\HMM‘HI‘WI \ \ WW HM HM...) no | (DCD-b K PREFERERTIAL S‘EGRESATIQN OF THE Y CHROMQ$GME [N SFERMATQGENEgiS OF DROSQPHILA MEL/ANOGASTER The“: {'00 fin Degree of M. S. MECEEGAN STM’E UNWEEESETY Carola M. Cattami 1968 masts ‘ LIBRARY ‘ "I LMichigan State Univel'Sit E 7 f -——- Eyffififi, -_‘ a 1- 2' i '~ . W “UV 3": 15 A my: mm. ABSTRACT PREFERENTIAL SEGREGATION OF THE Y CHROMOSOME IN SPERMATOGENESIS OF DROSOPHILA MELANOGASTER By Carola M. Cattani Deviations from expected 1:1 sex ratio in organisms utilizing XY-sex determination are frequently observed. Aberrant sex ratios may be due to factors Operating prior to fertilization. causing changes in the primary sex ratio. or subsequent to fertilization. resulting in a change in the secondary sex ratio. It has been established that normally deviant sex ratio shifts noted in Drosophila melanoqaster are due to factors functioning prior to fertilization. A relationship between paternal age and sex ratio due to a pre-fertilization phenomenon has been described. With an increase in the age of the male parent a decrease in the frequency of recovery of the Y chromosome is found. The object of this study is to demonstrate preferential seg- regation of the Y chromosome as a possible explanation for the paternal-age-related sex ratio shift. 1 . .171. Carola M. Cattani In order to test the hypothesis proposed crosses were made to determine the frequency of recovery of the Y chromo— some. From a cross involving an attached-KY male and a normal female. critical evidence to support the hypothesis was ob- tained. This could be concluded since the Y chromosome is recovered with a decreasing frequency as the age of the male increases even when the Y chromosome is attached to an X chromosome. In all of the crosses carried out in this study this decrease in recoverability of the Y chromosome was rea- lized independently of the chromosome complement of the aging male. Whether this preferential nonrandom segregation is due to the size of the Y chromosome or to some mechanism within the chromosome or its centromere is. as yet. undetermined. PREFERENTIAL SEGREGATION OF THE Y CHROMOSOME IN SPERMATOGENESIS OF DROSOPHILA MELANOGASTER BY Carola M. Cattani A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1966 ACKNOWLEDGMENTS I would like to thank Dr. Armon F. Yanders for his proposal of this study. His guidance and patience. without which this work could never have been completed. were greatly appreciated. I also wish to express my thanks to Dr- G. B. Wilson and Dr. J. R. Shaver for the interest they have shown during the course of this study. ii ACKNOWLEDGMENTS LIST OF TABLES. LIST OF FIGURES Section INTRODUCTION. MATERIALS AND RESULTS . DISCUSSION. . SUMMARY . BIBLIOGRAPHY. TABLE OF CONTENTS iii Page ii iv 15 18 26 34 36 21. Table I. II. III. LIST OF TABLES SUMMARY OF THE RESULTS OF THE PROGENY COUNTS AND THE SEX RATIO EOR.THE ORoO'x 0R9? CROSS (CROSS I) . SUMMARY OF THE RESULTS OF THE PROGENY COUNTS AND THE SEX RATIO FOR BOTH THE ORdO'X CROSSES (CROSS II AND CROSS IIa) AND THE COMBINED DATA FOR THESE CROSSES. SUMMARY OF THE RESULTS OF THE PROGENY COUNTS AND THE SEx RATIO FOR BOTH THE {Ord’d’x 0R9? CROSSES (CROSS III AND CROSS IIIa) AND THE COMBINED DATA FOR THESE CROSSES. iv Page 22 23 24 LIST OF FIGURES Figure Page l.--Weighted linear regression lines for the com— bined data of the three crosses are presented here. . . . . . . . . . . . . . . . . . . . . 25 PREFERENTIAL SEGREGATION OF THE Y CHROMOSOME IN SPERMATOGENESIS OF DROSOPHILA MELANOGASTER INTRODUCTION Discernible discrepancies in the theoretical progeny ratios. presumed by the principles of random segregation and random fertilization. have been frequently noted. Various causative mechanisms resulting in aberrant ratios have been proposed. These mechanisms may act during meiosis. gameto- genesis or upon the zygote causing a reduction in number or the elimination Of a particular class of progeny. Deviations from the theoretical 1:1 sex ratio in or- ganisms utilizing X-Y sex determination have been studied. Through experimentation with Qrosophila it has been possible to Obtain clear and precise analysis of sex determination and the hereditary factors influencing it. In DrOSOphila normal females produce eggs which contain one set of autosomes and one X chromosome and males produce sperm containing one set Of autosomes and either an X or a Y chromosome. The principle 1 of random segregation supposes that equal numbers of X and Y bearing sperm will be produced. Assuming that fertilization also occurs at random. a sex ratio of 19:10' should be achieved. However. many deviations from the expected 1:1 sex ratio have been reported. According to Brehme (1943) aberrant ratios may be a result Of factors which affect the production or viability of X and Y sperm. factors affecting the balance between sex-Chromosomes and autosomes. lethal factors affecting the survival of one or the other sex and factors causing reversal of the sex of the individual. One of the first aberrant sex ratios to be studied was described by Morgan (1911). He found that female Droso- phila carrying the mutant for rudimentary wings are almost sterile when mated to mutant males. However. when these females were mated to normal males their Offspring were in the ratio of 2 females to 1 male. He attributed this dis- tortion in sex ratio to a lethal factor contained in the single X-chromosome of the lost males passed on to them by their mothers. Counce (1956) studied the embryology of the rudimentary mutant and found that it affects larval differ- entiation. Gershenson (1928) described a sex-ratio abnormally found in a wild population of Drosophila Obscura resulting in the production of almost all female progeny. He attrib- uted this abnormality to a sex-linked mutant whose action was analogous to that of a gametic lethal. in that it renders Y-bearing spermatozoa incapable of participating in fertiliza- tion. This phenomenon has been referred to as "sex-ratio.” Further analysis of the “sex-ratio" condition was carried out by Sturtevant and Dobzhansky (1936) and Wallace (1948) in Drosophila pseudoobscura. It was shown that “sex-ratio” was widely distributed both geographically and taxonomically. Cytological examination Of spermatogenesis by Sturtevant and Dobzhansky led them to conclude that in males carrying “sex- ratio" the X Chromosome undergoes an equational division at each meiotic division and at the same time the Y chromosome degenerates. Thus. all the products of spermatogenesis would receive an X chromatid. Gershenson suggested that Since there was an absence Of differential viability the "sex-ratio“ fac- tor would increase in the wild population and would be fatal to the race. Wallace (1948) pointed out that such an increase does not actually occur due to seasonal fluctuations in the frequency of “sex—ratio.“ Therefore. natural selection prevents the X chromosome bearing “sex-ratio" from replacing its normal homolog. Novitski (1947) described an anomalous sex ratio con- dition in DrOSOphila affinis which resulted in the production by certain affinis males of only male offspring. He referred to this phenomenon as “male sex-ratio.“ He stated that this condition has as its basis a genetic constitution involving the “sex-ratio" X Chromosome found in Drosophila pseudoobscura and a recessive gene on Chromosome B. Its mode of action may correspond to the "sex-ratio“ condition described for the 9b: scura group. Another type of sex ratio abnormality producing uni- sexual progenies has been extensively studied. The factor. sex ratio (S3). which results in an absence Of male progeny was first reported by Magni (1953) in Drosophila bifasciata and by Cavalcanti and Falcao (1954) in Drosophila prosaltans and by Carson (1956) in Drosophila borealis. ._B was found to be transmitted exclusively from mothers to daughters. which led to the conclusion that this abnormality was due to the transmission of a causative agent through the cyto- plasm Of the egg. This causative agent acted by killing the eggs fertilized by Y—bearing Sperm. Magni was able to effect a "cure" for SB by high temperatures and Cavalcanti and Fal- cao found strains containing nuclear genes which disrupt the transmission of ”sex-ratio" particles. Malogolowkin and Poulson(l957) reported the existence of a maternally inherited “sex-ratio" Condition in Drosophila willistoni which could be effectively transmitted by injec- tion of ooplasm from infected into normal females. Poulson and Sakaguchi (1961) were able to establish the presence of treponema-like spirochetes in the hemolymph of adult “sex- ratio” females which are completely correlated with the pro— duction of unisexual progenies. They concluded that the "sex-ratio" condition in Drosophila species is the consequence of a stabilized host—parasite relationship which is dependent upon the genotype of the host and Of the infectious agent. Aberrant sex ratios have also been reported in higher animals whose sex determination is dependent upon the hetero- gametic male. Varied hypotheses and correlations have been prOposed in an attempt to decipher the cause of these deviant ratios. Weir (1958) demonstrated by artificial insemination experiments that an abnormal sex ratio he Observed in mice was due to a distortion in the primary sex ratio and not to differential survival Of zygotes. He hypothesized that the X- and Y-bearing Sperm are already present in disproportion- ate numbers by the time they reach the ductus deferens. A correlation between blood pH and sex ratio in mice was also investigated (Weir. 1962). The Offspring of a high-pH mouse strain resulted in a higher percentage Of males. and a low- pH mouse strain was associated with a low percentage of males. This shift in sex ratio was shown by experimentation to be dependent upon the genotype of the male parent. No indica- tion of significant differential mortality of the zygotes was found in the results. That a positive association exists between mean male sex ratio of children and the longevity of their parents in man has been reported by Lawrence (1940). He also noted that there was no regular increase or decrease in the sex ratio with increasing ages of the parents. Contrary to this find- ing Novitski (1953). Novitski and Sandler (1956) and Novitski and Kimball (1958) reported a linear relationship between paternal age and secondary sex ratio in man. Analysis dem- onstrated that there is a decrease in male offspring as the age Of the father increases and that this decrease is inde- pendent Of the age of the mother. TE Two cases of age-dependent sex ratio shifts have also been reported in DrOSOphila. Hannah (1955) found that aging the maternal parent resulted in an increase in the proportion of male Offspring. Aging Of mature gametes renders them less viable than unaged mature gametea and certain genotypes proved to be more sensitive to the aging effect than other genotypes. Hannah suggests several mechanisms which may contribute to the modified sex ratio caused by aging: missegregation or loss of a Chromosome during meiosis. crossing-Over lagging. or nuclear— cytoplasmic interaction. An Opposite effect of aging in Eggs- Ophila was also described. Yanders (1965) demonstrated that aging of Drosophila males causes a shift in sex ratio toward a greater proportion of female progeny. This paternal-age- related sex ratio shift is analogous to that found in man. A possible explanation for the observed deviations from the expected 1:1 sex ratio in animals was proposed by Novitski (1951). He found that in Drosophila melanoqaster females the physically shorter Chromosome of a structurally heteromorphic pair is included in the functional egg nucleus more frequently than the longer Chromosome. The effect was demonstrated by the use of ring—X and attached—X Chromosomes and confirmed by experiments involving structurally dissimilar rod chromosomes. This preferential recovery was attributed to non-random disjunction of Chromosomes at the second mei- otic division of oogenesis. Novitski suggested that this phenomenon may be responsible for aberrant sex ratios where sex is determined by a heteromorphic pair of chromosomes. Deviations from theoretical ratios other than sex ratio have also been studied. In their analysis of segrega- tion in a translocation heterozygote. Novitski and Sandler (1957) postulated that not all of the products Of spermato— genesis are functional. They found that in certain translo- cation—bearing (T (1:4) BS) males complementary classes of gametes were not recovered in equal numbers. Egg count ex— periments gave no evidence for differential zygote mortality. Gamete lethality and sperm competition were ruled out as pos- sible causes Of the distorted segregation ratios. They pro— posed that there exists a regular Class of nonfunctional gametes and that particular chromosomes segregated preferen- tially into these non-functional meiotic products. Using the same Bar-Stone translocation males. Zimmer- ing and Barbour (1961) Observed an “age effect“ associated with the recovery of aberrant gametic ratios. Observations indicated that the first sperm released by young Bar—Stone males clearly gave aberrant ratios; however. those sperm re- leased later by the same males show almost no distortion. That physiological differences between groups of cells des- tined to give rise to different sperm batches exist was sug- gested by Zimmering and Barbour. They also speculated that their results might have some bearing on the finding by No- vitski and Sandler (1956) that the secondary sex ratio in humans is dependent upon paternal age. This could be ac- counted for by a shift in the relative frequencies of X and Y-bearing sperm with an increase in the age of the father. A cytogenetic study designed to elucidate the SD (segregation-distorter) phenomenon in Drosophila melanoqas— .£§£. which was described by Sandler. Hiraizumi. and Sandler (1959). was undertaken by Peacock and Erickson (1965). The SD locus is proximally located on the right arm of the second Chromosome and it functions as an extreme Case of meiotic drive. When males are heterozygous for SD. the SD+-bearing gamete is rendered nonfunctional. This phenomenon requires synapsis in order to function. The SD effect does not occur in females. Sandler et a1. (1959) presented a formal cyto- genetic model based On preliminary evidence in which the SD locus caused a break in its synapsed homologue. This break 10 resulted in the formation of a dicentric bridge at anaphase II due to sister strand reunion. ultimately causing death of the SD+-bearing cells. However. Peacock and Erickson's (1965) recent cytological Observations indicated that meiosis was completely normal and all the products Of meiosis proceeded through Spermiogenesis to yield motile sperm. Both SD and SD+-bearing Sperm were shown to be capable of entering the sperm storage organs of the female. Counts of stored sperm and Of progeny recovered from Similarly inseminated females were made in order to determine the possibility of selection occurring between the time of sperm storage and fertilization. These counts showed that only one-half of the sperm stored by a female from a mating with an SD male are capable of fertil- izing an egg. Those sperm not able to fertilize an egg pre— sumably carried the SD+ chromosome. The same situation with regard to half Of the sperm being nonfunctional was exhibited by the Oregon-R control group. This led the investigators to the conclusion that one-half of the products of spermato- genesis are regularly nonfunctional in DrOSOphila melanoqas- ter. This possibility had been anticipated by Novitski and Sandler (1957) in their work with the Bar—Stone translocation- gametes. They proposed the concept. upon which the above ll conclusion was based. that a regular class of nonfunctional gametes exists and that particular Chromosomes had certain probabilities Of being included in the functional sperm. Peacock and Erickson proposed an inequality of the two spin- dle poles at first anaphase such that one pole ultimately leads to the formation Of two functional sperm and the other pole yields nonfunctional sperm. Concerning the SD phenome— non it was concluded that SD influences the orientation of the second chromosome bivalent at metaphase so that the SD- bearing chromosome almost always moves to the pole which produces the two functional gametes. The finding made by Peacock and Erickson (1965) has provided satisfactory explanations for various sex ratio phe- nomena. Recent reexamination of the “sex-ratio“ phenomenon found in the Obscura group was carried out by Novitski. Pea- cock and Engel (1965). Cytological investigations revealed that. contrary to the earlier reports of Gershenson (1928). Sturtevant and Dobzhansky (1936) and Wallace (1948). there is no replication Of the X chromosome during meiosis. Mei- osis. however. was found to be quite abnormal in spermato- genesis Of "sex-ratio" males. During the second meiotic div— ision the Y chromosome loses its Characteristic appearance 12 and forms a Chromatin mass devoid of a centromere and lacking centromeric activity at anaphase II. This degeneration of the Y chromosome was also noted by the earlier investigators. Half of the meiotic products resulting from this abnormal meiosis lack a sex chromosome. Since the "sex-ratio“ effect results in the production of all female progeny the hypothe— sis that One-half of the products of spermatogenesis are reg- ularly nonfunctional afforded a plausible explanation for this phenomenon. Simply stated. at anaphase I the X Chromo— some preferentially moves to the functional pole leading to the production of two functional X-bearing gametes. Hanks (1965) suggests that the deviant sex ratios he noted in the wild-type strains of Drosophila melanoqaster. Oregon—R and Canton-S could possibly be explained on the ba- sis of the hypothesis that functional and nonfunctional poles exist at anaphase I. An excess of one sex could be the re- sult of the orientation Of the X-Y bivalent at metaphase 1 so that the chromosome recovered more frequently proceeds to the functional pole more Often than its homologue. Hanks further suggests that this explanation may account for normal deviant sex ratios in general. Yanders (1965) also adopted Peacock and Erickson's hypothesis to explain the paternal-age—related sex ratio l3 shift he found in Drosophila melanoqaster. He suggested that this shift was due to abnormal segregation of the X and Y Chromosomes to the "functional” and “nonfunctional” poles with an increasing tendency for the Y chromosome to go to the nonfunctional pole as the male ages. Yanders also pro- posed that this type of nonrandom disjunction. which produces a Change in the primary sex ratio. may be responsible for the relationship between paternal age and sex ratio in humans re- ported by Novitski (1953). Novitski and Sandler (1956). and Novitski and Kimball (1958). The study presented here is based upon the assumption that the mechanism proposed by Peacock and Erickson (1965). in which chromosomes segregate preferentially to functional and nonfunctional poles. does exist. Two alternative explan- ations Can be Offered for the shift in sex ratio related to paternal age noted by Yanders (1965). These explanations Or hypotheses involve the preferential movement of the X and Y Chromosomes to the functional and nonfunctional poles. The first hypothesis suggests that the X Chromosome shows a preference to move to the functional pole more frequently than it does to the nonfunctional pole. The second hypothe- sis assumes that it is not the X chromosome that demonstrates l4 preferential movement but it is the Y Chromosome that chooses to segregate nonrandomly. Yanders (1965) proposed that the shift in sex ratio is due to the second alternative explana- tion. The experiments undertaken in this study were con- ducted to analyze the plausibility Of the second hypothesis. This hypothesis is testable by experimentation if carried out in the following manner: 1. Wild—type males were mated to wild-type females to establish the activity of X and Y chromosomes in a normal situation. 2. To test between recovery of gametes and differential survival of zygotes wild—type males were crossed to attached-X females carrying a free Y chromosome. 3. The third cross utilizing attached-XY males and wild- type females contributed the critical evidence needed by demonstrating the movement of the Y chromosome when it is attached to an X Chromosome. MATERIALS AND METHODS Three stocks of Drosophila melanoqaster were used; Oregon-R (OR). wild-type strain. attached-X (y m f/y w f:=) and attached-KY (YS w y - YL y+/yw/O). The wild-type stock was Obtained from the University of Chicago and the attached- X and attached-XY stocks were obtained from Oak Ridge National Laboratores. Cultures Of these stocks were maintained in the laboratory by mass transfers in half pint milk bottles on nu- trient medium (modified after Carpenter. 1950). The cultures used for the crosses were transferred every other day and kept in a constant temperature room at 230C. All the males and females used in the crosses were collected as virgins. Collections were made within a 12 hour period thereby minimizing variation in age. Virgin females were aged on nutrient medium at 23°C for six days after eclo- sion. Virgin males were aged for a maximum of 24 hours after eclosion on nutrient medium at 23°C. The crosses were designed to Show a paternal-age— related sex ratio shift. Therefore. the male's age increased while the female's age was kept constant at the time of mating. 15 16 In the Day 1 mating each 24 hour aged virgin male was intro- duced into a vial containing 2 virgin females (aged 6 days). The mating vials were then placed in the constant tempera— ture room for 24 hours. At the end of this 24 hour period (mating period) the males were removed from the mating vials and transferred to a new vial containing 2 more 6 day aged virgin females (Day 2 mating). At this time the male was 48 hours old. The females from the Day 1 mating were removed from the mating vials and placed in separate vials where they were permitted to lay eggs for nine days. This process. transferring the males to two new females at 24 hour inter— vals. was carried out for 21 consecutive days. All the mat- ings and transfers were made without etherization. Progeny counts were made on all the vials. i.e.. the mating vials and the vials into which the females had been separated. The parental females were removed from the vials and discarded after they were permitted to lay eggs for nine days. This prevented the inclusion Of the parental females in the progeny counts. The number and sex of the offspring were recorded according to the parental male and his age in days at the time of mating. Counts Of the progeny were made every other day for ten days. Any fly remaining stuck in the 17 medium after a collection was removed. examined. and recorded also. Three crosses were run concurrently. The three crosses were as follows: Cross I ORd'd' x ORSRS2 /\ Cross II ORcfo' x m9? /\ Cross III XYO'O' X 0R9? Six males were used for each of the crosses carried out. Crosses II and III were repeated at which time ten males were used for each cross. The crosses were repeated in order to confirm the results Obtained from the first ex— periments. Weighted linear regression analyses were used to Obtain the slopes for the above crosses. RESULTS The results of the progeny counts and the sex ratio. i.e.. the proportion Of female progeny. for the crosses car- ried out are summarized in Tables I. II. and III. Curves Ob- tained from weighted linear regression analyses Of the results of the separate crosses are presented in Figure l. The lepe values of the curves are also given in Figure l. The Slope obtained from the data relating sex ratio to paternal age in Cross 1 (ORJHDCORQQ) was positive. i.e.. the proportion of female Offspring increased with the increas- ing age of the male parent. The value for the lepe was .0020. which is in agreement with the value of .0034 Obtained by Yan— ders (1965). In this cross the male progeny derived their Y chromosome from the male parent whose sex Chromosomes were of the normal XY composition. This increase in sex ratio could be explained equally well by either of the alternative hypo- theses proposed; i.e.. the X chromosome may be demonstrating a preference for the functional pole or the Y chromosome may be preferentially Choosing the nonfunctional pole more Often as the male ages. 18 19 The combined data Of Crosses II and IIa (OROOTXLXXYQQ) resulted in a negative slope (—.0048). This indicated that the relative frequency of female progeny decreased with the increase in paternal age. In the OROO5CXXY$$ cross there was a free Y in the chromosome complement of the female parent. The male offspring are the recipients of the free Y from the female parent and an X Chromosome from the male parent. The female Offspring received a Y chromosome from the male parent and an attached-X chromosome from the female parent. As one would expect from the data obtained from Cross I. the number of progeny receiving the Y chromosome from the parental male decreased as the male aged. However. in the OROOTX.XXY$$ cross the female progeny received a Y Chromosome from the parental male and therefore. the female Class of progeny de- creased. Essentially the same effect was recognized in this cross. i.e.. the recoverability of the paternal Y Chromosome decreases with an increase in the age of the male. as was seen in Cross I. The decrease in sex ratio Observed in Cross II and Cross IIa therefore may be interpreted by either of the proposed hypotheses. The sex ratio for the progeny in the OROOIX XXYQQ Crosses began considerably lower than that Of the OROO’XIORQE cross. Although a lowered sex ratio is expected. due to the 20 nature of the cross. this does not fully explain the .4209 sex ratio Observed for the Day 1 matings. Upon checking the stock cultures from which the attached-X females were Obtained a sex ratio of .4549 was Observed. The lower ratio was there- fore attributed to a reduced viability of the XXY zygotes. This factor did not Change the interpretation of the results since the expected decrease in sex ratio as the age of the male increased was realized. The slope obtained from the third Cross ()QYd‘dIX 0R2?) was also negative. The value for the combined lepeS of the crosses was -.0024. which points out that the relative fre- quency of female progeny decreased as the age of the male parent increased. In this cross the X and Y chromosomes of the male parent are attached and move during meiosis as a single univalent chromosome. The female progeny receive this univalent and also an X chromosome from the female parent re— sulting in the following chromosome complement: XXY. The male progeny are of the Chromosome complement XO. since they received an X chromosome from the female parent and a nullo gamete from the parental male. The negative slope for the sex ratio in the progeny of Cross III indicated that the attached-XY chromosome pref- erentially segregates to the nonfunctional pole. This cross 21 constitutes the critical test for support of the second al- ternative hypothesis since the Y demonstrates a preference for the nonfunctional pole during anaphase I of meiosis even when it is attached to an X chromosome. If the X Chromosome was the dominant factor in this particular Case of nonrandom segregation one would expect a higher recovery of the unival- ent—XY chromosome. since the X Chromosome is normally recov- ered more frequently. 22 TABLE I. SUMMARY OF THE RESULTS OF THE PROGENY COUNTS AND THE SEX RATIO FOR THE ORd'd‘XORQQ CROSS. (CROSS I) CROSS I 1 814 .5418 2 943 .5398 3 919 .5147 4 1225 .5069 5 1409 .5089 6 1296 .5355 7 886 .5034 8 964 .5394 9 1314 .5381 10 1087 .5317 11 1090 .5266 12 1040 .5260 13 869 .5374 14 441 .5533 15 696 .5805 16 618 .5485 17 718 .5794 18 200 .5900 19 311 .5595 20 183 .5191 21 494 .5162 23 TABLE II. SUMMARY OF THE RESULTS OF THE PROGENY COUNTS AND THE SEX RATIO FOR BOTH THE ORo’d'X BOISE? CROSSES (CROSS II AND CROSS IIa) AND THE COMBINED DATA FOR THESE CROSSES. Combined Data Of Cross II Cross IIa Crosses II & IIa Mating Num- Frequency Num- Frequency Num— Frequency Period ber of $2 ber of ber Of 1 424 .3939 906 .4040 1330 .4008 2 578 .4204 1372 .4125 1950 .4149 3 742 .4259 853 .4174 1595 .4213 4 698 .4226 1265 .4119 1963 .4157 5 442 .4457 1319 .4321 1761 .4355 6 660 .4515 775 .3265 1435 .3840 7 621 .3961 1219 .3519 1840 .3668 8 404 .4530 1183 .3297 1587 .3611 9 219 .3379 733 .3820 952 .3718 10 234 .4316 660 .3970 894 .4060 11 303 .3498 548 .4179 851 .3937 12 268 .3843 453 .4305 721 .4133 13 254 .3937 424 .4222 678 .4115 14 76 .3289 349 .3095 425 .3129 15 79 .3671 368 .3694 447 .3624 16 96 .3854 151 .3046 247 .3360 17 98 .3571 209 .2153 307 .2606 18 --- ----- 169 .2426 169 .2426 19 7 2857 100 .4100 107 .4019 20 65 .4615 127 .4016 192 .4219 21 20 .3000 3 .3333 23 .3043 24 TABLE III. SUMMARY OF THE RESULTS OF THE PROGENY COUNTS AND THE SEX RATIO FOR BOTH THE X‘Ycfi'd‘x ORQSie CROSSES (CROSS III AND CROSS IIIa) AND THE COMBINED DATA FOR THESE CROSSES. Cross III Cross IIIa g:::;::dlgit: IIIa Mating Num- Frequency Num- Frequency Num- Frequency Period ber of 99 ber of $2 ber of $9 1 8 .3750 188 .4787 196 .4745 2 234 .4744 1279 .5035 1513 .4990 3 293 .5734 901 .4639 1194 .4908 4 701 .4964 1196 .5426 1897 .5256 5 856 .4872 1389 .5061 2245 .4989 6 417 .5252 1274 .5000 1691 .5062 7 356 .4522 1269 .4728 1625 .4683 8 280 .4393 1183 .4531 1463 .4505 9 419 .4654 1097 .5114 1516 .4987 10 364 .5440 1426 .5007 1790 .5095 11 580 .5362 653 .5314 1233 .5337 12 477 .4654 564 .5018 1041 .4851 13 720 .4306 850 .5318 1570 .4853 14 296 .4966 898 .4554 1194 .4657 15 181 .4033 643 .4883 824 .4697 16 274 .4781 784 .4949 1058 .4905 17 390 .4538 1044 .4550 1434 .4547 18 --- ----- 586 .4249 586 .4249 19 231 .5498 243 .4691 474 .5084 20 12 2500 65 .4462 77 .4156 21 372 .4597 86 .4070 458 .4498 25 vmoof lllll ammo x xOKOmAwn meoof III: mmccmwn x xovpmo omoo . ..... ammo x ..0930 "MBOHHOM mm mum mwcfla mmmnu How mmsHm> mmoam USE .mmmmouo woman on» no mumc COCHQEOU USO How MOCAH COflmMUHmUH MOUCHH OmuflmHUSII.H musmflm A madam: um mama :H Um< Hmcuoumm Hmommammioamaflmaflfl Emma one mm Ho r~_ _ . . _.._._. Somm. -mnm. -oom. .-m~m. 10mm. 1mhm. _Awallllllwa~w4: -ooe. -mme. mamv. .ome. -mnv. -oom . mdq||||||dflan4| .omm. -mhm. Ammmc Ream SGIBWSJ go Kouenbeig DISCUSS ION An attempt was made to demonstrate the relationship of preferential segregation Of the X and Y Chromosomes to the age of the male parent by the use of attached—X and attached -XY stocks. The experiments carried out indicated that re- covery of the Y Chromosome is age dependent regardless of the Chromosome complement of the aging male. In all the crosses an inverse relationship was observed between increasing pa- ternal age and the frequency of recovery of the Y chromosome. The results of the experimental cross involving an attached- XY male and a normal female indicated that the Y chromosome is recoverable less frequently as the male ages even when it is attached to an X Chromosome. This leads us to believe that the Y Chromosome plays the dominant role in the meiotic behavior Of the XY bivalent. It has already been established that normally aberrant sex ratios are dependent upon the male parent (Yanders. 1965; Hanks. 1965). Such deviations from a 1:1 ratio may be due to factors operating before fertilization. causing a change in 26 27 the primary sex ratio. or after fertilization. affecting the secondary sex ratio. That the deviations noted are due to a prezygotic phenomenon rather than zygotic lethality was con- cluded by both Yanders (1965) and Hanks (1965). Additional evidence supporting this conclusion was Obtained from two of the crosses carried out in this work. in which wild-type Ore- gon-R males were mated to both free X and attached-X females. If the aberrant sex ratios were dependent upon differential lethality of one of the sexes. the effect of paternal age on sex ratio should be the same in both crosses. and the regres- sion lines calculated from the data should be in the same di- rection. That is tO say. if an increase in the number of fe- male progeny is noted in one cross it should also be noted in the second cross. This. however. is not the Case. The free X cross resulted in an age-dependent increase in the proportion of female progeny and the attached-X cross re- sulted in an age-dependent decrease in the proportion of fe- male progeny. This strongly indicates that the Shift in sex ratio is due to a prezygotic phenomenon rather than prefer- ential lethality of one Of the sexes during embryonic devel- opment. 28 A number of theories have been developed which might be utilized to explain the decrease in recovery of the Y Chromosome related to paternal age. Novitski (1951) des- cribed a type of nonrandom disjunction in Drosophila females which is dependent upon the size of homologues. When two were of unequal size. the smaller homologue was recovered more frequently. Novitski suggested that this meiotic phen- omenon may be the cause of off-sex ratios in animals whose sex is determined by a heteromorphic pair of Chromosomes. An age effect was Observed in the recovery of abnor- mal gametic ratios resulting from a Bar—Stone translocation [T(1:4) BS] in Drosophila males by Zimmering and Barbour (1965) who ascribed it to distorted segregation ratios. They found that the shorter homologue was recovered more frequently in younger males; however. in older males. the distorted ratios tended to disappear. indicating that both homologues were recovered with almost equal frequencies. They proposed that this may be the result of physiological differences between the groups of cells destined to give rise to the different sperm batches. They speculated that the change in the sec- ondary sex ratio in humans correlated with the increasing age of the father (Novitski and Sandler. 1956) might be in- terpreted on the same basis. They conjectured that the 29 relative frequencies of X- and Y-bearing sperm Shift with the increase in age Of the father so that younger fathers have a greater probability Of having sons than do Older fathers. The Y chromosome in man is the shorter homologue and tends to be recovered more frequently from younger males than the longer X chromosome. but both homologues tend to be recovered with nearly equal frequencies from Older males. The shift in sex ratio related to paternal age in Drosophila is in the same direction as that found in humans; i.e.. as the parental male ages the sex ratio shifts toward a greater proportion of females. Though the shift is in the same direction the explanation suggested by Zimmering and Barbour (1961) cannot be applied to Drosophila. In Droso- phila the Y Chromosome is the larger of the heteromorphic sex chromosomes; and. in accordance with Zimmering and Bar- bour's hypothesis. the larger homologue is recovered less frequently in the progeny of young males. But. contrary to the effect noted by them. the frequency of recovery Of the larger homologue decreases linearly as the male ages. This linear decrease was noted in the results of all the crosses. Novitski and Sandler's (1957) concept Of a regular class of non-functional gametes proposed to explain the un— equal recovery of gametes from certain translocation-bearing 30 [T(1:4) BS] males was the basis for Peacock and Erickson's (1965) conclusion in their study on the nature of the segre- gation distortion phenomenon. From their investigation they were able to conclude that Drosophila melanoqaster regularly forms two functional and two nonfunctional sperm from each primary spermatocyte. They attributed this to the inequal- ity of the spindle poles at anaphase in Meiosis I. whereby one pole ultimately produces two nonfunctional sperm and the other pole two functional Sperm. Hanks (1965) suggested that the above hypothesis might be used to explain the normally deviant sex ratios he observed in two wild—type strains of Drosophila melanoqaster. He suggested that the homologue recovered in excess. in this case the X chromosome. has a slightly greater probability of segregating to the function- al pole during anaphase 1 than the other homologue. Yanders' (1965) observations on the paternal-age— related sex ratio shift in Drosophila melanoqaster were also explained on the basis of Peacock and Erickson's hy- pothesis. He interpreted the shift as an increase in the probability that the Y chromosome will go to the nonfunc— tional pole at anaphase l as the male ages. The results obtained in the present work are consistent with his inter— pretation. 31 The results of the crosses performed strongly indi— cate that the Y chromosome "preferentially" segregates to the nonfunctional pole more often as the age of the male in- creases rather than the X Chromosome “preferentially“ segre- gating to the functional pole more often. Critical evidence supporting this conclusion was obtained from the cross in- volving the attached-XY male. The decrease in female progeny of the chromosome complement XXY with the increase in the age of the male is evident even when the Y chromosome is attached to an X chromosome. An acceptable hypothesis for this result would be that the Y chromosome demonstrates a preference for the nonfunctional pole. Since the above conclusion was arrived at by the use of an attached-XY male. it is feasible to exclude the possi- bility that this effect on segregation is due to the use of a univalent as opposed to a bivalent? That univalents do not show random segregation was suggested by Peacock (1965). By use of attached-XY males he found that the frequency of recoverable nullo gametes is slightly higher than that of univalent attached-XY gametes. It appeared that the unpaired chromosomes showed polarized movement in the primary sperma- tocycte. This oriented segregation of univalents could be 32 explained by preferential movement of the univalent to the functional pole. Peacock inferred that this nonrandom segre- gation of univalents might be a function of chromosome compo- sition and structure. as well as total genotype. His findings therefore do not exclude the proposition of preferential seg- regation of the Y chromosome. In all of the data presented. the shorter chromosomes. and in the univalent the nullo gametes. were recovered with a higher frequency. Chromosome Size. therefore. may be the important factor in the phenomenon of nonrandom segregation. It would be more interesting. however. to assume that the preferential segregation of the Y chromosome is due to some mechanism which influences the alignment or movement of the Chromosomes at anaphase I. for this is subject. in principle. to experimental test. This factor could be contained within the entire Y chromosome or it may be limited to a specific portion of it. such as the centromere. The results presented here support either hypothesis. In all the crosses the Y Chromosome showed a preference for the nonfunctional pole as the male aged indicating that the mechanism or factor may be in the entire chromosome. Since the attached-XY stock used was composed of both the X and Y chromosomes but pre— sumably only a Y centromere (Lindsley and Novitski. 1958) 33 it is possible that the Y centromere itself. rather than the entire chromosome. may be the factor governing the preferen- tial movement of the Y Chromosome to the nonfunctional pole. Experiments utilizing Y chromosomes with centromeres of dif- ferent origins may provide sufficient evidence to solve the above problem. S UMMA RY Preferential segregation of the Y chromosome in sperm- atogenesis of Drosophila melanoqaster is proposed as a possible explanation for the observed Change in sex ratio related to paternal age. In order to test this hypothesis three differ- ent crosses were carried out: 1) ORo’of X 0R9? 2) ORo’d' X XXYQS? 3) 9Y8? X ORS352 In all of the crosses performed the recoverability of the Y chromosome decreased as the age of the male parent in- creased. These results were interpreted on the basis of Peacock and Erickson's (1965) hypothesis that "functional" and "nonfunctional" poles exist at anaphase l of meiosis and upon Yanders' (1965) observations that the paternal-age- related sex ratio shift he noted is due to abnormal segrega- tion of the sex chromosomes. 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