Fluorescent body distribution in spermatozoa in the male with exclusively female offspring*

Transcription

1 FERTILITY AND STERILITY Copyright 1988 The American Fertility Society Vol. 49, No. 4, April 1988 Printed in U.S.A. Florescent body distribtion in spermatozoa in the male ith exclsively female offspring* Pal E. Bibbins Jr., Ph.D.t Larry I. Lipshltz, M.D.:j: Jonathan B. Ward Jr., Ph.D. Marvin S. Legator, Ph.D. University of Texas Medical Branch, Galveston, and Baylor College of Medicine, Hoston, Texas Florescent (F) body distribtion as determined in a grop of men ho did not have a fertility problem, bt rather had fathered exclsively female offspring. The stdy as designed to analyze spermatozoa for the freqency of zero F-body (X-bearing) and one F-body (Y-bearing) spermatozoa. Semen samples ere separated (processed) for Y-bearing spermatozoa enrichment and reanalyzed for florescent body distribtion. The stdy consisted of 5 control (1 males) samples (nprocessed), 35 preseparation (35 patients) samples (nprocessed), and 18 postseparation (18 patients) samples (processed). A significantly higher freqency (P <.5) of zero F -body spermatozoa ere observed in the preseparation samples hen compared ith the control samples. The presence of more spermatozoa ithot florescent body correlates ith the occrrence of more female births. Fertil Steril 49:67, 1988 Attempts have been made to distingish beteen X and Y chromosome-bearing spermatozoa by looking for differences in size and srface properties of the to types of spermatozoa. 1 It is no possible to distingish X and Y chromosome-bearing spermatozoa folloing deoxyriboncleic acid (DNA)-binding of florochromes. 2 The analysis of florescent (F)-body distribtion in spermatozoa is based on the principle of differential staining of metaphase chromosomes ith qinacrine dihydro- Received Jly 1, 1987; revised and accepted December 3, * Spported in part by a contract from the Texas Veterans Agent Orange Assistance Program (Texas Department of Health). t Reprint reqests: Pal E. Bibbins, Jr., Ph.D., Division of Reprodctive Endocrinology, Department of Obstetrics and Gynecology, University of Loisville School of Medicine, 436 Medical Dental Research Bilding, 511 Soth Floyd Street, Loisville, Kentcky :j: Department of Urology, Baylor College of Medicine. Division of Environmental Toxicology, Department of Preventive Medicine and Commnity Health, University of Texas Medical Branch. 67 Bibbins et al. F-body cont in male ith female progeny chloride 3 and its high affinity for the hman Y chromosome. 2 The distal portion of the long arms of the Y chromosome is rich in adenine and thymine, thereby enhancing qinacrine florescence. 4 The distal portion of the Y chromosome floresces ith great intensity and can be observed as a bright florescing spot in interphase nclei. 5 We have analyzed florescent Y-body distribtion in a grop of men ho have fathered three or more girls ithot a male birth. Folloing the separation of spermatozoa for sex preselection, the ratio of spermatozoa bearing X and Y chromosomes as redetermined. Stdy Poplation MATERIALS AND METHODS The stdy grop consisted of men ho did not have a fertility problem, bt rather had fathered three or more girls ithot a male birth. The control grop consisted of men ho had fathered both boys and girls. Each control sbject provided five Fertility and Sterility

2 samples to analyze the stability of florescent body distribtion over time. Sample Collection and Processing Male sbjects provided semen samples to the fertility clinic for a rotine semen analysis, F -body distribtion analysis, and sperm separation. A 2- to 3-day sexal abstinence period as reqired before each sample (control or test) as collected for analysis. Samples ere collected by mastrbation into sterile containers. All of the test sbjects ere examined by the collaborating physician. Staining Procedre Air-dried smears ere made for florescent analysis.6 The slides ere fixed for 15 mintes ith absolte methanol. The slides ere stained for 6 mintes in a florochrome soltion consisting of the folloing components: (1) 5 mg qinacrine dihydrochloride, (2) 5 ml absolte ethanol, and (3) 5 ml distilled ater. 6 The slides ere rinsed in distilled ater, monted ith ph 5.5 Mcilvaine's bffer, 7 cover-glassed, and left ndistrbed for 3 mintes in the dark. Florescent Body Analysis Zero F-body, one F-body, and to F-body freqencies ere determined by examination of qinacrine-stained slides on a Zeiss Universal Microscope (Carl Zeiss, Inc., Ne York) ith epiflorescence, hich as eqipped ith a narro excitation band at 436 nm and a 4 7-nm barrier filter. Spermatozoa ere analyzed for the occrrence of to F bodies in a sperm head, hich is thoght to represent the presence of to Y chromosomes. 6 Each spermatozoon had to meet the folloing criteria in order to be scored: (1) normal size, (2) normal shape, (3) intact ith complete membranes, and (4) attached tail.6 Once the qality of each spermatozoon as established, the florescent body had to meet the folloing criteria in order to be scored: (1) it had to fall ithin the intact sperm head, (2) it had to lie in the focal plane ofthe sperm, and (3) it had to form a distinct point of light rather than a large area of diffse florescence, hich sally characterized overlapping chromatin. The F -bodies had to be approximately the same size and staining intensity. 6 Five hndred spermatozoa ere analyzed for each sample, and the percentage containing zero, one, and to F bodies as determined. Slide Analysis Preseparation and postseparation slides ere sbmitted for analysis. Preseparation and control slides ere made directly from the nprocessed ejaclated samples. Postseparation slides ere made after a portion of the ejaclated sample as separated by a modified techniqe of the "sim-p procedre" 8 9 sing Biggers, Whitten, and Whittingham (BWW) bffer 1 in order to recover a sample that had been enriched for spermatozoa containing a Y chromosome. The freshly ejaclated sperm sample as alloed to liqefy. The spermatozoa ere ashed and pelleted by centrifgation. The spernatant as decanted and the pellet as layered over ith BWW. The spermatozoa ere alloed to migrate from the pellet for 1 hor at 37 C, after hich the spernatant as removed containing an enrichment for Y chromosome-bearing spermatozoa. The sim-p procedre orks on the principle that Y chromosome-bearing spermatozoa are carrying less genetic material than X chromosome-bearing spermatozoa; therefore, Y bearing spermatozoa have less mass and can sim p from the pellet faster than the X-bearing spermatozoa. Slides for F-body analysis ere coded and scored blind. Statistical Evalation We tested the data for normalcy in distribtion by analyzing for goodness-of-fit sing the Kolmogorov-Smirnov One-Sample test, 11 and determined that the data are not normally distribted. A nonparametric (distribtion-free) method as sed for statistical analysis becase, ith this method, it is not necessary to assme that the observations are normally distribted. This method is also sed hen the distribtion is nknon. 12 In practice, data are often non-normal in distribtion or the sample size is not large enogh to gain the benefit of the central limit theorem. An inherent characteristic of nonparametric statistics is that they deal ith ranks rather than vales of the observations. The observations are arranged in an array, ranks are assigned from 1 to n, and one deals only ith positive integers. 12 Statistical analyses comparing the preseparation and control samples, preseparatiqn and postseparation samples, and postseparation and control samples ere performed sing the Kolmogorov Smirnov To-Sample test. The Kolmogorov-Smirnov To-Sample test is a test of hether to independent samples (one vari- Vol. 49, No.4, April1988 Bibbins et al. F-body cont in male ith female progeny 671

3 able, to grops) have been dran from the same poplation (or from poplations ith the same distribtion). This test is sensitive to any kind of difference in the distribtions from hich the to samples ere dran: differences in location (central tendency), in dispersion, and skeness. This test is concerned ith the agreement beteen to cmlative distribtions for to sets of sample vales. If the to samples have been dran from the same poplation distribtion, then the cmlative distribtions of both samples may be expected to be fairly close to each other, ith random deviations from the poplation distribtion. If the to-sample cmlative distribtions are "too far apart" at any point, this sggests that the samples come from different poplations. Ths, a large enogh deviation beteen the to distribtions is evidence for rejecting the nll hypothesis. 13 All analyses ere performed by compter sing the Statistical Package for the Social Sciences (SPSS) The parameters are reported as mean vales ± standard deviations. Cmlative relative freqency graphs ere constrcted to demonstrate zero and one F-body distribtions in the control, preseparation, and postseparation samples. Cmlative relative freqency, also knon as cmlative percentage (PCT), gives that percentage of individals having a measrement less than or eqal to the pper bondary of the class interval. The cmlative relative freqency is expressed as a proportion of the total nmber of participants for a particlar vale. Cmlative relative freqency plots make it possible to rapidly compare entire freqency distribtions. 17 Cmlative relative freqency graphs ere constrcted plotting zero F -body or one F -body conts per 5 spermatozoa conted verss the cmlative freqency (PCT) for the nmber of individals ith a particlar cont. RESULTS The stdy consisted of the analysis of 13 semen samples for the distribtion of F Y -bodies. The samples consisted of 5 control samples (1 patients), 35 preseparation samples (35 patients), and 18 postseparation samples (18 patients). The samples ere analyzed, making comparisons beteen the control and preseparation samples, the preseparation and postseparation samples, and the control and postseparation samples for zero, one, and to F-body conts (Table 1). The preseparation samples ere significantly different (P <.5) hen compared ith the control samples and postseparation samples for zero, one, and to F-body conts. No significant differences ere observed for zero, one, and to F -body conts hen the postseparation and control samples ere compared (Table 1). Cmlative relative freqency graphs for zero F body and one F-body conts ere constrcted to demonstrate the differences in distribtion hen comparing the control, preseparation, and postseparation samples. The zero F -body cont distribtion graph shoed that the preseparation samples contained more spermatozoa ithot an F body than the control grop. After separation, the postseparation zero F -body conts ere similar in distribtion to the control samples (Fig. 1). The distribtion graph for one F-body cont demonstrated that the preseparation grop contained significantly feer spermatozoa ith an F -body than Table 1 F-Body Distribtion in the Preseparation, Postseparation, and Control Samples No. of No. of Grop sbjects samples X-bearing cont Y-bearing cont 2Y -bearing cont Pre" Postb Control' P valesd P vales P vales' mean SD mean SD mean SD Statistical analysis as performed sing the Kolmogorov- Smirnov To-Sample test ith significance set at the.5 level. "Preseparation samples (nprocessed samples). b Postseparation samples (Y -body-enriched samples). 'Control samples (nprocessed samples). 672 Bibbins et al. F-body cont in male ith female progeny d P vales for comparison of control and preseparation samples. P vales for comparison of preseparation and postseparation samples. f P vales for comparison of control and postseparation samples. Fertility and Sterility

4 l!r-t> CONTROL e>-o PRE-SEPARATION..._.POST-SEPARATION l!r-t> CONTROL o-<> PRE-SEPARATION..._.POST-SEPARATION i3!!:. (j 7 z :::> 6 a: 5 "- > 4 ~ :::> 3 ::2: :::> ZERO F-BODY COUNT 1 9 I= 8!!:. >- 7 z :::> 6 a: 5 "- > 4 ~ :::> 3 ::2: :::> ONE F-BODY COUNT Figre 1 Graph of cmlative relative freqencies for zero F-body conts comparing the distribtions observed in the control, preseparation, and postseparation samples. This graph shos that the preseparation samples have a significantly higher cont of zero F-body spermatozoa as compared ith the control samples. After separation, the zero F-body conts for the postseparation samples are similar to the control samples. the control grop. After separation, the postseparation samples ere similar to the control grop in one F-body distribtion (Fig. 2). Several patients sbmitted more than one semen sample for analysis (Table 2), hich demonstrates the stability of the ratio of zero F -body to one F body over time. Similar stability as observed in the control grop for zero F -body and one F -body freqencies (Table 3). DISCUSSION It as not possible to differentiate spermatozoa ith a Y chromosome from those ith an X chromosome18 ntil the discovery that the distal portion of the long arms of the Y chromosome cold be stained ith qinacrine dihydrochloride.2 With normal segregation of the X and Y chromosomes dring meiosis, a spermatogonial cell shold give rise to to spermatozoa bearing Y chromosomes and to spermatozoa bearing X chromosomes.19 This translates into 5% ofthe spermatozoa shold have an F -body (Y -bearing) and 5% shold not have an F-body (X-bearing). There are slightly more X- than Y-bearing spermatozoa in hman semen, as indicated by or control grop (Table 1). Vol. 49, No.4, April1988 Figre 2 Graph of cmlative relative freqencies for one F-body conts comparing the distribtions observed in the control, preseparation, and postseparation samples. This graph shos that the preseparation samples have a significantly loer cont of one F-body spermatozoa as compared ith the control samples. After separation, the one F -body conts for the postseparation samples are similar to the control samples. Other investigators have observed similar reslts.2 21 The florochrome staining of hman spermatozoa ith qinacrine dihydrochloride is based on the principle of differential staining of metaphase chromosomes. 3 Qinacrine achieves its staining ability by intercalating into the DNA of the chromosomes. The heterochromatic regions of the Table 2 Stability of Zero and One F-Body Freqencies in the Preseparation Samples Sample no. Code no. F-body S-11 a b S-12 a b S-13 a b S-14 a b S-15 a b The difference beteen the sm of the percent zero F-body and percent one F -body, and 1% is the to F -body freqency. Percent zero F-body. b Percent one F-body. Bibbins et al. F-body cont in male ith female progeny 673

5 Table 3 Stability of the Zero F-Body and One F-Body Freqencies in the Control Grop %Zero F-body sample no. %One F-body sample no. Code no C C C C C C C C C C The difference beteen the sm of the percent zero F-body and percent one F-body, and 1% is the to F-body freqency. chromosomes are abndant in adenine and thymine, thereby enhancing qinacrine florescence. 4 The distal portion of the hman Y chromosome floresces ith great intensity becase of the richness of adenine and thymine, and the high affinity that qinacrine has for this portion of the Y chromosome.2 The presence of "adventitios" bodies (bacteria or artifacts) reslts in the occrrence of more sperm exhibiting one or to F -bodies. By sing the criteria established by Kapp and Jacobson, 6 most F objects that do not closely resemble F bodies can be readily eliminated. If a variety of random arrangements of celllar strctres ere observed as florescent bodies, one old anticipate that the freqency of zero F -body and one F -body spermatozoa old vary considerably more than e have observed. The variation in zero F -body and one F body freqencies is qite stable, as e have observed in the control grop (Table 3). F-body freqency in somatic cells is consistent ith the presence of the Y chromosome in metaphase preparations. A single F body is fond in interphase nclei from 46,XY male cells, none are fond in 46,XX female cells, and to F bodies are fond in 4 7,XYY male cells. 2 If F bodies are related to "adventitios" bodies or arrangements of celllar strctres, they also shold be seen in interphase nclei from normal females. In or F -body distribtion stdy, e analyzed the ratio of spermatozoa ith one F -body to spermatozoa ithot an F-body in males ho had fathered three or more girls, ithot the birth of a male offspring, to determine hether the ratio of X bearing to Y -bearing spermatozoa as normally distribted. The control grop as compared ith the test grops sing one randomly picked sample vale from each participant, and the average vale for each participant for the five samples sbmitted. 674 Bibbins et al. F-body cont in male ith female progeny No difference in significance as observed. A higher-than-normal percentage of spermatozoa did not have F -bodies in the preseparation samples, indicating that the percentage of spermatozoa ith an X chromosome as significantly higher than the control grop. In all of the preseparation samples, the percentage of spermatozoa ithot an F -body as significantly higher than the control grop. The percentage of zero F-bodies as as high as 79.2%. Several patients ere analyzed several times (Table 2), and a consistently higher percentage of spermatozoa as seen ithot an F -body. This old indicate that a permanent alteration has occrred in the ratio of X-bearing to Y-bearing spermatozoa. Dmoski et al. 21 have reported observing a significantly loer percentage of Y -bearing spermatozoa in males (n = 1) ho have fathered exclsively female children hen compared ith males ho have fathered both boys and girls. The qestion raised by or stdy is hy has this grop of males fathered exclsively female offspring and hy are they prodcing more spermatozoa ithot an F-body (X-bearing)? To hypotheses ill be presented that ill give zero F -body and one F -body freqencies similar to those observed in the test grop. These hypotheses may serve to explain hy the test grop is prodcing more spermatozoa ithot an F-body. The first hypothesis addresses the isse of nondisjnction. Nondisjnction of the sex chromosomes can occr at either the first or second meiotic division. First meiotic nondisjnction of either the X or Y chromosomes, or second meiotic nondisjnction of the X chromosome in spermatozoa, ill give F -body reslts the same as in normal meiosis. Second division meiotic nondisjnction of the Y chromosome ill increase the nmber of spermatozoa ithot an F -body; hoever, the nmber of spermatozoa ith to F bodies ill increase.19 Or preseparation to F- Fertility and Sterility

6 body freqency as loer than the control grop. The loer nmber of spermatozoa ith one F -body may have reslted in feer spermatozoa ith to F -bodies. With normal meiosis and second meiotic nondisjnction of the Y chromosome occrring in the spermatogonial cells, for types of gametes old be formed ith the folloing freqencies: X (112), Y (1/4), YY (118), and (118). This old reslt in the formation of for types of zygotes after fertilization: 46,XX, 46,XY, 47,XYY, and 45,X. Of the spermatozoa, 62.5% ill not have an F body, and 25% ill have one F-body. This ratio of zero to one F -body is similar to the reslts of the test grop; hoever, there old have to be one spermatogonial cell exhibiting second division meiotic nondisjnction of the Y chromosome for each spermatogonial cell dividing normally. This old reslt in 12.5% of the spermatozoa having to F -bodies, hich is dissimilar to or to F -body freqency of.74%. Meiotic nondisjnction of the Y chromosome ill reslt in an increased chance of chromosome anomalies in the embryo. 19 There ere no reports of chromosomal disorders in the children fathered by the males in or test grop. The second hypothesis is concerned ith the chromosomal composition of the spermatogonial cells. If the patients in the test grop had a probable 46,XY I 46,XX spermatogonial cell mosaicism ith to thirds of the cells being 46,XY and the remaining cells 46,XX, the folloing gametes old be formed ith the folloing freqencies: X (66.7%) andy (33.3%). Fertilization ill reslt in the folloing zygotes: 46,XX and 46,XY. These vales are similar to or F -body distribtions for the preseparation samples; zero F-body (67.9%), one F-body (31.4%), and to F-bodies (.7%). In order to confirm this event as a possible case for the exclsive birth of female offspring in the test grop, cytogenetics old have to be done on the spermatogonial cells. The disproportionately higher percentage of spermatozoa prodced ithot an F-body (X-bearing) by the males in the test grop correlates ith the exclsive birth of more female offspring. Acknoledgments. We thank Ms. Dianna Lokey for her technical assistance. REFERENCES 1. Beatty RA: F -bodies as Y chromosome markers in matre hman sperm heads: a qantitative approach. Cytogenet Cell Genet 18:33, Zech L: Investigation of metaphase chromosomes ith DNA binding florochromes. Exp Cell Res 58:463, Caspersson L, Farber G, Foley SE, Kdynoski J, Modest EJ, Simonsson E, Wagh U, Zech L: Chemical differentiation along metaphase chromosomes. Exp Cell Res 49:219, Smner AT: The natre and mechanisms of chromosome banding. Cancer Genet Cytogenet 6:59, Pearson PL, Bobro M: Florescent staining of the Y chromosome in meiotic stages in the hman male. J Reprod Fertil 22:177, Kapp RW Jr, Jacobson CB: Analysis of spermatozoa for Y chromosomal nondisjnction. Teratogenesis Carcinog Mtagen 1:193, Mcilvaine LC: A bffer soltion for colorimetric comparison. J Biol Chern 49:183, Drevis L-: The 'sperm-rise' test. J Reprod Fertil 24:427, Marmar JL: Seminal flid problems. In Crrent Therapy of Infertility , Edited by C-R Garcia, L Mastroianni, RD Amelar, L Dbin. Philadelphia, BC Decker, 1984, p Biggers JD, Whitten WK, Whittingham DG: The cltre of mose embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel Jr. San Francisco, WH Freeman, 1971, p Siegel S: Nonparametric Statistics for the Behavioral Sciences. Ne York, McGra-Hill, 1956, p Kzma JW: Basic Statistics for the Health Sciences. Palo Alto, CA, Mayfield Pblishing Company, 1984, p Siegel S: Nonparametric Statistics for the Behavioral Sciences. Ne York, McGra-Hill, 1956, p Kim JO, Kohot EJ: Analysis of variance and covariance: sbprograms ANOV A and oneay. In SPSS-Statistical Package for the Social Sciences, Edited by NH Nie, CH Hll, JG Jenkins, K Steinbrenner, DH Bent. Ne York, McGra-Hill, 1975, p Nie NH, Hll CH, Jenkins JG, Steinbrenner K, Bent DH: Description of sbpoplations and mean difference testing: sbprograms, breakdon and T-TEST. In SPSS-Statistical Package for the Social Sciences, Edited by NH Nie, CH Hll, JG Jenkins, K Steinbrenner, DH Bent. Ne York, McGra-Hill, 1975, p Hll CH, Nie NH: SPSS Update: Ne Procedres and Facilities for Releases 7 and 8. Ne York, McGra-Hill, 1979, p Kzma JW: Basic Statistics for the Health Sciences. Palo Alto, CA, Mayfield Pblishing Company, 1984, p Qinlivan WLG, Sllivan H: The ratios and separation of X and Y spermatozoa in hman semen. Fertil Steril 25:315, Evans HJ: Properties of hman X andy sperm. In Genetics of the Spermatozoon, Edited by RA Beatty, S Glecksohn Wallsch. Edinbrgh, Bogtrykkeriet Form, 1971, p Barlo P, Vosa CG: TheY chromosome in hman spermatozoa. Natre 226:961, Dmoski WP, Gaynor L, Rao R, Larence M, Scommegna A: Use of albmin gradients for X andy sperm separation and clinical experience ith male sex preselection. Fertil Steril 31:52, 1979 Vol. 49, No.4, April1988 Bibbins et al. F-body cont in male ith female progeny 675