technique - PubAg

ELSEVIER

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

T. Kawarasaki'% G. R. Welch2, C.R. Long2, M. Yoshida3and L. A. Johnson2 'Shizuoka Swine and Poultry Experiment Station, Kikugawa-cho, Shizuoka-ken, Japan 2Germplasm and Gamete Physiology Laboratory, Agricultural Research Service US Department of Agriculture, Beltsville, MD, 20705, USA 3Faculty of Agriculture Shizuoka University, Shizuoka, Japan Received for publication: April 20, 1998 Accepted: June 15, 1998 ABSTRACT 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 sperma~tozoahas 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. © 1998by ElsevierScience Inc.

Key words: flow sorting, X and Y chromosome-bearing spermatozoa, FISH, swine, semen Theriogenology 50:625-635, 1998 © 1998 by Elsevier Science Inc.

0093-691)(/98/$19.00 PII S0093-691X(98)00167-8

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INTRODUCTION The DNA content of X and Y chromosome-bearing sperm cells differs between domestic mammalian species, ranging from approximately 2.8 % in humans (13) to 7.5 % in chinchillas (10). This difference in sperm DNA content allows for the detection and separation of X and Y chromosome-bearing spermatozoa by coupling flow cytometric DNA analyses to standard cell sorting methodology (9, 11). The difference in DNA content between porcine X- and Y-bearing spermatozoa is 3.7% (8), and porcine X and Y spermatozoa are separated by flow cytometry on the basis of their DNA content (6, 8). Altered sex ratios in offspring were confirmed from births following surgical insemination into the isthmus of the oviduct in the pig (6) and rabbit (9), and by in vitro fertilization in the pig (19) and bovine (1). Flow sorted spermatozoa have also been microinjected, and flow sorted spermatozoa from cows, pigs, and sheep have given rise to the development of pronuclei in hamster oocytes (8). Sorted sperm purity has been confirmed by DNA reanalysis (9, 10), using PCR in cattle (21) and florescence in situ hybridization (FISH) in humans (12). Laboratory validation of the proportions of X- and Y- bearing spermatozoa is essential to field use of this technology. The standard method for verifying sorted X and Y sperm purity has been to reanalyze sorted sperm aliquots. Recently, we developed a rapid double FISH method using both porcine chromosome 1 and Y probes produced by PCR that allow for simultaneous detection of chromosome 1- and Y-bearing sperm cells within a few hours (16). Fluorescence in situ hybridization (FISH) is an effective method for estimating the purity of sorted X- and Y-bearing spermatozoa since it can be used to examine a large number of sperm cells by viewing sperm chromosomes directly without reliance on flow cytometry. To date, the purity of flow sorted X- and Y-bearing spermatozoa has not been verified by in situ hybridization in the pig. In this study we show that the FISH in combination with chromosome Y- and 1- specific DNA probes is useful for estimating the purity of X and Y sperm cells sorted by flow cytometry. The purity of the sorted X- and Y-bearing porcine spermatozoa and the accuracy of reanalysis of the sorted porcine sperm DNA content by flow cytometry previously reported (6) were confirmed in our study using the FISH method for viewing the chromosome content of thousands of individual sperm cells directly.

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MATERIALS and METHODS Flow Cytometric Cell Sorting Semen was collected from mature Yorkshire and Landrace boars using the gloved-hand technique (7). The sperm rich fraction was filtered during collection into a warmed thermos bottle. Sperm concentrations were determined by hemocytometer. An aliquot of semen was diluted with Beltsville TS (BTS; 7) to give a concentration of 15 x 106 sperm / ml. The fluorochrome, Hoechst 33342 (Calbiochem-Behring, La Jolla, CA, USA) was added to achieve a concentration of 6.25 ~ M. The samples were then incubated for 45 min at 32°(3 (9). Just prior to flow sorting, the samples were filtered through 37- t.z m nylon mesh. Propidium iodide (PI) was added to gate out dead spermatozoa (12). The spermatozoa were sorted using a flow cytometer/cell sorter (Epics V, Coulter Corporation, Miami, FL, USA) modified specifically for sorting and analysis of spermatozoa for DNA content. Modifications have been previously described (11). Flow Cytometric Reanalysis to Determine Sort Purity Approximately 100,000 sperm cells containing the X or Y chromosome were sorted (6) using the same staining and sorting criteria as for FISH except that the spermatozoa were collected in 0.6 ml microcentrifuge tubes. Sorted samples were then pulse-sonicated once to remove sperm tails, restained to approximately the original Hoechst concentration and then reanalyzed on the flow cytometer. Resulting DNA histograms were analyzed by fitting a pair of Gaussian distributions for purity determination (10). Preparation of Sperm Nuclei The preparation of sperm nuclei for presorting control spermatozoa was based on the method described by Kawarasaki et al. (16). Semen samples (5 x 107/0.5 ml) were washed once with an equal volume of phosphate buffered saline (PBS, pH7.4) containing 6 mM ethylenediaminetetraacetic acid, disodium salt (EDTA, Wako, Osaka, Japan) for 3 min at 410 x g. Washed spermatozoa were resuspended in 1 ml of PBS containing 2 mM dithiothreitol (DTT) at 37 °C for 15 min with slow rotation. After a wash with PBS, samples were fixed at room temperature for 10 min with acetic alcohol (methanol and acetic acid, 3:1, v/v), centrifuged, placed in fresh fixative, and dropped onto the center of a circle (15 mm in diameter) inscribed with a glass pen on clean microscope slides.

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The preparation of sperm nuclei from flow sorted cells was modified to accommodate sorted spermatozoa. Approximately 40,000 spermatozoa were sorted onto a l-cm2 area of the microscope slide using a Lief bucket (17) as the slide holder and sort collection well. After a sufficient number of spermatozoa was sorted onto the slide, the bucket was centrifuged for 10 min at 300 x g, and the supernatant was drawn off The bucket was then disassembled, and the slides were removed and allowed to air dry(13). The slides with sorted spermatozoa were treated with PBS (pH 7.4) containing 3 mM EDTA for 5 min and then with PBS containing 2 mM DTT for 10 min in a stain bottle at 30°G. They were then fixed with acetic alcohol for 30 min at 30°C and allowed to air dry. Preparation of DNA Probe by PCR Porcine Y-specific primers (15) were designed according to the porcine Y-specific DNA sequence (18). Porcine chromosome 1-specific primers (16) were designed according to Sus scrofa domestica Mcl satellite DNA sequence data (5). This DNA sequence data was obtained from GenBank Nucleotide Sequence Databases under the accession number X51555. The PCR labeling digoxigenin and biotin were mainly performed as described by Kawarasaki et al. (16). Slight modifications were made as follows: the template DNA was prepared from PCR products purified by low melting temperature agarose gel (Wako, Osaka, Japan) electrophoresis, using the method of Wieslander (22); the PCR amplification for Y chromosome template were performed using primers (5'-TCATGGACCAGGTAGGGAAT-3' and 5'-GAAAGACACGTCCTTGGAGA-3')(14) located outside the area of the primers for Y chromosome-specific probe and for chromosome 1 using the same primer for amplification of the probe; after PCR amplification for labeling, the amplified Dig-labeled Y chromosomespecific PCR product (75 ~ 1 distilled water were added) and biotin labeled chromosome 1specific DNA product were precipitated directly by ethanol without purification. All labeled DNA pellets were dissolved in 0.3 ml hybridization buffer containing 30 Ix I of 20 x SSCP (3 M NaC1, 0.3 M sodium citrate and 0.18 M NazI-IPO4, pH 7.8), 120 ~ 1 of deionized formamide (Wako, Osaka, Japan), 60 ~ 1 of 50% dextran sulfate (Oncor, Gaithersburg, MD, USA), 6 ~1 of salmon sperm carrier DNA (10 mg/ml; Sigma Chemical Co., St. Louis, MO, USA) and 84/~ 1 of distilled water. The hybridization mixture was denatured at 75 °C for 10 min, stored at 4 °C and used for in situ hybridization. Fluorescence In Situ Hybridization The biotin labeled chromosome 1 specific DNA probe was diluted 8 times with the denatured hybridization buffer. Dig-labeled Y chromosome specific DNA probe and the biotin labeled chromosome 1 specific DNA probe were mixed with 1:1 ratio (v/v). The methods for hybridization and washing were the same as described previously (16). The

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mixture, 70 p l of FITC labeled anti-Dig (5 p g/ml; Boehringer Mannheim, Mannheim, Germany) and avidin-Texas Red (1.25 ~t g /ml; Boehringer Mannheim) in buffer one containing 1% BSA, 0.3% Triton X-100 was applied to each slide under parafilm and the slides were incubated for 15 min at 38.5 °C in a moist chamber. The slides were then mounted with 20 u 1 of 9:1 mix of glycerol and PBS containing 4',6-diamidino-2phenylindole (DAPI; 0.125 p g/ml; Sigma) and 10 mg / ml 1,4-diazo bicyclo [2,2,2] octane (Sigma) and 10 p I of 9:l mix of glycerol and PBS containing 1 mg/ml p-phenylenediamine (Wako). The slides were sealed with nail polish. The sperm were observed on slides using a laser scanning confocal microscope system (LSM; Carl Zeiss, Thornwood, NY). Fluorochromes were excited using appropriate combinations of excitation and barrier filters and an Argon/Krypton laser for FITC and Texas Red X conjugates or UV laser for excitation of DAPI. Images were obtained using 100 X oil immersion objective. Optical sections of each field of view were obtained individually and digitally recombined into composite images using LSM software. After sinur percentage transformation (20) , the percentages of putative Y-bearing spermatozoa for each sperm population were tested statistically by the least squares method

(4). RESULTS Sorted X and Y sperm populations were estimated by FISH using DNA probe specific chromosome Y and 1. The X and Y sorted sperm population of each samPle were estimated by the reanalysis of flow cytometry simultaneously. In the FISH, more than 500 spermatozoa were examined. The existence of the signal for chromosome Y and 1 was confirmed. The results ofFISH applied to spermatozoa before sorting are presented in Table 1. To obtain the putative rate of Y-bearing spermatozoa, the number of sperm cells with the Y chromosome signal was divided by the number of sperm cells with the chromosome 1 signal (Texas Red). The putative rate of Y-bearing spermatozoa ranged from 50.9 to 54.2% among 4 boars, and the average putative rate of Y-bearing spermatozoa was 53.1% (Figure 1). The results for FISH applied to spermatozoa after sorting and reanalysis data for Xbearing spermatozoa are presented in Table I. To obtain the putative rate of X-bearing sperm cells, the number of spermatozoa without the Y chromosome signal but with the chromosome 1 signal was divided by the number of spermatozoa having the chromosome 1 signal (Texas Red). The putative rate of X-bearing spermatozoa ranged from 86.2 to 88.6% among 4 boars, with the average putative rate being 87.4% (Figure 2). In the reanalysis by

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flow cytometry, the putative rate of X-bearing spermatozoa ranged from 85.0 to 90.0% among 4 boars, with the average putative rate benig 87.0%. The results of FISH and the reanalysis data for Y-bearing spermatozoa are presented in Table 1. To obtain the putative rate of Y-bearing spermatozoa, the number of cells with the Ychromosome signal was divided by the number of spermatozoa having the chromosome 1 signal ( Texas Red ). The putative rate of Y-bearing spermatozoa ranged from 83.5 to 88.3% among 4 boars, with the average rate being 85.9% (Figure 3). In the reanalysis by flow cytometry, the putative rate of Y-bearing spermatozoa ranged from 80.5 to 87.5% among 4 boars, with the average rate being 84.8%. As expected, the percentages of Y-bearing spermatozoa in both X and Y sorted sperm populations were significantly altered (P< 0.0001) from those before flow sorting. There were no differences between FISH and flow cytometric reanalysis in Y-bearing spermatozoa percentages of X (P=0.87) and Y (P=0.60) sorted sperm populations. The percentage of Ybearing spermatozoa in all sperm populations before and after sorting was not influenced by Individual boar (0.42