrysogaster, Melomys cervinipes, and Pseudomys australis, the two ventral processes also fluoresced brightly. In P. australis, fluorescence in the apical hook of sperm heads was largely localized to its upper and lower surfaces. The sperm of N. alexis did not show consistent positive fluorescence. The localization of fluorescence in these spermatozoa after staining with DiOC₆(3) was mainly restricted to regions where a large accumulation of perinuclear theca material lies beneath the plasmalemma. The reason for this remains to be determined, but DiOC₆(3) may be useful for quickly demonstrating areas of abundant perinuclear thecal material in sperm heads of eutherian mammals by light microscopy.     

Movement characteristics of ejaculated sperm from cynomolgus monkeys (Macaca fascicularis) analyzed by manual and automated computerized image analysis

Sperm motility is an important indicator of male reproductive function. An automated computerized system was used to measure the movement characteristics of cynomolgus monkey sperm. Swimming velocities were in good agreement with data derived from tracking sperm heads manually with a digitizer, but sperm counting by the system was erroneous. In some ejaculates, there were two subpopulations of sperm with different curvilinear velocities, linearities of swim-paths, and lateral movements of the heads.     

Full-term development of golden hamster oocytes following intracytoplasmic sperm head injection

The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.     

Radiation-induced DNA content variability in mouse sperm

Mouse sperm collected from the cauda epididymidis 35 days after acute testicular X-ray exposure and fluorescently stained for DNA show dose-dependent increases in the coefficient of variation (CV) of flow cytometrically obtained fluorescence distributions. By comparing dose-response curves obtained with three protocols which overcome the optical and cytochemical difficulties of sperm measurement in different ways we conclude the response is due to X-ray-induced DNA content variability. In the range between 0 and 600 rad the dose dependence of the square of CV of the DNA content variability, delta CV2D, is described by delta CV2D = Bx + Cx2, with 0 less than or equal to B less than or equal to 0.23 X 10(-2) and C = (0.44 +/- 0.06) X 10(-4). The dose x is measured in rad and delta CVD is expressed in percent. Computer modeling of the shapes of the fluorescence distributions show that at 600 rad 30 to 40% of the sperm have abnormal DNA content. Some have errors as large as two whole chromosomes, but it is not clear whether they are due to whole chromosome nondisjunction or a finer fragmentation of the genome. Exposures to benzo(a)pyrene and mitomycin C cause no detectable DNA content variability. We conclude mouse sperm DNA content measurements are not sensitive to small amounts of aneuploidy and as such will only be useful in detecting agents that produce substantial DNA content variability. Another animal with a smaller number of chromosomes might be more favorable. These sperm measurement techniques may find additional application in other areas of reproductive biology, such as the determination of the relative numbers of X and Y chromosome-bearing sperm in semen that may be artificially enriched in one population.