Y-bearing Sperm Research Articles

Sexing mammalian sperm for production of offspring: the state-of-the-art

Predetermination of sex in livestock offspring is in great demand and is of critical importance to providing for the most efficient production of the world's food supply. With the changes that have taken place in animal agriculture over the past generation the application of sex preselection to production systems becomes increasingly necessary. The current technology is based on the well-known difference in X- and Y-sperm in the amount of DNA present. The method has been validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content and embryo biopsy for sex determination. The technology incorporates modified flow cytometric sorting instrumentation to sort X- and Y-bearing sperm. Resulting populations of X or Y sperm can be used in conjunction with IVF in swine and in cattle for the production of sexed embryos to be transferred to eligible recipients for the duration of gestation. It can also be used for intratubal insemination and for deep-uterine and conventional insemination in cattle. This semipractical sexing method, though currently impractical for some production systems (where large numbers of sperm are required for fertilization) could be used to provide a more flexible progeny-producing option in many livestock operations. Improvements in the production rate of sexed sperm continue as new technology is developed. High-speed sorting is one of the newer technological advances and is being used in our laboratory to increase sorted sperm throughput. With our original technology we sorted 350,000 sperm/h. We now sort 6 million of each sex, under routine conditions. Sorting only the X population results in about 18 million sperm/h. Improvements in the technology will no doubt lead to much greater usage of sexed sperm, depending on the species involved. Insemination of lower sperm numbers in cattle has proven to be an effective means of utilizing the sexing technology. Solving the problems associated with inseminating low sperm numbers in the pig would be advantageous to the utilization of sexed sperm for some type of deep artificial insemination. Such a development would also enhance the economy of using lower sperm numbers with conventional artificial insemination (AI) and aid the swine industry worldwide. The use of sexed sperm for non-ordinary applications such as endangered species, laboratory animals, hobby or pet species is also of interest and will become a part of the move to be more reproductively efficient in the next millennium. Sexed sperm on demand over the next several years will provide livestock producers with many options in seeking to improve efficiency of production and improve quality of products to enhance consumer acceptability.

Segregation of sex chromosomes in spermatozoa of 46,XY/47,XXY men by multicolour fluorescence in-situ hybridization.

The sex chromosome disomy and diploidy rates on ejaculated spermatozoa from two patients with mosaic Klinefelter's syndrome were estimated, using X/Y/15 multicolour fluorescence in-situ hybridization (FISH). A 8/18 dual fluorescence in-situ hybridization analysis was also carried out. In triple FISH, a total of 1691 (patient 1) and 811 (patient 2) spermatozoa were analysed. Frequencies of cells with hyperhaploidies for sex chromosomes were 2. 01% and 3.45% for patients 1 and 2 respectively, with both patients showing a significantly increased incidence of 24,XY and 24,XX disomies and only patient 2 showing a significantly increased incidence of 24,YY disomy in comparison to the control (P < 0.001). The 46,XX diploidy rate in patient 1 was also significantly higher than the control (P < 0.01). The ratio of X-bearing to Y-bearing spermatozoa differed from the expected 1:1 ratio for only patient 1 (1.18:1). There was no significant difference for chromosomes 8, 15 or 18 disomy frequencies in comparison to those estimated in the control population. These results support the hypothesis that some 47,XXY cells are able to go through meiosis and form spermatozoa with an abnormal gonosomal complement. Thus, there is an increased risk, for these 46,XY/47,XXY men, of producing offspring with a gonosomal abnormality.

Sexing of porcine embryo by in situ hybridization using chromosome Y- and 1-specific DNA probes

This study was carried out to determine if a rapid, simultaneous detection system using chromosome Y- and 1-bearing boar spermatozoa was applicable for sexing embryos. Porcine embryos were recovered from gilts and sows 4 to 6 d after mating, and whole embryos or biopsy cells were mounted on a glass slide with a small amount of fixative (methanol: acetic acid: distilled water = 9:1:4). The samples were then stained by means of a fluorescence in situ hybridization (FISH) procedure developed specifically for the detection of Y-bearing spermatozoa. Hybridization was performed using digoxigenin (dig)-labeled chromosome Y- specific DNA, and biotin-labeled chromosome 1-specific DNA sequences were detected as a signal of FITC and Texas Red on nucleus visualized DAPI-stain. Proportions of whole embryos labeled with chromosome 1-probe were 17 and 97% at the 3 to 16 and ≧32 cell stage, respectively. Of the 93 biopsied embryos analyzed by FISH, 85 embryos (91 %) could be accurately classified as male or female. Of the 65 biopsied embryos, 60 embryos (92%) had a clear blastocoele and a inner cell mass after 48 h of culture in vitro, and these embryos were evaluated as available embryos. One out of 4 recipient gilts which received sexed embryos at transfer farrowed 12 piglets of the expected sex. The results of this study demonstrated that porcine embryos at the ≧32 cell stage can be sexed within 2 h using the FISH method. Moreover further development of the FISH technique could make it an effective tool for the study of early porcine embryos and for the control of porcine sex.

Sex ratio distortion in bovine sperm correlates to recombination in the pseudoautosomal region.

A total of 2122 single sperm from 35 bulls belonging to six different paternal half-sib groups were analysed with respect to two markers in the bovine pseudoautosomal region (PAR) and sex-specific loci on the X and Y chromosomes, respectively. A segregation ratio significantly different from 1:1 was observed in a test over all families, with a higher proportion of X-bearing gametes (53.5%). The analysis of recombination conducted separately for X- and Y-bearing sperm showed that X-bearing sperm cells possess highly significant individual and between-family variability in recombination rate, whereas Y-bearing sperm show linkage homogeneity. To test whether the two phenomena are related, different logistic regression models were fitted to the data. The results show that sex ratio significantly correlates with changes in recombination rate among X-bearing but not among Y-bearing sperm. Different hypotheses to explain these observations are discussed.

Non-Mendelian Segregation of Sex Chromosomes in Heterospecific Drosophila Males

Interspecific hybrids and backcrossed organisms generally suffer from reduced viability and/or fertility. To identify and genetically map these defects, we introgressed regions of the Drosophila sechellia genome into the D. simulans genome. A female-biased sex ratio was observed in 24 of the 221 recombinant inbred lines, and subsequent tests attributed the skew to failure of Y-bearing sperm to fertilize the eggs. Apparently these introgressed lines fail to suppress a normally silent meiotic drive system. Using molecular markers we mapped two regions of the Drosophila genome that appear to exhibit differences between D. simulans and D. sechellia in their regulation of sex chromosome segregation distortion. The data indicate that the sex ratio phenotype results from an epistatic interaction between at least two factors. We discuss whether this observation is relevant to the meiotic drive theory of hybrid male sterility.

Separation of sperm through a 12-layer percoll column decreases the percentage of sperm staining with quinacrine.

Previous methods of enriching sperm with a higher percentage of Y-bearing sperm have been questioned because the claims that Y enrichment was present were based on quinacrine staining of the Y chromosome, and the enrichment was not confirmed by polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) techniques. A technique was evaluated that theoretically could increase the percentage of X-bearing sperm by isolating a fraction of the "heaviest" sperm by passing them through 12 layers of discontinuous Percoll gradient. Initially 12 specimens were checked both before and then after separation with 12 layers of Percoll for percentage of Y sperm. The median for baseline Y percentage was 49% and after processing the percentage of Y dropped to 10%. An additional 19 specimens were checked after separation only. The median was 19%. The sample with the lowest preseparation % of quinacrine staining sperm was 45% and the highest was 54%. After 12-layer Percoll, the lowest percentage was 3% and the highest was 24%. There have been claims that quinacrine staining can falsely increase apparent Y-bearing sperm enrichment following certain separation procedures, e.g.. albumin separation, by nonspecific staining of autosomal chromosomes. If anything, then, it should falsely decrease X-bearing sperm enrichment. Thus, 12-layer Percoll separation may actually enrich for X-bearing sperm or possibly this procedure somehow nonspecifically inhibits the ability of quinacrine to stain the Y chromosome.

Multicolor fluorescence in situ hybridization analysis of meiotic chromosome segregation in a 47,XYY male and a review of the literature

The frequencies of aneuploid and diploid sperm were determined in a 47,XYY male using multi-color fluorescence in situ hybridization (FISH) analysis, and compared with those from 10 control donors. A total of 30,078 sperm from the patient was scored, 15,044 by two-color FISH for chromosomes 13 and 21, and 15,034 by three-color FISH for the sex chromosomes using chromosome 1 as an internal autosomal control for diploidy and lack of hybridization. The frequencies of X-bearing (49.73%) and Y-bearing sperm (49.46%) in control males were not significantly different from the expected 50% (chi(2)-test for goodness of fit). The ratio of 24,X (50.60%) to 24, Y sperm (48.35%) in the patient, however, was significantly different from the controls (P = 0.0144, chi(2)-test for independence) and from the expected 1:1 ratio (P = 0.0055, chi(2)-test for goodness of fit). There was no significant increase in the frequency of diploid sperm when compared with the controls (chi(2)-test for independence). Significantly increased frequencies were found for 24,YY (0.07% vs. 0.02%, P = 0.0009) and 24,XY (0.44% vs. 0.29%, P = 0.0025), but not for 24,XX (0.05% vs. 0.05%, P > 0. 05), 24,+13 (0.07% vs. 0.07%, P > 0.05) or 24,+21 sperm (0.21% vs. 0. 18%, P > 0.05) in the 47,XYY male when compared with control donors (chi(2)-test for independence). Our results support the theory that loss of the extra Y chromosome occurs during spermatogenesis in most cells. In this XYY patient there was a significant increase in the frequency of sperm with sex chromosomal abnormalities but no suggestion of an inter-chromosomal effect on autosomes. All 3-color FISH studies in the literature demonstrate a significantly increased risk of gonosomal aneuploidy in XYY males, with the risk being on the order of 1%.

Spontaneous frequencies of aneuploid and diploid sperm in 10 normal Chinese men: assessed by multicolor fluorescence in situ hybridization

Many studies have been published establishing the background frequencies of disomic and diploid sperm in normal men by fluorescence in situ hybridization (FISH) analysis, with highly significant variance among the reports. Besides interdonor heterogeneity and differences in the experimental protocols used, the question of inherent differences in chromosome malsegregation and meiotic arrest among different geographic and ethnic groups of donors has been raised. In this study, multicolor FISH analysis was carried out on semen samples from 10 nonsmoking, nondrinking Chinese men from the People’s Republic of China. The results were compared to FISH data on 10 nonsmoking, nondrinking Canadians under the same experimental conditions, in the same laboratory. A total of 200,497 sperm was scored in the Chinese donors and compared to 202,320 sperm from Canadian donors. Approximately 10,000 sperm per chromosome probe per donor were analyzed. The mean hybridization efficiency was 99.99%. The frequencies of X-bearing and Y-bearing sperm were not significantly different from the expected 50% for each individual and for the combined data from all donors (49.73% vs. 49.46%, P = 0.3946). The mean disomy frequencies (range) were 0.07% (0.02%–0.12%) for chromosome 13, 0.18% (0.09%–0.19%) for chromosome 21, 0.05% (0.01%–0.09%) for 24,XX, 0.02% (0.01%–0.06%) for 24,YY, and 0.29% (0.13%–0.49%) for 24,XY. The mean diploidy frequency (range) was 0.38% (0.22%–0.73%) for 13-21 hybridizations and 0.32% (0.07%–0.70%) for XY hybridizations. Highly significant interdonor heterogeneity was found for diploidy (P = 0.0000) and for XY disomy (P = 0.0011), but no age effect was observed in any category of disomic or diploid sperm. The data reported here show no marked differences in disomy and diploidy frequencies between the mainland Chinese and Canadian groups, if donor heterogeneity is taken into account.

Sex-ratio meiotic drive in Drosophila simulans is related to equational nondisjunction of the Y chromosome.

The sex-ratio trait, an example of naturally occurring X-linked meiotic drive, has been reported in a dozen Drosophila species. Males carrying a sex-ratio X chromosome produce an excess of female offspring caused by a deficiency of Y-bearing sperm. In Drosophila simulans, such males produce approximately 70-90% female offspring, and 15-30% of the male offspring are sterile. Here, we investigate the cytological basis of the drive in this species. We show that the sex-ratio trait is associated with nondisjunction of Y chromatids in meiosis II. Fluorescence in situ hybridization (FISH) using sex-chromosome-specific probes provides direct evidence that the drive is caused by the failure of the resulting spermatids to develop into functional sperm. XYY progeny were not observed, indicating that few or no YY spermatids escape failure. The recovery of XO males among the progeny of sex-ratio males shows that some nullo-XY spermatids become functional sperm and likely explains the male sterility. A review of the cytological data in other species shows that aberrant behavior of the Y chromosome may be a common basis of sex-ratio meiotic drive in Drosophila and the signal that triggers differential spermiogenesis failure.

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.

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 presorted 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.

Clinical experience with flow cytometric separation of human X- and Y-chromosome bearing sperm

Numerous methods to separate human X- and Y-bearing sperm have been reported with unconfirmed separation after DNA analysis and inconsistent birth results. Successful flow cytometric separation of sperm resulting in alteration of the sex ratio of young born has been demonstrated in several animal species. Flow cytometric separation of human X- and Y-bearing sperm (MicroSort ®) a has been confirmed after DNA analysis by fluorescence in situ hybridization (FISH). Pregnancies and births have resulted from the use of MicroSort after intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

Preliminary study of the incidence of disomy in sperm fractions after MicroSort flow cytometry.

Using triple colour fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the disomy level for chromosome 21 and the sex chromosomes in flow cytometric sorted sperm samples. There were no statistically significant differences in the disomy rates when comparing the sorted samples (either for X- or Y-bearing spermatozoa) with non-sorted samples. There were no diploid spermatozoa observed in any sample group after MicroSort processing.

Effect of timing of artificial insemination on sex ratio

For a number of years, the time of insemination or mating during estrus has been believed to influence the sex ratio of offspring, with early insemination resulting in more females and late insemination, more males. Possible mechanisms of altering the sex ratio include facilitating or inhibiting the transport of either X- or Y-chromosome-bearing sperm through the reproductive tract, preferential selection of sperm at fertilization, or sex-specific death of embryos after fertilization. In livestock species, there is evidence for preferential selection of X- or Y-bearing sperm, based on the maturational state of the oocyte at fertilization. In deer and sheep, early and late insemination appears to skew the sex ratio toward females and males, respectively. In cattle, conflicting reports on the effect of time of insemination on sex ratio make the premise less clear. Many of the published studies lack adequate observations for definitive conclusions and/or are based on infrequent observations of estrus, making it difficult to assess the effect of time of insemination on sex ratio. It is likely that any effect of time of insemination on sex ratio in cattle is relatively small. Evidence is accumulating that treatments used for synchronization of estrus or ovulation in cattle may influence the sex ratio.

Sex preselection: High-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X- bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.

Sex selection and preimplantation genetic diagnosis

Sex selection and preimplantation genetic diagnosis

Simultaneous detection of X- and Y-bearing bull spermatozoa by double colour fluorescence in situ hybridization.

Double colour fluorescence in situ hybridization with sex chromosome probes was applied on sperm cells of five Swedish Holstein-Friesian bulls. It was demonstrated that cosmids with strong fluorescence signals and scraped chromosomes can successfully be used as markers in this type of study. X and Y segregated as expected according to a 1:1 ratio, and there were no interindividual variations. There was a tendency for there to be more Y- than X-bearing spermatozoa, but this bias was assumed to be due to the markers used. Disomic spermatozoa occurred with a frequency of more than 0.1 % (0.067% XX, 0.029% YY, and 0.029% XY), which is considerably lower than the frequency in humans. Diploid sperm cells occurred with a frequency of 0.05 %.

Relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte.

The aim of this study was to explore how some reproductive methodologies may affect the sex ratio. We first confirmed the association between the maturation stage of bovine oocytes at the time of in vitro fertilisation (IVF) and the sex ratio of in vitro-derived embryos. Secondly, we studied whether the time of insemination, prior to or after ovulation, could alter the sex ratio in sheep. In the first experiment, bovine oocytes were matured in vitro for 16 h; then oocytes were either fertilised in vitro immediately after extrusion of the first polar body or IVF was delayed for 8 h. The proportion of cleaving embryos and their development to the 8-cell stage was enhanced with delayed insemination. Moreover, delaying IVF produced a male-to-female sex ratio of 1.67:1.00, which was significantly different from the expected 1:1 ratio (p < 0.05), whereas more female embryos were produced when oocytes were fertilised in vitro immediately after polar body extrusion (sex ratio of 1.00:0.67; p < 0.05). In the second experiment, 380 ewes were inseminated at different times before or after ovulation, producing 537 lambs. Significant differences in the sex ratio were obtained when we compared the sex of the offspring of ewes inseminated during the 5 h preceding ovulation (more females) with those inseminated during the 5 h after ovulation (more males). Our results suggest that the differential ability of X- or Y-bearing spermatozoa to fertilise oocytes depending either on time of insemination or oocyte maturation state, may be due, at least partially, to 'intrinsic' differences in the physiological activity of X- or Y-bearing spermatozoa before fertilisation.

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

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. 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. We found a difference in volume of X- and Y-bearing bovine sperm heads matching the difference in DNA content (3.5-4%). 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.

The ratio of X- and Y-bearing sperm in ejaculates of men with three or more children of the same sex.

The present study evaluated the proportions of X-bearing and Y-bearing sperm within the semen of donors who were the declared fathers of three or more sons or daughters. The proportions of sperm were determined using dual-color fluorescence in situ hybridization to identify the X and Y chromosomes. The only difference observed was in semen volume. There was no increase in the proportion of Y-bearing sperm for men with only sons (49.7 +/- 1.3%) or of X-bearing sperm for men with only daughters (44.8 +/- 2.6%). A preponderance of either sons or daughters in a family cannot be explained simply by an altered ratio of X-bearing and Y-bearing sperm in the father's semen.