Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation

In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored. This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised. Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA. These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific. In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa.     

A method for chromosome analysis of ram spermatozoa using zona-free hamster oocytes

A method for displaying ram spermatozoan chromosomes using the interspecific zona-free hamster oocyte penetration was described to distinguish X- and Y-bearing spermatozoa. Semen samples from four rams were frozen and stored in liquid nitrogen. After thawing the samples, motile spermatozoa were collected by the swim-up method and treated with ionophore A23187 for the purpose of facilitating their capacitation. Slides were prepared by the gradual fixation-air dry method. The rates of oocyte penetration, first cleavage metaphase, and the number of ova that were karyotyped successfully were 67.9, 60.8 and 40.6%, respectively. The overall success rate (number of spermatozoa karyotyped/number of oocytes used for insemination) was 47.9%. A total of 1009 spermatozoa were analyzed, and the ratio of X- and Y-bearing spermatozoa was 508:501.     

A chromosomal method to distinguish between X- and Y-bearing spermatozoa of the bull in zona-free hamster ova

The interspecific in-vitro fertilization system using zona-free hamster ova was adopted to distinguish chromosomally between X- and Y-bearing bull spermatozoa. Frozen semen samples were thawed and washed with modified BWW medium including 0.3% BSA (pH 7.2) to remove cryoprotective medium. Motile spermatozoa were recovered from the semen by the 'swim-up' method. These spermatozoa were treated with ionophore A23187 to facilitate capacitation. Adequate capacitation of spermatozoa was found by their movement patterns and the insemination was performed at the optimum time. The fertilized ova were cultured in Medium TC199 with 10% FBS including podophyllotoxin and vinblastine until they reached first cleavage metaphase. Chromosome slides were prepared by our gradual fixation-air drying method. The success rate of the method was 56% of the number corresponding to the zona-free ova used. Altogether 1116 spermatozoa from 4 different bulls were successfully analysed and the ratio of X- and Y-bearing spermatozoa was 542:574 (P greater than 0.3).     

Differences in motility of human X- and Y-bearing spermatozoa.

Human Y-bearing spermatozoa, as identified by the quinacrine staining technique, were found to be significantly more motile than X-bearing spermatozoa under laboratory conditions. This difference is consistent with current estimates of the difference in mean head DNA content.     

Rapid and simultaneous detection of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization

This study was carried out to develop a rapid and simultaneous detection system of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Dig)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chromosome and chromosome 1-specific DNA was applied to the preparation, immediately denatured at 75°C for 8 min, hybridized for 5 min at 37°C and overall FISH steps were done within a few hours. When double FISH with Dig-labeled chromosome Y-specific and biotin-labeled chromosome 1-specific probes was applied to sperm nuclei pretreated with dithiothreitol, the average of 50.9% of sperm nuclei had the Dig-signal, 99.2% of the sperm nuclei had the biotin-signal and the average of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bearing spermatozoa ranged from 49.8% to 52.8% among 5 boars and the average putative rate of Y-bearing spermatozoa was 51.0%. The results indicated that a rapid and simultaneous FISH with chromosome Y- and 1-specific porcine DNA probes produced by PCR made possible more accurate assessment of Y-bearing porcine spermatozoa. © 1996 Wiley-Liss, Inc.     

Increased frequency of X-bearing sperm in males from an infertility clinic: analysis by two-color fluorescence in situ hybridization

Semen samples from 34 men visiting the Lübeck infertility clinic were investigated using a two-color FISH method to determine the ratio of X- and Y-bearing sperm. The overall ratio was significantly shifted to a preponderance of X-containing sperm. A statistical comparison with seven reports from the literature which included 53 normal probands demonstrated in our patients a significant tendency of a preponderance of X-bearing sperm and significantly less Y-bearing sperm. Furthermore, the Lübeck sperm samples are remarkably more heterogeneous in respect to their variability of X- and Y-bearing spermatozoa than in the other mentioned studies with normal probands. These phenomena have to be evaluated in further studies on groups of infertile males showing similar infertility histories.     

Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.     

CAG repeat lengths in X- and Y-bearing sperm indicate that gender bias during transmission of Huntington's disease gene is determined in the embryo

The size of the CAG tract at the Huntington's disease (HD) locus upon transmission depends on the gender of the parent. However, the basis for the parent-of-origin effect is unknown. To test whether expansion and contraction in HD are "imprinted" in the germ cells, we isolated the X- and Y-bearing sperm of HD transgenic mice. Here we show that CAG repeat distributions in the X- and Y-bearing spermatozoa of founding fathers do not differ. These data show that gender-dependent changes in CAG repeat length arise in the embryo.     

Separation of human X and Y spermatozoa by free-flow electrophoresis

Free-flow electrophoresis is a fast and promising method for gamete separation. Pretreated seminal plasma-free human spermatozoa were injected continuously as a fine stream into the buffer medium of the separation chamber flowing perpendicular to the forces of an electrical field, which separated the spermatozoa according to their differences in electrophoretic mobility. For characterization of the two classes of spermatozoa before and after separation, quinacrine mustard staining was used to identify the Y-chromosome-bearing spermatozoa carrying the fluorescent body (F-body). Human spermatozoa moved toward the anode and were separated into two main peaks. The faster moving fraction contained nearly 80% Y-bearing spermatozoa and the slower peak consisted mainly of pure X-bearing spermatozoa. Whereas sperm viability as determined by eosin staining was nearly unchanged, sperm motility was reduced after free-flow electrophoresis.     

A method for cytogenetic analysis of boar spermatozoa using hamster oocytes.

In this paper, the authors detail a method for displaying boar spermatozoa chromosomes using heterospecific zona-free hamster oocyte penetration technique. Semen samples from two Large-White boars having a normal spermogram were studied. The first one had a normal karyotype (38,XY), the second carried a reciprocal translocation rcp(3;7)(p1,3;q2,1). After in vitro fertilization by capacitated sperm, culture and cytogenetic analysis of hamster eggs we obtained metaphase spreads of spermatozoa chromosomes. The ratio of X- and Y-bearing spermatozoa was 49.2% and 50.8%, respectively.     

Selecting a mammalian species for the separation of X- and Y-chromosome-bearing spermatozoa

All (524) male karyotypes in An Atlas of Mammalian Chromosomes (Hsu & Benirschke, 1967-1977) were visually estimated for the chromatin difference between the X- and Y-chromosome-bearing spermatozoa. After more exact measurement of axial chromatid length for 100 karotypes, 24 species were found in which the difference between X and Y chromosomes was greater than 6.2%. It is suggested that such species would be the best for attempts to separate the X- and Y-bearing spermatozoa.     

Cytogenetic Analysis of Spermatozoa in a Patient with Chromosome Constitution 45,X/46,X,r(Y) by Intracytoplasmic Injection into Mouse Oocytes

The chromosome set of human spermatozoa was studied by intracytoplasmic injection into mouse oocytes. A total of 85 metaphase plates of male pronuclei of a patient with chromosome constitution 45,X/46,X,r(Y) and 108 metaphase plates of patients with normal sperm parameters (control group) were examined. The ratio between X- and Y-bearing chromosomes in the 45,X/46,X,r(Y) patient and in the control group did not differ from 1 : 1. A significant increase in the rates of diploidy, hypoploidy, hyperploidy of sex chromosomes, and chromosome structure rearrangements in spermatozoa of the patient in comparison with spermatozoa in the control group was recorded.     

Cytogenetic analysis of spermatozoa in a patient with chromosome constitution 45,X/46,X,r(Y) by intracytoplasmic injection into mouse oocytes

The chromosome set of human spermatozoa was studied by intracytoplasmic injection into mouse oocytes. A total of 85 metaphase plates of male pronuclei of a patient with chromosome constitution 46,X,r(Y)/45,X and 108 metaphase plates of patients with normal sperm parameters (control group) were examined. The ratio between X- and Y-bearing chromosomes in the 46,X,r(Y)/45,X patient and in the control group did not differ from 1:1. A significant increase in the rates of diploidy, hypoploidy, hyperploidy of sex chromosomes, and chromosome structure rearrangements in spermatozoa of the patient in comparison with spermatozoa in the control group was recorded.     

On the selective elimination of Y-bearing sperm

The potential use of antibodies that selectively recognize either X-bearing or Y-bearing sperm is self-evident. Thus our attention was directed to the fact that under optimal conditions, H-Y antibody lyses 50% of mouse spermatozoa. Accordingly, we asked whether expression of H-Y antigen is haploid in spermatozoa from XY male mice heterozygous for the autosomal dominantSxr gene, for if H-Y expression were haploid, H-Y antibody would be expected to kill 75% of spermatozoa derived from these XY,Sxr/- males. However, maximal lysis remained at the 50% level, which indicates that haploid expression of H-Y antigen and the potential immunoselection of Y-(or X-) bearing spermatozoa are unlikely.     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Cytogenetic analysis of human spermatozoa using intracytoplasmic sperm injection into mouse oocytes

The chromosome complement of human spermatozoa has been analyzed after their intracytoplasmic injection into unfertilized mouse oocytes. A total of 427 metaphase plates have been obtained, including 176 metaphase plates from spermatozoa with normal head morphology (108 and 68 spermatozoa from patients with normal (the control group) and abnormal spermogram parameters, respectively), and 251 metaphase plates from spermatozoa with abnormal heads (76, 91, 67, and 17 spermatozoa with large, amorphous, elongated, and round heads, respectively). The frequency of chromosome abnormalities in the control group is 26.1%, with hyperploidy, hypoploidy, and structural aberrations accounting for 7.4, 12.3, and 6.4% of the abnormalities, respectively. In none of the groups did the ratio between the numbers of X- and Y-bearing spermatozoa significantly differ from 1 : 1. The diploidy frequency was significantly higher in spermatozoa with large and amorphous heads compared to the control group (2.36, 3.29, and 0%, respectively). None of the groups of spermatozoa differed from the control group with respect to the frequency of structural aberrations. The type of the abnormal head morphology has been found to be correlated with the sperm chromosome complement.     

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.     

Microisolation and microcloning of bovine X-chromosomes for identification of sorted buffalo (Bubalus bubalis) spermatozoa

Flow-cytometry sorting technology has been successfully used to separate the X- and Y-chromosome bearing spermatozoa for production of sex-preselected buffalo. However, an independent technique should be employed to validate the sorting accuracy. In the present study, X-chromosomes of bovine were micro-dissected from the metaphase spreads by using glass needles. Then X-chromosomes were then amplified by PCR and labelled with Cy3-dUTP for use as a probe in hybridization of the unsorted and sorted buffalo spermatozoa -chromosome. The results revealed that 47.7% (594/1246) of the unsorted buffalo spermatozoa were positive for X- chromosome probe, which was conformed to the sex ratio in buffalo (X:Y spermatozoa=1:1); 9.6% (275/2869) of the Y-sorted buffalo spermatozoa and 86.1% (1529/1776) of the X-sorted buffalo spermatozoa showed strong X-chromosome FISH signals. Flow cytometer re-analysis revealed that the proportions of X- and Y-bearing spermatozoa in the sorted X and Y semen was 89.6% and 86.7%, respectively. There were no significant differences between results assayed by flow-cytometry re-analysis and by FISH in this study. In conclusion, FISH probe derived from bovine X- chromosomes could be used to verify the purity of X and Y sorted spermatozoa in buffalo.     

Direct chromosomal analysis of human spermatozoa: preliminary results from 18 normal men.

Chromosomal analysis of 240 spermatozoa from 18 normal men was performed using in vitro fertilization of zona-free golden hamster eggs. The frequency of chromosome abnormalities in this population was 9.2% (22/240). Of the abnormal complements, 18 were aneuploid (13 hyperploid and five hypoploid) and four had a chromosome break. The sex ratio of Y-bearing to X-bearing sperm was .68. The frequency and type of sperm chromosome abnormalities is compared with those seen in spontaneous abortions.     

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.