Difference in volume of X- and Y-chromosome-bearing bovine sperm heads matches difference in DNA content.

BACKGROUND: To investigate the possibilities of sperm head volume as a sorting criterion for gender preselection, we determined the magnitude of the difference in volume of X- and Y-chromosome-bearing bull sperm heads. MATERIALS AND METHODS: Bovine sperm heads were sorted on the basis of their DNA content in X- and Y-chromosome-bearing fractions, using an existing flow-cytometric technique. Images of sperm heads of both populations were recorded using Differential Interference Contrast (DIC) microscopy. After reconstructing the DIC images, the area and the optical thickness of sperm heads of both populations were determined. RESULTS: We found a difference in volume of X- and Y-bearing bovine sperm heads matching the difference in DNA content (3.5-4%). CONCLUSIONS: Our findings indicate that volume can be used as a criterion to distinguish X- and Y-chromosome-bearing sperm, making development of a technique to sort X- and Y-chromosome-bearing sperm based on head volume theoretically possible. A strong advantage of such a technique over the existing technique based on DNA content would be that X- and Y-chromosome-bearing sperm cells could thus be sorted without subjecting them to any staining.     

Difference in sperm head volume as a theoretical basis for sorting X- and Y-bearing spermatozoa: potentials and limitations

Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered.     

Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA

Laboratory validation is essential in developing an effective method for separating X and Y sperm to preselect sex. Utilizing sexed sperm from a particular experiment to test fertility and achieve the subsequent phenotypic sex without knowing the likely outcome at conception is too costly for most applications. Further, research advances need to be built on an ongoing assessment with respect to the collection of data to continue progress towards achieving a successful outcome. The Beltsville Sperm Sexing Technology, which is based on the sorting of X- and Y-bearing sperm through the process of flow-cytometric sperm sorting, is also well suited for validation in the laboratory by "sort reanalysis" of the sperm X- and Y-bearing fractions for DNA content. Since the sexing technology is based on the use of Hoechst 33342, a permeant nuclear DNA stain for sorting X- and Y-bearing sperm, it also can be the marker for determining the proportions of X and Y populations by sort reanalysis. The process consists of using an aliquot of the sorted sperm and sonicating to obtain sperm nuclei. The uniformity of the nuclear staining is re-established through the addition of more Hoechst 33342. Separate analysis of each aliquot produces a histogram that is fitted to a double gaussian curve to determine proportions of X and Y populations. The relative breadths of the distributions of DNA of X- and Y-bearing sperm within a species affects interpretations of the histogram. Sort reanalysis is consistently repeatable with differences in X/Y DNA equal to or greater than 3.0%. This information on sex ratio of the sperm then provides the precise tool by which one can predict the outcome in terms of sex, from a particular sample of semen. Simple analysis of unsorted sperm to determine the proportions of X- and Y-bearing sperm based on DNA content is also an effective tool for validating sperm-sex ratio, whether it is in a sample assumed to be 50:50 or predicted to be something other than 50:50. This simple analysis provides for a check on the potential sex ratio of any sample of semen.     

Mitochondrial Membrane Potential and DNA Stainability in Human Sperm Cells: A Flow Cytometry Analysis with Implications for Male Infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.     

Identifying non-sperm particles during flow cytometric physiological assessment: a simple approach

Flow cytometry is now being used more frequently to determine sperm functional characteristics during semen assessment for artificial insemination. With this methodology, viable and potentially functional cells are detected as unstained events differentiated from non-sperm events through their light-scattering characteristics. However, it can be shown mathematically that identification of sperm on the basis of light scatter leads to significant overestimation of unstained viable cells and underestimation of responding cells in tests of sperm function (subpopulations expressing different fluorescence patterns). We have developed a simple and cost-efficient flow cytometric approach for identifying non-sperm particles that can be carried out in parallel with functional assessments. Our method is based on the sperm's osmotic intolerance. Diluted in water, lethal osmotic shock causes major damage to the cell membranes, and all sperm will stain with propidium iodide (PI). Particulate material which is not PI-positive can then be quantitatively evaluated by FACS analysis and the results substituted in mathematical equations to provide true values for sperm counts and subpopulations. In practical tests, the percentage of non-sperm particles determined by this technique was closely comparable to the figure obtained either by SYBR14^®/PI staining or by PI/CFDA staining. As well as being valuable with respect to tests of sperm function, the procedure is also suitable for obtaining accurate sperm counts during routine semen evaluation.     

Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis

The only known and measurable difference between X- and Y-chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations High resolution two-dimensional (2-D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2-D electrophoresis. Up to 1,000 protein spots per gel could be detected and quantified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X- and Y-chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation. © 1996 Wiley-Liss, Inc.     

Preselection of sex of offspring in swine for production: current status of the process and its application

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.     

Separation of X- and Y-chromosome-bearing murine sperm by free-flow electrophoresis: evaluation of separation using PCR.

The effectiveness of separation of murine X- and Y-bearing sperm by free-flow electrophoresis was evaluated by the polymerase chain reaction (PCR). The ratio of X- and Y-bearing sperm from cauda epididymis was analyzed before and after free-flow electrophoresis. A Y-chromosome-specific sequence (pY353/B) and an autosomal sequence (myogenin) were used to estimate the ratio between X- and Y-sperm in the separated fractions. Cauda epididymal mice sperm were separated into two peak fractions under the electric field. Each peak fraction contained sperm of normal shape, however, the motility of the sperm was extremely diminished after separation by electrophoresis. DNA was extracted from 10(4) sperm from each fraction and from the unseparated sperm, and Y-chromosome specific PCR was performed. The PCR experiment revealed that fraction No. 16 (the peak near the cathode) was a Y-sperm rich fraction, whereas fraction No. 22 (the peak near the anode) was a Y-sperm poor one. These results suggested that murine X- and Y-sperm could be successfully separated by free-flow electrophoresis. Analysis of the chromosome-specific sequence by PCR was demonstrated to be a direct and adequate method to evaluate the separation of X- and Y-sperm.     

Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

Incidence of chromosome aberrations in mammalian sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting

The separation of two sperm populations is possible using the technique of flow sorting, provided that a significant difference exists in the DNA content of X- and Y-bearing sperm. In order to ascertain whether or not chromosome damage was induced in sorted sperm, chromosome preparations were made from isolated sperm that had been microinjected into hamster eggs. While egg chromosomes exhibited a low frequency of chromosome aberrations, ranging from 4 to 7%, a large proportion of sperm cells exhibited chromosome damage. Between 29% of unstained and unsorted sperm and 38% of stained and unsorted sperm exhibited some type of chromosomal abnormality and this proportion increased to 50% in sorted sperm. If only damaged sperm nuclei are considered, the two unsorted sperm groups had a mean of 0.6 breaks, 0.8 triradial exchanges, and 0.2 quadriradial exchanges per nucleus. However, sorted sperm, which were stained with a fluorochrome and exposed to UV-laser irradiation, exhibited a mean of 2.9 breaks, 2.6 triradial, and 1.9 quadriradial exchanges per nucleus in which damage occurred. These observations indicate that the treatments and manipulations to which sperm nuclei are subjected during flow sorting cause chromosomal aberrations, and that exposure of the cells to UV-laser irradiation contributes substantially to the chromosome damage observed.     

Flow cytometric sorting of non-human primate sperm nuclei

Pre-determination of the sex of offspring has implications for management and conservation of captive wildlife species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort spermatozoa of non-human primate species for use with assisted reproductive technologies. The objectives of this study were to: (i) determine the difference in DNA content between X- and Y-bearing spermatozoa (ii) sort sperm nuclei into X- and Y-enriched samples; and (iii) assess the accuracy of sorting. Spermatozoa were collected from two common marmosets (Callithrix jacchus), seven hamadryas baboons (Papio hamadryas) and two common chimpanzees (Pan troglodytes). Human spermatozoa from one male were used as a control. Sperm nuclei were stained (Hoechst 33342), incubated and analyzed using a high-speed cell sorter. Flow cytometric reanalysis of sorted samples (sort reanalysis, 10,000 events/sample) and fluorescence in situ hybridization (FISH; 500 sperm nuclei/sample) were used to evaluate accuracy of sorting. Based on fluorescence intensity of X- and Y-bearing sperm nuclei, the difference in DNA content between X and Y populations was 4.09 +/- 0.03, 4.20 +/- 0.03, 3.30 +/- 0.01, and 2.97 +/- 0.05%, for marmoset, baboon, chimpanzee and human, respectively. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94 +/- 0.9 and 93 +/- 0.8%, 94 +/- 0.7 and 94 +/- 0.5%, 91 +/- 0.9 and 97 +/- 0.6%, 94 +/- 0.6 and 94 +/- 0.9%, for marmoset, baboon, chimpanzee and human, respectively). These data indicate the potential for high-purity sorting of spermatozoa from non-human primates.     

Clinical aspects of sperm DNA fragmentation detection and male infertility

Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. Currently, there are four major tests of sperm DNA fragmentation, including the Comet, Tunel, sperm chromatin structure assay (SCSA) and the acridine orange test (AOT). The Comet assay is a light microscope technique where the sperm cells are mixed with melted agarose and then placed on a glass slide. The cells are lysed and then subjected to horizontal electrophoresis. The Tunel assay, another light microscope technique, transfers labeled nucleotide to the 3'OH group of a broken DNA strand with the use of terminal deoxynucleotidyl transferase. The fluorescence intensity of each scored sperm is determined as a "yes" or "no" for sperm on a light microscope slide or by channels of fluorescent intensity in a flow cytometer. The light microscope-based AOT, uses the metachromatic properties of acridine orange to stain sperm cells. The SCSA treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA as measured by flow cytometry (FCM) as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). The SCSA method has defined a 27-30% DNA fragmentation index (DFI) as the point in which a man is placed into a statistical category of taking a longer time to in vivo pregnancy, intra uterine insemination (IUI) and more routine in vitro fertilization (IVF) cycles or no pregnancy. Current data suggest that intracytoplasmic sperm injection (ICSI) may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.     

Selection and separation of X- and Y- chromosome-bearing mammalian sperm

Preselection of the gender of offspring is a subject that has held man's attention since the beginning of recorded history. Most scientific hypotheses for producing the desired sex of offspring address separation of X- and Y-bearing sperm, and most have had limited, if any success. Eight of these hypotheses and their experimental verifications are discussed here. Three hypotheses are based on physical characteristics of sperm, one on supposed differences in size and shape, another on differences in density, and a third on differences in surface charge. There has been no experimental verification of differences based on size and shape, and the results from attempts to verify separation of X- and Y-bearing sperm based on density have been mixed. Electrophoresis may provide a method for separating X-and Y-bearing sperm, but it is currently unproven and would be of little practical utility, since sperm motility is lost. A fourth hypothesis employs H-Y antigen to select preimplantation embryos. This method reliably produces female offspring, but does not permit the selection of male offspring and does not work on sperm. There are two applications of the theory that X- and Y-bearing sperm should be separable by flow fractionation. Flow fractionation using thermal convection, counter-streaming sedimentation, and galvanization is highly promoted by its originator but has not gained wide acceptance due to lack of independent confirmation. Flow fractionation by laminar flow is said to provide up to 80% enrichment of both X- and Y-bearing sperm; however, this method also has not been confirmed by other workers or tested in breeding trials. The sixth theory discussed is that of separation through Sephadex gel filtration. This method may provide enrichment of X-bearing sperm, but, again, other experimenters have not been able to adequately confirm the enrichment. The best-known approach to sperm separation is that employing albumin centrifugation, yet even with this method, not all researchers have been able to confirm a final fraction rich in Y sperm, and trials in animals have given contradictory results. The most reliable method for separating X- and Y-bearing sperm is use of flow cytometric and flow sorting techniques. These techniques routinely separate fractions with a purity greater than 80% and can be above 90%. Unfortunately, these methods do not always work for human samples. Furthermore, as with electrophoretic approaches, the methods identify and separate only chemically fixed sperm and provide limited biological applications. Generally accepted experimental laboratory procedures for verification of proportions of X- and Y-bearing sperm are lacking. Staining of sperm with the fluorochrome dye quinacrine will identify a structure known as the “F-body” in human sperm and sperm from a few primates. The dye does not work other mammalian sperm. Its validity as a measure of sperm genotype is the topic of debate. We have used two methods to verify claims of separation of sperm. flow cytometry, and in vitro fusion. One can use flow cytometry to test the efficiency of separation of sperm samples. We tested seven commercial methods for the separation of bovine sper, and none were found of result in enrichment. We also used in vitro fusion of human sperm to denuded hamster ova to test enrichment of Y-bearing sperm from the albumin separation process. out results demonstrated no Y-bearing-sperm enrichment from this process. Scientific problems impeding the success of separation seem to be under investigation with an ever-increasing rate. Hybridization probes for DNA sequences specific to the X or Y chromosome may be the next appropriate technology to test of the selection and separation of X- and Y-chromosome-bearing mammalian sperm.     

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.     

Cell Sorting daring Pattern Formation in Dictyostelium

Formation of the prestalk-prespore pattern in Dictyostelium was investigated in slugs and submerged clumps of cells. Prestalk and prespore cells were identified by staining with vital dyes, which are shown to be stable cell markers. Dissociated slug cells reaggregate and form slugs that contain a prestalk-prespore pattern indistinguishable from the original pattern. The pattern forms by sorting out of stained prestalk cells from unstained prespore cells. Sorting also occurs in clumps of dissociated slug cells submerged in liquid or agar. A pattern arises in 2 h in which a central core of stained cells is surrounded by a periphery of unstained cells. Sorting appears to be due to differential chemotaxis of stained and unstained cells to cAMP since exogenous cAMP (>10⁻⁷ M) reverses the normal direction of sorting-out such that stained cells sort to the periphery of the clumps.
Isolated portions of slugs regenerate a new prestalk-prespore pattern. Posterior isolates regenerate a pattern within 2 h due to sorting of a population of vitally stained 'anterior-like' cells present in posteriors. Anterior-like cells do not sort in intact slugs due to the influence of a diffusible inhibitor secreted by the anterior region. During posterior regeneration this signal is absent and anterior-like cells rapidly acquire the ability to sort. Anterior isolates regenerate a staining pattern more slowly than posterior isolates by a process that requires conversion of stained prestalk cells to unstained prespore cells.
The results suggest that pattern formation in Dictyostelium consists of two processes: establishment of appropriate proportions of two cell types and establishment of the pattern itself by a mechanism of sorting-out.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing greater than or equal to 97% G1 cells, greater than or equal to 80% S cells, and 70-75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60-70%, which was comparable to the 60-80% usually found for asynchronous 9L cells. The percentage of cells in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In genereal, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the greater than or equal to 97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G2 phases was direct elutriation with the long collection method.     

Separation of sperm and vaginal cells based on ploidy, MHC class I-, CD45-, and cytokeratin expression for enhancement of DNA typing after sexual assault.

BACKGROUND: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility (MHC) class I, CD45 and cytokeratin expression. METHODS: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. RESULTS: The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified. CONCLUSIONS: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape.     

Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′-dihexyloxacarbocyanine iodide)

We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′-dihexyloxacarbocyanine iodide (DIOC₆) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae-free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC₆ staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC₆ (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC₆-positive organelles. Sea urchin and surf clam sperm stained with DIOC₆ fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC₆ were less motile than unstained sperm, they were capable of inseminating only zonae-free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.     

History of commercializing sexed semen for cattle

Although the basic principles controlling the sex of mammalian offspring have been known for a relatively long time, recent application of certain modern cellular methodologies has led to development of a flow cytometric system capable of differentiating and separating living X- and Y-chromosome-bearing sperm in amounts suitable for AI and therefore, commercialization of this sexing technology. After a very long history of unsuccessful attempts to differentiate between mammalian sperm that produce males from those that produce females, a breakthrough came in 1981 when it was demonstrated that precise DNA content could be measured. Although these initial measurements of DNA content killed the sperm in the process, they led to the ultimate development of a sperm sorting system that was capable, not only of differentiating between live X- and Y-sperm, but of sorting them into relatively pure X- and Y-sperm populations without obvious cellular damage. Initial efforts to predetermine the sex of mammalian offspring in 1989 required surgical insemination, but later enhancements provided sex-sorted sperm in quantities suitable for use with IVF. Subsequent advances in flow sorting provided minimal numbers of sperm sufficient for use in AI. It was not until the flow cytometric sorting system was improved greatly and successful cryopreservation of sex-sorted bull sperm was developed that efficacious approaches to commercialization of sexed semen could be implemented worldwide in cattle. A number of companies now offer sex-sorted bovine sperm. Innovative approaches by a diverse group of scientists along with advances in computer science, biophysics, cell biology, instrumentation, and applied reproductive physiology provided the basis for commercializing sexed semen in cattle.     

Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.