mums ON THE EFFECTS on: 2:; AND Y CHROMQSDMES ON PROTEIN svmnssls mu SPERMATDZOAL DEVELOPMENT 2N memosopmm MELANoGAmn 1 "Mask 5:)»: 9M Dear“ of Ph. D. MCI-{MAN EITATE UNIVERSITY 3&5 Byung Yuan 1961 ft .-4¢Q ‘ ’.-~ “3..an This is to certify that the thesis entitled Studies on the Effects of X and Y Chromosomes on Protein Synthesis and Spermatozoal Development in Drosophila melanogester presented by Sei Byung Yoon has been accepted towards fulfillment of the requirements for wk :6 MW Major professorc’ Date August 4L 1961 0-169 LIBRAR Y Michigan State University a” a... w. m. (5 ML .nLinyti _ STUDIES ON THE EFFECTS OF X AND Y CHROMDSOMES ON PROTEIN SYNTHESIS AND SPERMATOZOAL DEVELOPMENT IN DROSOPHILA MELANOGASTER by Sei Byung Yoon AN ABSTRACT Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1961 3:. fix Approved ABSTRACT STUDIES ON THE EFFECTS OF X AND Y CHROMOSOMES ON PROTEIN SYNTHESIS AND SPERMATOZOAL DEVELOPMENT IN QROSOPHILA MELANOGASTER Sei Byung Yoon PART I. EFFECTS OF THE X AND Y CHROMOSOMES ON PROTEIN SYNTHESIS. The effects of the X and Y chromosomes on protein synthesis in Drosgphila melanogaster were analysed by immunogenetic methods util- izing agar-diffusion techniques. For this purpose a whole array of coiso- genic stocks was derived containing variable numbers of X and/or Y chromosomes. It was found in all cases that males possessed two sex specific antigens designated 3 -l 8: 3 -2, which were absent in females, and that females possessed two sex specific antigens designated-3 -l & i? -2, which were absent in males. The differences in antigenic specificity in these cases were independent of the presence or absence of the Y chromosome but were attributable to a dosage effect of the X chromosomes. Another antigen (Y-l) was found in all stocks and its specificity was the same regardless of the stock or sex tested. However, the antigen manifested itself in different forms. Oregon R flies exhibited a "complete" form of the antigen, whereas in a stock called YA it was present in an "incomplete" form. Y-l fron.males and females of the YA stock is capable of inducing the formation of antibody and combining with it but is not capable of forming a visible precipitate, while Y-l from males and females of the Oregon R stock is also capable of forming precipitate. The two stocks differ only in the genotype of females: Oregon R females are X/X, while YA females are XX/Y. It is suggested that the difference between the complete and incomplete forms of the Y-l antigen is attributable to differences in number of combining sites. Genetically, a matroclinous effect is exhibited whereby in order for a fly to possess incomplete Y-l its mother must have had a Y chromosome. This effect is apparently initiated in the oocyte, is independent of the previous history of the cytoplasm, and is perpetuated through meiosis, fertilization, and ontogeny. The respective roles of euchromatin and heterochromatin in protein synthesis are discussed. PART II. EFFECTS OF THE Y CHROMDSOME ON SPERMATOZOAL DEVELOPMENT. The effects of the Y chromosome upon the development of spermatozoa in DrosoPhila melanogaster were observed. These obser- vations were made on both ig_zi!g_and in vitgg systems. Techniques were developed in order to make this study. Deve10ping spermatozoa from the testes of Oregon R wild and X/O males were compared. It was found that spermatozoa in X/O males never reached maturity. On the basis of these Observations, an artificial classification of develOping sperm, utilizing a number of parameters, is suggested. lass C chambe that a locomo served invest. obsem and uni The motility and locomotion of spermatozoa from the sperm mass of seminal vesicles of males and the sperm ball from.the genital chamber of impregnated females were studied.in'zitgg. It was found that a portion of female oviduct or unfertilized eggs would initiate locomotion by attracting the spermatozoa. This locomotion was ob- served only in sperm from the sperm mass. In the course of this investigation, a possible case of an ig_!it£g fertilization was observed. The effects upon motility of spermatozoa, utilizing immunized and unimmunized globulin fractions from rabbit sera, were studied. STUDIES ON THE EFFECTS OF X AND Y CHROMOSOMES ON PROTEIN SYNTHESIS AND SPERMATOZOAL DEVELOPMENT IN DROSOPHILA MELANOGASTER by Sei Byung Yoon A THESIS Submitted to ‘ Michigan State university in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1961 ACKNOWLEDGEMENTS Sincere appreciation is gratefully extended to Dr. Allen S. Fox for his continued stimulation and guidance in this work. Moreover, the interest by Dr. Fox in the intellectual and technical development of the author is also greatly appreciated. In addition, the author wishes to acknowledge his gratefulness for the personal interest and critical comments on this thesis by Dr. Jean B. Burnett and Mr. Morton S. Fuchs. Appreciation is extended to Prof. James R. Burnett and Dr. Charles G. MEad for their aid in the preparation of diagrams and photographs. The assistance in language by Miss Eileen A. Sweeney is also gratefully acknowledged. ii TABLE OF CONTENTS PART I. EFFECTS OF THE X AND Y CHROMOSOMES ON PROTEIN SYNTHESIS. I.INTRODUCTION... ..... II. MATERIAIS AND METHODS III . VI. PART II. EFFECTS OF THE Y CHROMOSOME ON SPERMA'IOZOAL II. III . A. GENETIC METHODS. . B. DIIUNOIMICAL METHOIB REULTS . . . . A. SEX-SPECIFIC ANTIGENS . . . . . . B. Y-l ANTIGEN . . . . C. PARTIAL CHARACTERIZATION OF ANTIGENS . DISCtBB IO] 0 0 0 0 0 0 0 0 0 O O O O O 0 BINARY REFERENCES . DEVELOPMENT . INTRODUCTION.............. SPERMATOZOA IN THE MALE GENITAL ORGANS . . A.ME'1‘HOIB ... B Q om RVATIONS O O I I O O I O O O O O O O SPERMATOZOA IN THE FEMAIE GENITAL ORGANS . A. mm o o o ,o e o o". o. o o o e o B. OBSERVATIONS . . . iii Page qoxoxr—i 15 15 21. 3h 36 1+2 m. 53 56 56 56 66 66 66 VI. VII. VIII. SPERMA'IOZOAEXERQ.................... A. SPERM MAss FROM SEMINAL VESICLE OF MALE B. SPERM BALL FROM GENITAL CHAMBER OF FEMALE c . IOCOMO‘I'ION OF SPERMATOZOA ........... EXPERIMENTAL MODIFICATION OF SPERMA‘IOZOAL MOTILITY . A. TREATMENTWITH WUNEGIOBULIN . . . . .. ..... B. TREATMENTWITHFLYEX'I'RACTS .......... AGGLUTINATION or SPERMATOZOA BY NORMAL AND IMMUNE GIOBULIN . DISCUSSION......................... REFERENCES... ....... ...... iv 72 72 77 79 83 83 81+ 89 9o 97 99 PART I. EFFECTS OF THE X AND Y CHROIDSOMEB OF PROTEIN SW18 . I. INTRODUCTION Ever since the cytological characterization of hetero- chromatin by Heitz in 1928, the biological role of this part of the chromosomal material has been the subject of much experimentation as well as speculation. The functions assigned to it have ranged from a genetically inert substance (Muller 1932) until the present time where it has been charged.with so many responsibilities in the metab- olism of the cell that it takes on considerable importance in many theories of cellular behavior. Indeed, so many widespread effects have been attributed to heterochromatin that it becomes difficult to define its biological role. It is therefore evident that a clear statement of its biolOgical function,which will explain its varying effects as well asia further elucidation of its mode of action, is a fUndamental problem facing modern biology today. The distinction between euchromatin and heterochromatin, while vague and ill-defined at the genetical level, is clear at the cytological level. Heitz (1928) described heterochromatin on the basis of its differential staining reactions in interphase or prOphase nuclei. In DrosOphila melanogaster the Y chromosome and the chromo- central regions of the X chromosomes and autosomes are stainable during interphase and early prephase, while euchromatin stains lightly or not at all. The heterOpycnosis exhibited by heterochromatin has been attributed variously to differences between nucleic acid content of the heteropycnotic regions and the rest of the chromosomal complement l 2 (Devan 19%) or to a difference in coiling (Wilson and Boothroyd l9hl and 191m; Ris 1915; and Coleman l9h3). Cytochemical studies have indicated that chromosomes appear to be integrated fabrices of both nucleic acids and proteins. Further enzymatic studies using purified nucleases and proteases have demonstrated that both RNA and DNA are present in euchromatic as well as heterochromatic regions of the salivary chromosomes in Drosophila (Kaufmann et al., 1951). Heitz (1933) distinguished three types of heterochromatin, alpha, beta and intercalary. Alpha heterochromatin is that found in somatic interphase chromosomes, while the beta type is found in chromocentral regions, the Y chromosome and the proximal third of the .X chromosome. Intercalary heterochromatin is indistinguishable cytologically from euchromatin, but is presumably present as single bands or blocks distributed in the euchromatic regions of chromosomes. Its existence has been inferred from a similarity of behavior with beta heterochromatin, i333 it exhibits stickiness, high breakability 'when exposed to x-rays, and the ability to affect euchromatic loci in variable ways. However, the author agrees with Fuscaldo, (1960) who says "the difficulty of characterizing these regions cytologically casts.doubt on the advisability of differentiating intercalary regions as distinct from the euchromatin." Cooper (1959) has reviewed the suggested effects of hetero- chromatin and has separated them into eight categories: 1) the direct action of heterochromatin on the genie material and on gene action. 2) The stabilizing action on kinetochores and chromosome ends, on pairing at meiosis and chiasma localization. 3) Hetero- chromatin is active transchromosomally and in influencing variegation. h) It has metabolic activity in mediating nucleic acid synthesis. 5) It; is active in governing mitosis and 6) in development by regulating growth rates and differentiation. 7) It is suspected of having a role in sex determination and in providing the means for gene dupli- cations which may acquire new functions. Finally, 8) heterochromatin has been charged with being the "seat" of the unorthodox in genetic systems. The most direct experimental evidence for determining the biological role of heterochromatin comes from the laboratory of J. Schultz. Schultz (1956) found that heterochromatin in the form.of the Y chromosome is active in the metabolism of the nucleic acids during the formation of the egg. It changes the base constitution of RNA in the mature egg cyt0plasm but affects neither the total amount of RNA nor the concentration of the nucleoside precursors (Ievenbook, Travaglini and Schultz 1958). The behavior of the Y chromosome is equated by Schultz to all of the heterochromatin. It was postulated by Schultz (1956) that heterochromatin provides the basis of a feedback system in nucleic acid synthesis. In view of present day knowledge concerning the role of RNA in protein synthesis Schultz's work has far reaching significance. It provides a direct bridge connecting the biological function of heterochromatin to protein synthesis. Fox (1959) went a step further. Taking into consideration that genes determine primary structure as demonstrated by Ingram (1956) in the case of the amino acid sequence in the hemoglobins, Fox suggested that heterochromatic regions are concerned with the final stages of protein synthesis during which the tertiary structure of the protein is established. In other words, if particular euchromatic genes are responsible for determining the sequence of amino acids in a protein, 3,3, for specifying primary structure, a possible array of tertiary structures would be provided. Heterochromatin would then Specify the particular tertiary configuration assumed by the protein. Fuscaldo (1960) provided striking confirmation of Fox's hypothesis. WOrking with Drosophila melanogaster she analyzed, immunochemically, the white-variegated position effect to determine the effect of eu- heterochromatic rearrangements on protein structure and specificity. In all cases an alteration of the relationship of heterochromatin to the white locus resulted in a change in the preperties of an antigen, differing in nature from the changes resulting from simple mutation at the white locus. In the present work an investigation was undertaken to study the effect of the Y chromosome upon the antigenic structure of various stocks of Drosophila melanogaster utilizing agar-diffusion techniques. This sensitive method allows one to detect subtle differences in protein structure which would be missed by other procedures. It was heped that an immunochemical analysis of this sort would shed light on the relationship of heterochromatin to protein structure. In addition, perhaps COOper's (1956) suggestion that the differential heterochromatic effects are due to disturbances in the euchromatic- heterochromatic balance could be answered. II. MATERIAIS AND METHODS A. Genetic methods: Coisogenic stocks were derived and maintained according to Fbx (1958, 1959). All stocks were raised on standard corn meal medium, enriched with brewer's yeast, seeded.with living yeast, and incubated at 25°C. Genetic abbreviations used in describing stocks and matings are given in Table l. 1. Oregon R (Series I): isogenic wild stock. This stock was originally isogenized by Dr. J. Schultz and subsequently maintained by Dr. Allen S. Fox for 168 generations by single pair brother-sister matings (Table 2). 2. YA: coisogenic with Oregon R. Females possess an attached X chromosome: the autosomes and Y chromosome are the same as Oregon R (Fbx, 1956). Coisogenicity was maintained with Oregon R by single pair matings for 81 generations according to the breeding system in Table 2. 3. Primary X-Yarm stocks: coisogenic with Oregon R. Oregon R males were mated.with YA females. Exceptional yellow males which arose as the result of crossing-over between the attached X chromosomes and the Y chromosome of YA females were obtained (Stern, 1927, 1929: Anderson, 1925). Eight such exceptional males were isolated (Table 3) and stocks established (YEA,Y2B,Y20 ..... Y2K). These stocks, which contain either the short or the long arm of the Y chromosome attached to the X, were tested to determine which arm was present (Tables h and 5) (Kaufmann, 1933)- h. Secondary X‘Yarm stocks: coisogenic with Oregon R. Ubing.M-5 Oregon R females (coisogenic with Oregon R: Table 6) and primary X-Yarm males (Y2A,Y2B,Y2C.....Y2H), secondary X'Yarm stocks (Y2a,Y2b,Y2c.....Y2h) were derived and maintained as shown in Table 7. These stocks made available females homozygous for the X'Yarm chromosomes present in males of the corresponding primary x.Yarm stocks. 5. YS'X°YL/O: coisogenic with Oregon R. The YS:X'YL chromosome was inserted (Muller, 1936) into the Oregon R background and maintained coisogenic with Oregon R except for the YS°X°YL chromosome. Tests (Stern, 1929) assured the absence of free Y chromosomes in YS°X'YL/O males. 6. YS°X'YL/X: coisogenic with Oregon R. An X chromosome L of the Oregon R females was replaced by the YS'X'Y chromosome of the Y3°X'YL/O stock (Fox, 1956). B. Immunological methods: All stocks were cultured at 25°C. in half-pint milk bottles on standard corn meal medium enriched with brewer‘s yeast. The desired genotypes, which provided antigenic material for these studies, were collected from contaminant-free bottles, starved for 12 hours, quickly frozen (-7o°c.), 1yophilized, and stored at -12°c. To prepare the flies for immunological use, a 21 homogenate (w/v) of each genotype was prepared in an all-glass, Potter-Elvehjem homogenizer using cold buffered saline (0.85% NaCl buffered at pH 7.h with 0.005 Miphosphate). Homogenate aliquots were stored in the deep freeze until needed for immunization. Whole homogenate in quantities of 2 ml., h ml., 8 ml., and 8 ml., respectively, was injected into rabbits intraperitoneally on alternate days. If preliminary bleeding gave evidence of serum with insufficiently high titer (using the precipitin-ring technique), an additional 8 m1. injection was given after another week. The rabbits were exsanguinated from the heart one week after the last dose. Antiserum was separated, l/l0,000 (w/v) merthiolate added, and stored in the deep freeze. Additional merthiolate (1/10,000) was added prior to use. Specific antigen components of the different genotypes were investigated by means of the Ouchterlony agar-diffusion technique (Ouchterlony l9h9: Oudin 1952) with slight modifications (as suggested by Dr. Allen 8. Fox). USing aseptic technique, petri dishes with 8 filter paper strips (1 g” xé?) distributed around the periphery, containing 30 ml. of filtered 2% (w/v) Difco-Bacto agar solution in standard buffered saline (merthiolate 1/10,000 added), were prepared. After solidification, wells were cut in the agar by the use of a template and then each well was sealed at the bottom with one drop of melted agar. The geometrical arrangement of the wells is shown in Figure 1. The clear supernatant obtained from the centrifugation of the whole homogenate (3,200 xg, 30 min., 5°C.) was used as the antigen. Antigen solutions were placed in wells 1, 2 and 3, and antiserum.in well An. Each well holds 0.15 ml. of test solution. The wells of a plate were all filled and refilled simultaneously, and incubated at 25°C. A total of ten refillings was sufficient for development of a definitive pattern of precipitate lines. 9 Inhibiting antigens (BJBrklund, 1952) were prepared by dialyzing the fly supernatants against distilled.water, 1y0philizing the non— dialyzable fraction, and restoring it to one tenth the original volume in standard buffered saline. 0.15 ml. of inhibiting antigen was placed in the An.well and allowed to diffuse into the agar. Two or three dosesn were required to completely inhibit the formation of precipitate lines of common antigenic components in the Ouchterlony test that followed. The last dose of absorbing antigen was administered three days prior to the start of the test. 10 Table 1. List of genetic abbreviations used in tables and text. Abbreviation Full Genotype 79‘— ‘ x X'YL y-YL X‘YB ‘ y-YS Mrs scsi’B InS wa sc8 YS°X'YL YS'X In En B y-YL Cy In(2L)Cy, In(2R)Cy, cn2 Pm In(2LR)Pm,ds33k H H Sb In(3R)Mo,3b sr Table 2. Rating system used in maintaining Oregon R and YA stocks. Oregon 3 YA 1N ‘_ \ I___—‘—‘—““‘-—+ ... x/x and x/ Y X/Y x Y \and X/Y x/x MAI xkhnd x/ Y X/X aZE—I——_‘—X;Y—‘I———I~‘fi—I—_XyY x\\\\\XX;Y\\\3nd X/Y etc. ll Table 3. Derivation and maintenance of primary X'Yarm stocks. XX/Y (mt) ' x X/Y (Oregon R66) xvyarm/y ,3 XX/Y (YA$$) (Single, exceptional yellow males) X'Yam/Y x iii/y (YA $9.) X'Yam/Y x XX/Y (YAS?) etc. Table A. Tests to identify Yarm in primary X'Yarm stocks. Test hating 1 Test Mating 2 X'Yarm/Y x yf:: /sc'YL X°Yarm/Y x yfz: /YSYS#2 X'Yam/sc-YL x x/x (Oregon R) X'Yam/YSYS#2 x x/x (Oregon R) Sterile if X'Yam = X‘YL Fertile if X'Yam = X'YL Fertile if X‘Yam ;_- X'YS Sterile if X°Yam ; X-YS Table 5. Results of tests identifying Yarm in primary X'Yarm stocks. l2 Stock Test Mating 1 Test Mating 2 Identity of Yarm Y2A Sterile Sterile neither X'YS or X-YL Y2B Fertile Sterile X'YS YQC Fertile Sterile X'YS Y2D Fertile Sterile X'YS YQE Fertile Sterile X'YS YQF Sterile Fertile X'YL Y2G Fertile Sterile X'YS Y2K Sterile Fertile X'YL Table 6. Derivation and maintenance of M-S Oregon R stock. W X/X (Ore M-S/ +: Cy/+: Sb/+ M- 5/ + M-S/M-S gon R ) Ne—NQ—Xe— X // M-S/M-S M-5/. M-s/M-s M-S/+ M-S/M-S c—x a—x M—S/Y M-S/Y M~5/Y X/Y (Oregon R) M-S/Y X/Y (Oregon R) M—5/Y: Cy/Pm: Sb/H +/Y: Pm/+: H/+ x X/Y (Oregon R) etc. it 13 Table 7. Derivation and maintenance of secondary X°Yarm stocks. M-S/M-S (M-S Oregon R$J x X'Yarm/Y (Primary x Yarms) and X.Yarm/X.Yarm x X.Yarm/Y arm and xty /X'Yarm x X‘Yarm/Y etc. 1h Figure l. A 12" stainless steel or pure nichrome wire (B & 8 gauge 000), for positioning filter strips, is placed on bottom of dish. Geometry of Ouchterlony agar-diffusion plate: 90-mm. petri dish, filled with 30 ml. of 2 percent agar in 0.85 percent NaCl buffered at pH 7.h with 0.00S.M Phosphate. Dimensions in inches. III. RESULTS OBTAINED A. Sex-specific antigens: Ouchterlony tests of antisera, produced by injection into rabbits of the antigenic preparations from a variety of female genotypes, demon- strate the presence in females of a minimum of two antigenic components ( $4 and $4) which are not present in males. Figures 2 and 3 illus- trate a typical plate demonstrating the basis of this conclusion. Antibodies to these female-specific components have been observed in a number of antisera against female extracts, but some such antisera have exhibited only one of the specific antibodies and some have lacked both (Column 2, Table 8). Antisera produced against male antigenic preparations, on the other hand, have never exhibited female-specific antibodies (Column 2, Table 9). All antigenic extracts from female genotypes have produced the female-specific precipitate lines in Ouchterlony tests performed with apprOpriate antisera (Column 3, Table 8). Antigenic extracts from male genotypes have never exhibited these precipitate lines (Colman 3, Table 9). In a similar fashion, antisera produced against male antigenic preparations demonstrate the presence in males of a minimum of two antigenic components ( 6—1 and 3-2) which are not present in females (Figures h and 5). Column 2 of Table 9 indicates that antibodies to one or both of these male-specific antigens have been found in all anti- Isle sera, while column 2 of Table 8 shows that such antibodies have never been found in anti-female sera. Similarly, column 3 of Table 9 shows that these male-specific antigens have been demonstrated as precipitate lines in Ouchterlony tests of antigenic extracts from a variety of male genotypes, while column 3 of Table 8 shows that none 15 16 of a wide variety of female genotypes have resulted in the formation of these precipitates when tested with appropriate antisera. The distribution of female-specific and male-specific antigens, demonstrated by the presenceof specific antibodies in appropriate antisera. and by the formation of precipitate lines in Ouchterlony tests, has been confirmed by the results of BJOrklund inhibition tests. The example giverl in Figure 6 shows that concentrated extracts of YA males are incapable of inhibiting antibodies to $~l and 3-2, and that these antigens are present in Oregon R females (X/X) and YA females (ii/Y). Column h of Tables 8 and 9 summarizes the results of other tests of this sort. In no instance do the results of the BJOrklund inhibition tests contradict the results discussed above. They further suggest that the antigenic differences between the sexes in Drosgphila are qualitative, i,g, that the antigens 32-1 and $-2 are absolutely absent in males and that 8 -l and 6-2 are similarly absolutely absent in females. This conclusion is strengthened by additional experiments where increase of the con- centration of inhibiting antigen up to lOO-fold has neither removed nor shifted the position of the sex-specific precipitate lines. Tables 10, 11 and 12 show the distribution of the sex specific antigens of various genotypes. It is evident that the presence or absence of these sex specific antigens is a function of the number of X chromosomes and is completely independent of the presence or absence of the Y chromosome. For example, all flies which contain two X chromosomes have 3-1 and 5,3-2 antigenic components and do not have the 3 -l or 8 -2 components even though the Y chromosome may be present (ii/Y). Flies which contain one X chromosome do not possess 3-1 or FIR-2 components but instead exhibit only the 6 -l and 3 -2 antigens even though the Y chromosome may not be present (X/O). 17 Figures 2 and 3 --- Photograph and diagram of Ouchterlony plate develOped with anti-X/X (Oregon R 1??) serum (An well). Antigen wells filled with extracts of genotypes indicated in Figure 3. Lines 3-1 and 2-2 :female specific components. Figures h and 5 --- PhotOgraph and diagram of Ouchterlony plate develOped with anti-X/Y (YA 6’5) serum (An well). Antigen wells filled with extracts of genotypes indicated in Figure 5. Lines 3 -l and 3-2 :male specific components. Line Y-l : antigen exhibiting maternal influence of Y chromosome. 18 Figure 2. Figure 3 . Figure 1+. Figure 5. l9 ‘\ Figure 6. --— Demonstration that the female specific components ( Si-l and S¥-2) are present in females but are absent in males. Inhibiting antigen : X/Y (YA) male extract (An well). Antiserum : anti--X/X serum (An well). Antigens : well #1, X/Y #2, X/x #3. fi/Y Figure 7. --- Demonstration that Y chromosome exerts maternal influence on antigen Y-l. Serum : anti-YAg’o" , same as in Figure 1+. Antigens : Well #1, YA 99 (iii/Y) #2, Oregon R Q?— (X/X) #3, 23/0 3S?(YA cytoplasm) of generation 3 (Table 18) Y-l is represented by the heavy line closest to the antigen wells. Note that it is incomplete opposite well #1, but complete Opposite wells #2 and.#3. Figure 6 . Figure 7 - 2O Table 8. Summary of Results of Ouchterlony and BJBrklund inhibition tests with female genotypes. Antigens inferred Antisera Antigens demon- demonstrated strated as pre— from inhibition Genotype to contain cipitate lines in of corresponding antibodies to Ouchterlony tests antibodies 3-13-2 91-13-22 3-1 3-2 3-1 3-2 3-1 3-2 9-13-2 X/X (Oregon R) - - + + - - + + fi/YMA) - - + + - - + . SOT/O (See Table 18) - - + — — — f X'Y'Y/X’Yz (Y2a) - .. x-YS/X~YS (Y2b) .. _ X°YS/X'YS (Yec) - - X'YS/X-YS (de) - - x-YS/x-YS (Y2e) ' - - X'YL/X-YL (Y2f) - - X-YS/X-YS (Y2g) - - X-YL/X-YL(Y2h) - - - - - - Blank places indicate that apprOpriate tests were not performed. 22 Table 9. Summary of results of Ouchterlony and BjBrklund inhibition tests with male genotypes. Antisera Antigens demon- Antigens inferred Genotype demonstrated strated as pre- from inhibition to contain cipitate lines in of corresponding antibodies to Ouchterlony tests antibodies 8-1 6123-1 &-2 $1 6-2 ¥-l 2-2 3-1 3-2 3:1 3-2 X/Y (oregon R) ‘ - “’ - 0' ‘l’ - - 1- x/Y (YA) + . - .. . . - - , _ _ X/O (See Table 19) + + - - YS-x-YL/O - - - - + . - - - YS°X‘YL/Y (See Table 19) - + - - X°YL/Y (Y2f) + 9 - - 1- + - — r f Blank places indicate that appropriate tests were not performed. Table 10. Distribution of sex-specific antigens in Oregon R and YA stocks. Stock Sex Genotype Sex-specific antigens ’8-1 8-2 3-1 59-2 F X/X - - r * Oregon R M X/Y r + - - F iii/Y - - . . YA M X/Y 1' t ‘ " 23 Table 11. Distribution of sex-specific antigens in various females. Genot e Sex-s ecific anti ens yp Total sex chromosome p 8 constitution 3—1 6-2 ¥~1 3-2 EOE/O XX - - . x-Y7/X°Y? XXY? Y2 - - X'YS/X'YS xxxSyS _ - X‘YL/X'YL XXYLYL - - Blank places indicate absence of appropriate evidence. Table 12. Distribution of sex-specific antigens in various males. fir Sex-specific antigens Genotype Total sex chromosome constitution ’8—1 2-2 3—1 2—2 ){/O X + + - - YS'X'YL/O XY - + - - Y3°X‘YL/Y XYY + . - - x.Y8/Y XYYS + q- - - 2h B. Y-l antigen: Figure h exhibits a well-defined precipitate line,labeled Y-l, opposite antigen wells which contained antigens extracted from Oregon R males and females but not Opposite the well which contained the extract of YA males. This precipitate line has been observed in Ouchterlony tests of the majority of antisera studied, including antisera to both Oregon R males and females and to both YA males and females (Table 13). These antisera demonstrate the existence of an additional antigen, Y-l, not distributed in a sex-specific manner. The example given in Figure A and the data of Table 13 pose an apparent paradox, for while the Y-l antibody is present in antisera to YA males and YA females these antisera fail to form the corres- ponding precipitate line with YA antigenic extracts. The results suggest that the Y-l antigen is present in both the YA and Oregon R stocks, though perhaps in a different form with regard to the prOperty of forming precipitate with the Y-l antibody. In order to confirm this inference BJBrklund inhibition tests were used. When YA inhibiting antigen was used against any antiserum, inhibition of the Yol antibody resulted and the Y-l line was not formed with any of the antigens tested (Table it). This means that Y-l antibody contained in the antiserum must have been absorbed by a corresponding antigen present in the inhibiting antigen. Therefore, it is evident that the Y-l antigen must also be present in the YA stock. Since inhibition antigens are concentrated ten-fold by dialysis and 1y0philization, Fbx (1958) has considered the possibility that the failure of YA extracts to form the Y-l precipitate line in Ouchterlony 25 tests is attributable to reduced concentration of le in the YA stock. He reports, however, that YA extracts fail to produce the Y—l line in Ouchterlony tests even when concentrated ten-fold, thus demonstrating that the effect is not a simple quantitative one. The properties of the Y-l antigen, as exhibited by males and females of the Oregon R and YA stocks, are summarized in Table 15. The antigen is present in all four genotypes, and is capable of inducing antibody formation in the rabbit in all four cases. As far as can be told from the results of the ijrklund inhibition tests, its antigenic specificity is the same in all four genotypes. It differs only in its ability to form precipitate with the corresponding antibody. To describe this difference, the terms "complete antigen" and "incomplete antigen" are used in reference to Y-l in the Oregon R and YA stocks respectively. From.a genetic point of view, it is immediately apparent that the presence or absence of a Y chromosome in an individual has nothing to do with whether the Y-l antigen will be complete or incomplete. Thus, Oregon R and YA males are genetically identical, but the former possesses complete Y-l while the latter possesses incomplete Y-l. Furthermore, there does not seem to be any dosage effect associated with the X chromosomes, since the males and females of each stock possess the same form of the antigen (Table 16). A clue to the explanation of this paradox comes from the recognition that YA flies and Oregon R flies have different mothers. The kind of Y-l found in an individual (male or female) depends on whether its mother was an Oregon R female or a YA female (Table 17). The inheritance of this difference in Y—l is matroclinous. 26 It is evident that a difference between Oregon R and YA females exists. An obvious difference is that YA females contain a Y chromosome while Oregon R females do not. Therefore, with respect to the inheritance of the Y-l antigen there are two possibilities: (l) the presence or absence of a Y chromosome in the oocyte prior to meiosis determines the kind of Y-l produced in the individual after fertilization, and.(2) the difference between them might be one involving cytOplasmic inheritance, i.e., a self-reproducing cytOplasmic system, independent of the Y chromosome, transmitted through the eggs (Sonneborn, l9h3). In order to distinguish between these two possibilities, two types of females were derived, and their progeny were tested for the form of the antigen. Table 18 portrays the derivation of females possessing YA cytoplasm but no Y chromosome. Conversely, Table 19 exhibits the derivation of females possessing Oregon R cytOplasm with a Y chromosome. Antigenic extracts of the progeny of these females (also indicated in Tables 18 and 19) were tested.with antisera known to possess Y-l antibodies. The results of the tests are summarized in Table 20, and an illustrative example is given in Figure 7. As may be seen from Table 18 and the upper part of Table 20, ‘2270 females whose cytOplasm is derived from their YA (ii/Y) mothers nevertheless produce prOgeny which exhibit the complete form of the Y-l antigen. ,It therefore suggests that the YA cytOplasm cannot transmit incomplete Y-l in the absence of the Y chromosome. This observation also demonstrates that the production of incomplete Y-l is not attribut- able to the attached X chromosomes present in YA females. 27 On the other hand, the insertion of a Y chromosome into Oregon R cytOplasm does not result in the production of incomplete Y-l (Table 19, lower part of Table 20). At first sight this would make it appear that the presence of a Y chromosome in a female is not sufficient for the production of incomplete Y-l in her progeny, but that both YA cytOplasm and the Y chromosome are required. It should be remembered, however, that the Y chromosome which has been inserted into Oregon R cytOplasm is not an intact Y, but rather than present in the YS'X'YL chromosome. The possibility must be considered that elements of the intact Y are missing in this chromosome, or that the integral organ- ization of the Y is essential for the maternal effect on the Y-l antigen. This problem was resolved by an examination of the form of the Y-l antigen in females of the secondary X'Yarm stocks. The cyto- plasm present in these stocks was derived through the M-5 Oregon R stock from Oregon R females (see Tables6 and 7). As may be seen in Table 21, the females of the Y2f stock (X'YL/X°YL) exhibit the incomplete form of Y-l. This Observation establishes that neither an intact Y chromosome nor YA cytOplasm are essential for the maternal effect on the Y-l antigen. It therefore appears that the presence of some specified part of the Y chromosome in an oocyte initiates a chain of events which results in the production of incomplete rather than complete Y-l. This chain of events does not depend on the previous history of the oocyte cytOplasm, nor is it interrupted by removal of the Y chromosome during meiosis. Table 13. 28 Presence or absence of precipitate line resulting from Y—l antigen in Oregon R and YA stocks. Antiserum Genotype Tested Y-l Precipitate Line Anti-x/x (Oregon R) X/X (Oregon X/Y (Oregon ii/Y (YA) x/Y (YA) R) R) Anti-x/Y (Oregon R) X/X (Oregon X/Y (Oregon ii/Y (YA) R) R) Anti-FFVY (YA) x/x (Oregon x/Y (Oregon ii/Y (YA) x/Y (YA) R) R) Anti-x/Y (YA) X/X (Oregon X/Y (Oregon ii/Y (YA) x/Y (YA) R) R) 29 Table 1h. Inhibition of Y-l precipitate line by antigenic extracts of Oregon R and YA flies. Antiserum Source of inhibiting Inhibition of Y-l antigen precipitate line Anti-X/X (Oregon) X/X (Oregon R) r EOE/Y (YA) . x/Y (YA) + Anti-x/Y (Oregon R) EOE/Y (YA) . Anti-iiyY (YA) X/X (Oregon R) * fi/Y (YA) . x/Y (YA) + Anti-x/Y (YA) x/x (Oregon R) + fi/Y (YA) . X/Y (Oregon R) r x/Y (YA) + Table 15. Distribution of the Y-l antigen as indicated by various criteria. Anti-Y-l Y-l Y-l Inferences Genotype in identified identified Presence or Farm of antiserum by by absence of Y-l precipitate inhibition Y-l X/X (Oregon R) v + r + Complete X/Y (Oregon R) t t t # Complete TOE/Y (YA) . - + - Incomplete xh(m) + - + o hmmkm 30 Table 16. Form of Y-l antigen in Oregon R and YA flies with respect to sex, X-chromosome dosage, and presence or absence of Y chromosome. Genotype Sex X-chromosome Y chromosome Y-l dosage, (presence or absence antigen x/x (Oregon R) F XX - Complete x/Y (Oregon R) M x + Complete EOE/Y (YA) F xx + Incomplete X/Y (YA) M X r Incomplete Table 17. Relationship of form of Y-l antigen to parental origin. ‘ Genotype _ Stock Mbther Of Father of Y-l antigen ' stock stock X/X Oregon R Oregon R Oregon R Complete X/Y Oregon R Oregoan Oregon R Complete 'ii/Y YA YA Oregon R Incomplete X/Y YA YA Oregon R Incomplete 31 Table 18. Mating system for derivation of females with YA cytoplasm but no Y chromosomes (in box) Ei/Y (YA) x YS-X'YL/O Generation 1 353/0 x YS°X-Y /O Generation 2 ... é”"”””’f”’f”””\\\\\\\\\\:;\$ I. XX/O Y -X'Y /0 Generation 3 Table 19. Mating system for derivation of females with Oregon R cytOplasm and Y chromosomes (in box). X/XWX YS-X-YL/O Generation 1 x/O YS-X'YL/XW x X/Y Generation 2 x/x YS «x-YL/x YS 'X'YL/Y x/Y Generation 3 Table 20. Relationship of form of Y-l antigen to maternal origin. 32 Cytoplasmic Presence or Form of constitution absence of Y-l Genotype Tested of mother Y chromosome antigen in mother TOE/Y (YA) YA Incomplete X/Y (YA) YA Incomplete if/O (Generation 3, Table 18) YA Complete YS-X‘YL/O (Generation 3, Table 18) YA Complete X/X (Oregon R) Oregon R Complete X/Y (Oregon R) Oregon R Complete x/x (Generation 3, Table 19) Oregon R Complete YS°X5YL/X (Generation 3, Table 19) Oregon R Complete YS°X'YL/Y (Generation 3, Table 19) Oregon R Complete X/Y (Generation 3, Table l9) Oregon R Complete 33 opoamfioo mpmaaSoo mpoamsoomH OpoameOo opmagaoo opoaano mpoaaeoo opoagaoo omwfipsm a-» no such mmamaom m».x\q».x m».x\m».x a».x\m».x m».x\m».x m».x\mw.x m».x\m».x mw.x\m».x we xxaw.x ooemoa mo manpoeow mm» mm» em» om» am» om» am» am» mooom .mxuoum shmh.x hasmmoomm mo mmamsmm ma commend a-» mo anon .Hm manna 3h C. Partial characterization of antigens: The presence of the sex-specific antigens and the Y-l antigen in the preparations used for BJOrklund inhibition tests demonstrates that these antigens are non-dialyzable, 1.3. of relatively lalge molecular weight. In addition, a number of proteolytic enzymes (trypsin, chymo- trypsin, protease) have been found to destroy all antigen activity (Fbx and Mead, unpublished). The sex-specific and Y-l antigens there- fore appear to be proteins, and further characterization has been achieved by studies of their solubility in water and dilute salt solution. The antigens were prepared as before except that aliquots were taken from which two fractions were derived. Fraction 1 consists of the antigen supernatant after dialysis against distilled.water. Fraction 2 is the precipitate formed during dialysis, which is then resuspended in an equal volume of phosphate buffered saline (0.85%, pH 7.h). Table 22 depicts the presence or absence of antigenic activity found in each of the three fractions when tested against apprOpriate antisera. It is clear that 8-1 and 8-2 antigens are found in the water soluble fraction and are therefore considered to be albumin. The 5?-l antigen is not found in the water soluble fraction (Fraction 1) but is found in Fraction 2, which indicates that it is a globulin. The Y-l antigen is also presentin Fraction 2, suggesting that it too is a globulin. 35 Table 22. Solubility of sex-specific and Y-l antigens in water and weak salt solution. Source of Antigens present Fraction Fraction 2&1 gf2 2:1 2:2 Y-l x/Y (Oregon R ) l + . - - 2 - - - . X/X (Oregon R) 1 - — - - 2 - - + r Fraction 1: Non-dialyzable, water-soluble fraction. Fraction 2: Non-dialyzable, water-insoluble fraction, resuspended in buffered 0.85% saline. Tests were not made for the presence of 9,2 in the fractions. IV. DISCUSSION On the basis of the immunogenetic analyses performed, it is evident that males possess two specific antigens which are not present in females, and that females have two other specific antigens which are absent in males. The difference between the antigenic constitution of males and females Leannot. be attributed to an autosomal difference since the autosomes are qualitatively and quantitatively the same in both sexes. Therefore, the explanation for the difference of the sex-specific antigens must reside in the sex chromosomes. Two possibilities exist: 1) the antigenic differences are attributable to the presence of the Y chromosome, and 2) the observed differences are attributable to the presence of two X chromosomes in the female and only one X chromosome in the male. The first postulation is eliminated by the fact that X0 males which lack the Y chromosome possess only the male-specific antigens and XXY females which have a Y chromosome exhibit only the female- specific antigens. This demonstrates that the presence or absence of a Y chromosome in an individual does not determine its antigenic compo- sition with reference to the sex-specific antigens. Furthermore, it is to be noted that the male antigens of YA and Oregon R stocks are the same. Thus, it is apparent that the presence of the Y chromosome in the mother does not result in a difference in the antigenic specificity of her prOgeny. 36 37 The second possibility seems to be established. In every instance where an individual has two X chromosomes the female sex-specific antigens are the only ones found, and if only one X chromosome is present the male sex-specific antigens are the only ones that can be demonstrated. It is apparent then that the shift in the number of X chromosomes results in a qualitative shift of antigenic specificity. Another consideration to be taken into account is that the proximal third of an X chromosome is heterochromatic, and it is a possibility that the difference in antigenic specificity resides in this component. That this is not the case is illustrated by the fact that the proximal third of the X chromosome and the short arm of the Y chromosome are homologous (Kaufman 1933; COOper 1959). Thus, an X/Y male would have two of these heterochromatic regions as would an X/X female. Since, as already stated, these flies have different sex antigenic components, the difference can not be assigned to the heterochromatic proximal third of the X chromosome. Further, X/O males, XY/Y males and XX/Y females all show specific sex antigen components consistent with the dosage of the euchromatic portion of the X chromosome. It has been established that both the male and female sex- specific antigens are proteins and the obvious conclusion.would be that the antigenic differences observed are a reflection of differences in protein biosynthesis which is under genetic control. That differences in protein synthesis under genetic control can be detected via changes in antigenic specificity is by no means a new idea. Indeed, Fox and co- workers (Fox 19h9; Fox and White 1953; Chovnick and Fox 1953; Barish and Fox 1956) reported differences in antigenic specificity attributable to 38 single gene differences and have even showed the appearance of new antigens as a result of interaction between non-alleles, between pseudoalleles, and between alleles (FOx, 1958). It appears from the work reported herein that the difference between antigenic specificities in males and females may not be solely a function of the dose of X- chromosomal genes, but also due to a difference in genic balance with respect to the autosomal genes. In other words, the shift in the specificity of sex-specific antigens between the sexes may be the result of a complex interaction between genes located on the X chromosome in the case of males, or two X chromosomes in the case of females, and those on the autosomes. This difference in dosage apparently alters protein syntheses. This idea is supported by the fact that complex interactions between the sex chromosomes and the autosomes have been known for many years in mechanisms of sex determinations (Bridges 1922). Considering the other aspect of the immunogenetic analysis, i,g, the formation of the Y-l antigen, a completely different situation is encountered. 0n the basis of the results it was determined that the Y-l antigen is present in both males and females. However, the form of the antigen is different between stocks even though the speci- ficity appears to be the same, although critical tests remain to be performed. Available evidence that the specificity of the Y—l antigen from any source is the same comes from the demonstration that 1) both forms of the antigen are capable of eliciting the formation of antibody, 2) both antigens are capable of cross reacting with antiserum to either, and 3) the inhibitory power of each antigen is the same against either antiserum. The only difference between the Y-l antigens in the two 39 stocks was that one (from Oregon R) was capable of combining with the antibody and subsequently forming a precipitate, while the other one from YA flies poSsessed only the ability of combination with the antibody without precipitate formation. At first glance it would seem that two possibilities could explain the inability of the Y-l antigen from YA stocks to form a visible precipitate: 1) the antigen found in the YA stock is haptenic, and 2) there is insufficient antigen present to produce a visible precipitate (2:23 this difference is simply one of a quantitative nature). The Y-l antigen in the YA stock induces antibody formation demonstrating that it is not haptenic. Many-fold concentration of the antigen does not confer an ability to form visible precipitate. This clearly illustrates that the inability of the Y—l antigen (from YA) to form a visible precipitate is not attributable to a concentration factor. Precipitate formation depends upon an Optimum concentration ratio between antigen and antibody. This Optimum ratio of concentrations or the equivalence point can be approached two ways. First, and most obvious, is the ratio of antigen molecules to antibody molecules.' Second, the equivalence point can be obtained from the ratio of combining sites between the antigen and antibody molecules. The former consideration, as previously indicated, is eliminated and only the latter possibility remains. That this is the correct interpretation is supported by the following fact: antibody obtained from rabbits immunized with either Oregon R or YA antigens has the identical ability of precipitate for- nation with Oregon R Y-l antigen. This suggests that the number of to combining sites on both antibodies must be the same and that only the number of combining sites on the antigens is different. Thus, antigen obtained from Oregon R has the correct number of sites so that the equivalence point is reached forming a visible precipitate. The antigen obtained from the YA stock has too few combining sites so that the equivalence point is never reached. From the genetic point of view it has already been established that the presence or absence of the Y chromosome in the mother determines what form the antigen will take in an individual. In view of previous reports by other workers this Observation has prime theoretical importance. Caspersson and Schultz (1938) demonstrated that if a Y chromosome is present in the oocyte there is an increase in absorbancy at 2,573 A probably due to RNA. waever, it was shown (Callan 19h8, Schultz 1956) that the total amount of RNA was essentially unaffected. levenbook and coaworkers (1958) showed that the presence of a Y chromosome in an oocyte changed the base ratios of the RNA in the egg. Therefore, it was concluded that the increase in ultraviolet absorption is due to changes in the base ratios of RNA between oocytes that have the Y chromosome and those that do not. The work presented herein indicates that the presence of the Y chromosome in an oocyte alters the structure of a protein. Taken together with previous work, and the strong evidence (Brachet 1957) that RNA is directly involved in protein synthesis, the conclusion that heterochromatin affects protein structure through an RNA intermediate is strongly suggested. As previously noted the specificity of the Y-l antigen seems to be the same in the complete and incomplete forms. Since the ML specificity of the Y-l antigen is not associated with either the number of X chromosomes, or with the presence or absence of the Y chromosome, it must be attributed to some part of the genome that is the same in both stocks, possibly the euchromatin of the X or the autosomes. Heterochromatin and euchromatin therefore appear to have different functions in protein synthesis, each possibly mediated by different RNA'S. V. SUMMARY 1. Immunogenetic analysis of the antigenic effects of the X and Y chromosomes in Drosophila melanogaster was performed utilizing agar- diffusion techniques. 2. Melee possessed two sex-specific antigens which were not present in the females. These antigenic components were designated. 6-l.and. 8-2. 3. Famales possessed two sex-specific antigens which were not present in the males. These antigenic components were designated ‘$-l and 5?-2. h. The differences in antigenic specificity between the sex-specific antigens was found to be independent of the presence or absence of the Y chromosome. 5. The differences in antigenic specificity between males and females were attributed solely to the difference in the number of X chromosomes. 6. Another antigen, Y-l, was found to have the same specificity (regardless of stock or sex) in all stocks, but manifested itself in different forms. 7. The two forms in which the Y-l antigen exist were termed complete Y-l and incomplete Y-l. The complete Y-l was capable of forming a precipitate with antibody while the incomplete was not. This difference was shown to exist between stocks and not between the sexes. 8. The difference between the two forms of the Y-l antigen was attributed to the presence or absence of a Y chromosome in the oocyte of an individual's mother. 9. The difference between the complete form of the Y-l and the incom- plete form are discussed and it is concluded that this difference is A2 1+3 probably a reflection of a difference in the number of combining sites per molecule. 10. The effect of the Y chromosome, which is heterochromatic, contrasts with that of euchromatin. It is suggested that heterochromatin and euchromatin have different roles in protein synthesis. VI. REFERENCES Anderson, E. G., 1925. Crossing-over in a case of attached X chromosomes in Drosophila melanogaster. Genetics, 10: hO3-hl7. Barish, N. and A. S. Fox, 1956. Immunogenetic studies of pseudo- allelism in Drosophila melanOgaster. II. Antigenic effects of the vermilion pseudoalleles. Genetics, hlzh5-57. Brachet, J., 1957. Nucleic acids in heredity and protein synthesis, in "Biochemical Cytology" (J. Brachet, ed.), pp. 226-286. Academic Press, New York. BJBrklund, B., 1952. Specific inhibition of precipitation as an aid in antigen analysis with the gel diffusion method. Proc. Soc. Experm. Biol. & Med., 79:319-32h. Bridges, C. B., 1922. The origin of variations in sexual and sex limited characters. Amer. Nat., 56:51-63. Bridges, C. B., 1939. CytolOgical and genetic basis of sex, in "Sex and interna1.SecretionP (C. B. Bridges), pp. 15-63. Williams & Wilkins, Baltimore. Callan, H. G., l9h8. Ribose nucleic acid in the Drosophila egg. Nature, 161: MO. Caspersson, T. and J. Schultz, 1938. Nucleic acid metabolism of the chromosome in relation to gene reproduction. Nature, 1h2z29h-295. hh #5 Chovnick, A. and A. S. Fox, 1953. Immunogenetic studies of pseudoallelism in Drosophila melanOgaster. I. Antigenic effects of the lozenge pseudoalleles. Proc. Nat. Acad. 301., 39:1035-10h3. Coleman, L. C., l9h3. Chromosome structure in the Acrididae with special reference to the X chromosome. Genetics, 28:2-8. Cooper, K. W., 1959. Cytogenetic analyses of major heterochromatic elements (especially Xh and Y) in Drosophila melanogaster and the theory of "heterochromatin". Chromosoma, 10:535-588. COOper, K. W., 1956. Phenotypic effects of Y chromosome hyper- ploidy in Drosophila melanogaster, and their relation to variegation. Genetics, h1:2h2-26h. Fox. A. 8., l9h9. Immunogenetic studies of Drosophila melanogaster. II. Interaction between the rb and v loci in production of antigens. Genetics, 3h:6h7—66h. Fbx, A. S. and T. B. White, 1953. Immunogenetic studies of Drosophila melanogaster. III. Further evidence of genic interaction in the determination of antigenic specificity. Genetics, 38:152-166. Fox, A. S., 1958. Genetics of tissue specificity. Annals N. Y. Acad. sci., 73:611-63h. FOx, A. S., 1959. Genetic determination of sex-specific antigens. JOurn. Nat. Cancer Inst., 23:1297—1308. Fox, A. 8., J. B. Burnett and M. S. Fuchs, 1959. ElectrOpho- retically separable tyrosinases and their interconversion in Neurospora crassa. Rec. Gen. Soc. Am., 28:70, and Genetics, kh:510 (Abstr.). A6 FOx, A. S., 1959. Problems in genetic control of protein synthesis. Science, 130:1hl7-lh18 (Abstr.). FUScaldo, K. E., 1960. An immunogenetic analysis of white variegated position effects in Drosophila melanogaster. Thesis for Ph. D. Michigan State University. Heitz, B., 1928. Das Heterochromatin der Moose. I. Jahrb. Wiss. Bot., 69:762-818. Heitz, E., 1933. Die somatische HeterOpyknose bein Drosophila melanogaster und ihre genetische Bedeutung. Z. Zellforsch., 20:237-287. Ingram, V. M., 1958. Abnormal human haemoglobins. Biochem. et Biophy. Acta., 28:539. Kaufmann, B P., 1933. Interchange between X and Y chromosomes in attached X females of Drosophila melanogaster. Proc. Nat. Acad. Sci., 19:830-838. Kaufman, B. P., M. R. McDonald and H. Gay, 1951. The distribution and interrelation of nucleic acids in fixed cells as shown by enzymatic hydrolysis. J. Cell Comp. Physiol. 38 (Suppl. l):71-100. Ievan, A., l9h6. Heterochromacy in chromosomes during their contraction phase. Hereditas, 32:hh9-h68. Levenbook, L., E. Travaglini and J. Schultz, 1958. nucleic acids and their components as affected by the Y chromosome of Drosophila melanogaster. 1. Constitution and amount of the ribonucleic acids in the unfertilized egg. Expt'l. Cell. Research, 15:h3-61. h? Muller, H. J. and T. S. Painter, 1932. The differentiation of the sex chromosomes of Drosophila into genetically active and inert regions. Z. indukt. Abstamm. - u. Vererb. Lehre, 62:316-365. Muller, H. J., 1936. Insertion of foreign chromosomes into homozygous host stock. DrOSOphila Inform. Serv., 6:8. Ouchterlony, 0., 19h9. Antigen-antibody reactions in gels. Acta Path. et Microbiol. Scandinav., 26:507-515. Oudin, J., 1952. Specific precipitation in gels and its application to immunological analysis. Mbthods in Medical Research, 5:335-378- Ris, H., 19h5. The structure of meiotic chromosomes in the grass- hOpper and its bearing on the nature of "chromosomes" and "lamp- brush chromosomes". Biol. Bull., 89:2h2-257. Schultz, J., 1956. The relation of the heterochromatic Chromo- some regions to the nucleic acids of the cell. Cold Spring Harb. Symp. Quant. Biol., XXI:307-328. Sonneborn, T. M., l9h3. Gene and cytoplasm. I. The determination and inheritance of the killer character in variety h of Paramaecium aurelia. 'Proc. Nat. Acad. Sci., 29:329-3h3. Stern, C., 1927. Ein genetischer und zytologischer Beweis fur Vererbung im Y Chromosome von DrOSOphila melanogaster. Z. indukt. Abstamm. - u. Vererb. Lehre, hh:187-231. Stern, C., 1929. Untersuchungen fiber Aberrationen des Y Chromosome von Drosophila melanogaster. Z. indukt. Abstamm. - u. Verebungslehre, 51:253-353- Wilson, G. B. and E. R. Boothroyd, 19h1. Studies in differential reactivity. I. The rate and degree of differentiation in somatic chromosomes in Trillium erectum. L. Canad. Jour. Res., C, 19:h00-h12. Wilson, G. B. and E. R. Boothroyd, 19%. Temperature induced differential contraction in the somatic chromosomes of Trillium erectum. L. Canad. JOur. Res., C, 22:105-119. PART II. EFFECTS OF THE Y CHROMOSOME ON SPERMATOZOAL DEVELOPMENT . 1+9 ABSTRACT Sei-Byung Yoon The effects of the Y Chromosome upon the develOpment of spermatozoa in Drosophila melanogaster were observed. These obser- vations were made on both in zigg_and in vitae systems. Techniques were develOped in order to make this study. Developing spermatozoa from the testes of Oregon R wild and X/O males were compared. It was found that spermatozoa in X/O males never reached maturity. On the basis of these Observations, an artificial classification of develOping sperm, utilizing a number of parameters, is suggested. The motility and locomotion of spermatozoa from the sperm mass of seminal vesicles of males and the sperm ball from the genital chamber of impregnated females were studied $2.X£E£2° It was found that a portion of female oviduct or unfertilized eggs would initiate locomotion by attracting the spermatozoa. This locomotion was ob- served only in sperm from the sperm mass. In the course of this investigation, a possible case of an in vitgg fertilization was observed. The effects upon motility of spermatozoa, utilizing immunized and unimmunized globulin fractions from rabbit sera, were studied. 50 TABLE OF CONTENTS Page I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . 53 II. SPERMATOZOA IN THE MALE GENITAL ORGANS ........ . . 56 A. METHODS ..................... . . 56 B. OBSERVATIONS ..... . . . ....... . . . . . . 56 l. TESTIS ............. . . ....... 56 2. SEMINALVESICLE ....... 59 3. ACCESSORY GLAND . . . .............. 63 1:. ANTERIOR EJACUIATORY DIET ...... . . . . . . 63 5. EJACULATORY BULB . . . ..... . ...... . . 63 6. POSTERIOR EJACULATORY DUCT ......... . . . 61: III. SPERMATOZOA IN THE FEMALE GENITAL ORGANS . ........ 66 A.METHODS..... ........ ..........66 ' B e OEHVATIONS e e e e o o e o e e e o o e e o o o o o o 66 1' GMITAL CW 0 e oooooooooooo e o e 66 2. VENTRALSEMINALRECEPTACLE............ 69 3.SFERMATHECA..... ....... .......69 1:.COMMONOVIDUCT ...... ............70 5 IATERAL OVIDUCT .............. . 7O 6. OVARY. ..... . . . ........... . . . 71 IV.SFERMAIOZOAgy_g§_q ...... ...........72 A. SPERM MASS FROM SEMINAL VESICLE OF MALE ....... 72 1. IN DROSOPHILA RINGER . . . . . .......... 72 2. TREATMENT WITH HYALURONIDASE . .......... 76 3.1'REAmENTwITHTRYPSIN......... ..... 77 51 52 Page B. SPERM BALL PROM GENITAL CHAMBER OF FEMAIE 77 1. INDROSOPHIIARINGER............... 77 2. TREATMENTVITR HYALURONIDASE‘. . . . . . . . . .. 78 3. TREA'IMENTWITHTRYPSIN........ ...... 78 c.IOcomTIONOFSPERMATOZOA 79 l.SPERMATOZOAFROMMALES..............79 2. SPERMATOZOAPROMPEMALES............. 8o 3. ATTRACTION OF SPERM BY OVIDUCT FRAGMENTS AND EGGS. 80 1+. POSSIBLEEVITROPERTILIZATION 82 v. FDCPERIMENTAL MODIFICATION OF SPERMATOZOAL mTILITY . .. 83 A. 'IREATMENTWITHDMUNEGLDBULIN............ 83 1.MATERIAISANDMETHODS...............83 B. TREATMENTVITEPLYEIE'RACTS ..... 81+ 1.MATERIAI.SANDMETHODS..............81+ 2.RESULTS........-.-............88 VI. AGGLUTINATION OF SPERMATOZOA BY NORMAL AND IMMUNE _.- ). GIOBULIN........................89 1.MATERIAISANDMETHODS..............89 2.RESULTS.....................89 VII. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 9o A.EEZQOBSERVATIONS.................9O B. ELILRQOBSERVATIONS .. ........ 93 c.ExPERmENTALmnBILIZATION.............9h VIII.SuZMARI...... ...... .............97 IX.REFERENCES........ ..... ..........99 I. INTRODUCTION .Male fertility in Drosophila melanogaster has been shown to depend on the simultaneous presence of two sets of fertility factors on the long and short arms of the Y chromosome respectively (Stern, 1929; Shen, 1932; Kaufmann, 1933; Brosseau, 1960). In the absence of any of these fertility factors males are sterile, although it generally has been believed that they produce morphologically normal, but non-functional, sperm. Shen (1932) reported that male flies lacking a Y chromosome produced sperm which were immotile. He inferred that the loss of motility was due to degeneration of the sperm before maturation. various authors (Shen 1932; Eadorn and Stern 1938) have reported that normal x/Y males produce sperm which are motile in the seminal vesicle and in the testis. Miller (1950) suggested that sperm.motility of the normal male may be induced by a secretion of the accessory gland. Therefore, it is generally accepted that the fertility factors of the Y chromosome have specific effects on the viability and motility of sperm cells, but that the biogenesis of these sperm is independent of the presence or absence of a Y chromosome (Shen 1932; Eadorn and Stern 1938). less of viability and consequent immotility may be the result of external factors. Miller (l9hl) reported that sperm of E, athabasca did not retain its viability in the ventral receptacle of 2, affinis females for as long a period of time as in the 53 51+ receptacle of females of their own species. Further, Oliver (19h2, l9h5) showed that the infertility of females, homozygous for the mutant lozenge-glossy, is due to the loss of motility of spermatozoa that are stored in the ventral receptacle. Therefore, the motility of sperm is apparently a function of a complicated interaction between a sperm cell and its progenitor's genotype as well as an interaction between it and its environment. This environmental effect may be quite subtle; for instance, sperm in the ventral receptacle of a homozyous glossy female are immotile while they are motile in heterozygous females. On the other hand, the environmental effect may be of a more general nature as Shown by the fact that Sperm in the ventral receptacle of a female of a different Species are less viable than in females of the same species. An investigation of the effects of the Y chromosome on sperm motility in an in 33533 system appeared to be a profitable method of approach to the problem of the causes of sperm immotility. This is so because an in 33339 system would allow one to perform a wider variety of physical and chemical manipulations than would be possible in the intact organism. It was hOped that by studying Sperm cells in_vi££g_it would be possible to differentiate effects caused respectively by the genotype, environment, and the inter- action between them. As a background for such studies it was necessary to study the develOpment and activity of sperm ig_vivo. 55 CT\ \J‘ l\) J?’ ‘0" Plate I. Male Reproductive System 1. 2. 3. 1+. 5. 6. 7. 8. of Drosoghila. Distal Portion Basal Gyre II ]Testis Basal Gyre I Seminal Vesicle Accessory Gland Anterior Ejaculatory Duct Ejaculatory Bulb Posterior Ejaculatory Duct Redrawn from Miller, A., 1950. "Biology of DrOSOphila“ (M. Demerec, ed.), p. 508. II. SPERMATOZOA IN THE MALE GENITAL ORGANS The necessity of obtaining living sperm for use in experi- ments described in later sections led to observations of the male and female genital organs and of the condition of the sperm contained therein. Except where indicated, these observations were made on flies of the isogenic Oregon R, Series I, wild stock. A. methods Virgin males were isolated within 8 hours of eclosion and aged on fresh medium. Observations were made on males varying in age from 0 day (day of eclosion) to 30 days. Dissection of the genital organs was performed in 0.01 ml. of DTOSOPhila Ringer solution (Ephrussi and Beadle 1936; Bodenstein 19h6) on a depression slide, aided by the use of a binocular dissecting microsc0pe at 27x magnification. The inset genital organs are readily obtained by holding the wings with one forceps while plunging a second forceps between the third and fourth abdominal segments and pulling the abdomen apart. The desired organs are then transferred to Ringer solution on a second depression slide and Observed at apprOpriate magnifications. B. Observations l. Tastis a. X/Y males Compact sperm bundles, running parallel to the long axis of the testis, fill the upper (distal) portion and basal gyre II 56 57 of the testis of flies of all ages. Glittering lines, representing the individual spermatozoa, are seen within the bundles, and extend continuously along their whole length. The spermatozoa in such extended bundles are never motile. In virgin males less than one day old, sperm bundles are usually not present in the basal gyres of the testis, particularly in the first gyre (region A of Shen, 1932). The basal gyres of h or 5 day old males contain numerous, convoluted sperm bundles. If the testis is dissected and the sperm bundles are placed in DrosoPhila Ringer Solution, depending on the region from which the Sperm bundles are taken, the following observations are made: The extended bundle in the distal portion and basal gyre II, when pulled apart, releases spermatozoa, which then become widely separated (Figure l and Figure 3). Sometimes exceptional structures are observed, which when pulled apart are seen to contain trans- luscent hyaline fluid instead of spermatozoa (Figures2 and h). In other respects, these structures seem to correspond to sperm bundles. The convoluted bundles in basal gyre II, when pulled apart, release spermatozoa which then become widely separated. The sizes of the exceptional structures and of the sperm bundles found in the distal portion and in basal gyre II are indistinguishable. The numbers of separated spermatozoa from.these compact bundles, either extended or convoluted, are also indistinguishable. The convoluted, compact bundle occasionally exhibits slow motility as a whole. Some convoluted bundles, found mainly in the area of the 58 Junction of basal gyre II and basal gyre I, have spermatozoa which are slightly separated from each other. Individual sperm- atozoa in these bundles already exhibit weak motility. In region-A, individual spermatozoa are widely separated and motile. The Sperm- atozoa are convoluted following the same pattern as the bundles. These observations are summarized in Table 1. Table 1. Motility and shape of sperm from )g/ymale Region of Testis Type of sperm bundle and Motility of grouping of spermatozoa spermatozoa Distal portion- Extended bundle. Not motile basal gyre II Compact sperm grouping. Basal gyre II Convoluted bundle. Absent, or Compact Sperm grouping. occasionally present Basal gyre I Convoluted bundle. weak or Separated sperm. vigorous motility. b. X/O males In X/O males, a compact structure, running parallel to the long axis of the testis, fills the upper portion and basal gyre II of the testis of flies of various ages. This structure consists of elongated, large units which are arranged parallel to each other and seem to be unbroken along their whole length. 59 The boundaries of each unit do not appear clear, although a demarcation can be distinguished. The size of the individual units appears the same. Each large unit displays individual spermatozoa-like structures within it, though they are difficult to see. The units themselves never exhibit motility, and are so brittle that a slight touch breaks them into pieces of varying lengths and widths. No individual spermatozoa are ever released from.any of these units. Convolution was never observed in any region of the testes from flies of various ages. These units seem to correspond to the sperm bundles or exceptional structures seen in X/Y males. In X/O males less than one day old, region-A occasionally contains one or two compact sperm bundles. The size of these bundles is the same as that of the large structures found in other regions of the testis. These bundles, when pulled apart, release sperm- atozoa. 'With regard to their diameter, the size of these sperm- atozoa is indistinguishable from that of normal spermatozoa obtained from X/Y males. The sperm bundle or the released spermatozoa never exhibit motility and are never convoluted. These observations are summarized in Table 2. 2. Seminal vesicle The seminal vesicle contains numerous, convoluted sperm bundles which glitter and wiggle in reflected light when placed in Drosophila Ringer solution. The size of the vesicle and the number of sperm.bundles therein increase with time. The rate of Table 2. Motility and Shape of sperm from x/O male. Region of Testis Type of sperm bundle and Motility of grouping of spermatozoa spermatozoa Distal portion- Extended bundle. basal gyre II No spermatozoa. Basal gyre II Extended bundle. No spermatozoa. Basal gyre I Extended bundle. Not motile Compact sperm grouping (found in male testis of less than one day old) increase is fairly slow for the first 3 days of the fly's adult life, but is remarkably accelerated for the next day or two. When both Junctions of the seminal vesicle (testis and testicular duct, vas deferens and ductus ejaculatorious) are cut, sperm bundles do not leak out spontaneously even though the vesicle may appear to be bloated. In order to obtain sperm from the seminal vesicle, an incision must be made. Following such an incision, the sperm flow out and the seminal vesicle collapses. The seminal vesicle seldom exhibits any kind of spontaneous contractile motion. Occasional spasmodic contractile motions were observed in the very enlarged seminal vesicle of old flies. In addition to X/Y flies, the following specially derived males were observed: X/O, X-YS/O, and.X°YL/0. No spermatozoa were 61 Figure l. ---Bund1es of spermatozoa from the distal portion of XZY male testis. Most of these bundles contain spermatozoa. Note extended form of bundles. Magnification 50x. Figure 2. ---Exceptional structures from the distal portion of m males testis. Some of these structures contain hyaline fluid. Magnification 50x. Figure 3. ---An enlargement of a sperm bundle showing individual spermatozoa. The outer membrane of the bundle has been torn allowing the spermatozoa to leak out. Magnification 500x. Figure 1+. ---An enlargement of an exceptional structure. The outer membrane has been torn and instead of spermatozoa leaking out, a hyaline fluid is seen. Magnification 500x. 62 Figure 1 . Figure 2 . Figure 3 . Figure 1+. 63 observable in the seminal vesicles of any of these males. Their seminal vesicles appear to remain infantile throughout their life span. It is evident that if a complete Y chromosome is not present no spermatozoa are discerned. 3. Accessory gland. No spermatozoa were ever observed in this organ. Wave-like contractions on the surface of this gland were observed to proceed toward the ejaculatory duct. When the accessory gland is dissected out, a somewhat cloudy and granular secretion flows out spontaneously at the Junction of the ejaculatory duct. This secretion sinks Slowly to the bottom of a depression slide and is soluble in Drosophila Ringer solution but coagulates in 80% ethyl alcohol. In the presence of this secretion, in Ringer solution, the duration of sperm motility is increased. HOwever, no increase in vibrational motion was observed. h. Anterior ejaculatory duct. Contrary to expectation, no sperm were ever observed in this duct. Regular peristaltic motion in the caudal direction is seen. The fluid formed in this duct is soluble in DrOSOphila Ringer and has no effect on sperm motility. 5. Ejaculatory bulb. Sperm were never observed in this organ. If the ejaculatory bulb is cut from the ejaculatory ducts, material leaks out only" through the posterior portion but not through the anterior portion. 6b. This secretion Sinks rather rapidly to the bottom of a depression slide and is insoluble in Drosophila Ringer. This material is not dispersed by either hyaluronidase or trypsin. It dissolves in ether and is stained bright red by Sudan IV indicating the presence of lipids. When an incision in the body of the bulb is made, the material which leaks out does not dry or harden in air. The secretion from the natural posterior orifice sometimes hardens and forms a solid cylinder. The secretion from this bulb does not affect sperm motility. 6. Posterior ejaculatory duct. No spermatozoa were ever observed in this duct. 65 Plate II. Female Reproductive System of Drosophila. l. Ovary 2. Lateral Oviduct 3. Common Oviduct h. Spermatheca 5. Ventral Seminal Receptacle 6. Genital Chamber Redrawn from Miller. A., 1950. "Biology of Drosophila" (M. Demerec, ed.), p. 520. ...J III. SPERMATOZOA IN THE FEMALE GENITAL ORGANS A. Methods Virgin females, h days old, are allowed to mate with twice the number of virgin males. At various intervals the females are isolated and examined. In order to obtain Sperm from an impregnated fly the following procedure is followed: a single female is transferred into 0.01 ml. of DrOSOphila Ringer on a depression slide. PresSing the abdomen bilaterally with a forceps allows one to grasp the protruded gon0pod with a second forceps, and the intact genital chamber can be removed with a gentle pulling motion. The sperm ball is obtained from the genital chamber by dissection and placed into Ringer solution. The sperm ball is an egg-shaped mass found only in the genital chamber of impregnated females. It is not to be confused with the sperm mass from the seminal vesicles of males. B. Observations l. Genital chamber: a. Maximum Contact time with maleS--10 minutes. The genital chamber appears full and translucent if a sperm ball has been deposited. If there is no sperm ball, the chamber is smaller and clearer. The sperm ball itself appears to be a solid mass composed of distinguishable, convoluted fibrous structures (the spermatozoa). 66 67 It is about half the size of an unexpanded genital chamber. The size of the sperm ball seems to be dependent upon the age of the male, for a sperm ball taken from a female mated with a 5-day old male is approximately 2% times larger than a ball from a female that is mated with a 3-day old male. In addition, the Shape of the ball is different depending upon the age of the male. A sperm ball taken from a female mated with a 3-day old male is approximately 'spherical, while a full Sized ball from a female mated with a 5-day old male appears to be ellipsoidal. The spermatozoa in the sperm ball are embedded in a translucent, mucoid substance. The spermatozoa themselves are more transparent than the embedding material. The sperm ball itself does not appear to be homogeneous. The spermatozoa are more concen- trated at the cephalad region with reference to the female, while the mucoid substance is more prominent toward the caudal direction. A similar mucoid substance, which does not contain spermatozoa and is not continuous with the sperm ball, is believed to be the sperm plug. The convoluted spermatozoa seem to be arranged in a regular pattern. There does not appear to be any movement. b. Maximum contact time with males-~20 minutes. The mucoid substance appears to have become liquefied at the edge of the cephalad region, and the spermatozoa in this region are more widely separated and exhibit motility $2.§2323 The caudal portion remains solid and the spermatozoa are still non-motile. 68 c. Maximum contact time with males-~25-30 minutes. The extent of the liquefied area has increased considerably. In actuality, liquefaction takes place simultaneously in two directions. On the surface, liquefaction starts at the cephalad end and progresses toward the caudal end. Simultaneously, liquefaction starting from the surface proceeds inwardly. In both directions a viscosity gradient exists. At this stage the caudal area is still predominately solid. The spermatozoa are actively motile at the surface of the cephalad region of the sperm ball and non-motile at the caudal end. The mbtility of the sperm seems to be a function of the viscosity of the mucoid substance. d. Maximum.contact time with males-~50 minutes. .Most of the sperm ball is liquefied and the majority of sperm at the surface are motile. In the central portion of the sperm ball, motility is weak or absent. The motility of the spermatozoa at the posterior end is not as vigorous as at the anterior end of the liquefied sperm.ball. At this time an occasional sperm ball devoid of spermatozoa in the central section can be detected. e. Maximum.contact time with males--60-65 minutes. Mbtile spermatozoa are seen at the junction of the genital chamber and the common oviduct. When an incision is made at the surface of the genital chamber and the chamber is pressed laterally, the sperm ball leaks out spontaneously. The sperm ball spreads radially and gradually becomes a round plaque. Centrifugal dispersion 69 and centripetal gathering of the spermatozoa are easily observed due to the slight alternate expansion and contraction of this plaque. However, the diameter of the plaque remains relatively unchanged. The spermatozoa are vigorously motile. f. Maximum Contact time with males-v70 minutes. Spermatozoa are rarely seen in the genital chamber. 2. Ventral seminal receptacle. 'a. Maximum contact time with males--10—2O minutes. No spermatozoa are found in this tube. b. Maximum contact time with males—-25-30 minutes. Motile spermatozoa are observable through this wall of the receptacle. Spermatozoa are more abundant in the distal region than in the proximal region. Individual spermatozoa lie parallel to the long axis of the tube. Occasional locomotion of individual spermatozoa was observed inside the tube. 3. Spermatheca. a. Maximum contact time with maleS~-lO-20 minutes. Spermatozoa are not present at this stage. b. Maximum contact time with males-~25-30 minutes. Convoluted, motile spermatozoa are observable through the tissue and appear to be revolving around the axis of the Spermatheca. This apparent revolution of the motile spermatozoa was not observed in 22252“ The motility of the sperm cells was greater in the sperm- atheca than in any other location. 70 h. Common oviduct. a. Maximum contact time with males--25-50 minutes. Spermatozoa are not yet seen in the common oviduct. b. Maximum Contact time with males--65 minutes. Motile spermatozoa are observable at the junction of the genital chamber and the common oviduct. Motility is not Vigorous. c. Maximum Contact- time with males--7O minutes. Most of the Spermatozoa are now found in this duct. Motility is not vigorous. Spermatozoa appear to be extended and lie parallel to the length of the duct. They are more or less scattered around inside the duct. d. Maximum contact time with males--80 minutes. Some spermatozoa are seen, but they are weakly motile. 5. Lateral oviduct. a. Maximum contact time with’males--65, 70 minutes. No Spermatozoa are observed in either lateral oviduct. b. Maximum contact time with males--80 minutes. Mbtile Spermatozoa are observable at this time in the lateral oviducts. The sperm appear to be extended, lie parallel to the length of the ducts, and are not very Vigorous. c. Maximum contact time with males--later than 80 minutes. At the anterior part of the oviduct a somewhat cloudy, amorphous elongated mass is seen. Near the junction of the ovaries, this mass appears cloudy and mucoid. The spermatozoa seem to be disintegrated. 6. Ovary. Maximum contact time with males--later than 80 minutes. No sperm were distinguishable within the ovary. 71 IV. SPERMATOZOA IN VITRO A. Sperm mass from seminal vesicle of male. Sperm from the seminal vesicle of the male will be referred to as the "Sperm mass" in contradictinction to the sperm ball obtained from the genital chamber of the female. 1. In DrOSOphila Ringer. In order to obtain active motile spermatozoa from the seminal vesicle, the dissection must take place in DrOSOphila Ringer solution. The resultant sperm mass cannot be transferred into fresh Ringer solution through the air because it then becomes non-motile. The sperm mass itself easily sticks to a forceps or glass rod and is very difficult to remove. When a clean incision is made, the sperm mass leaks out exhibiting-a regular arrangement of tightly coiled spermatozoa which seem to be approximately parallel to each other and which may be recognized by the layered appearance of coils. In other words, each coil may be likened to a coin, the arrangement of these coils is then analogous to the arrangement of coins in a roll. The seminal vesicle then may be thought of as containing numerous rolls. The axes of these rolls lie in a position such that they are parallel to the long axis of the seminal vesicle. Each coil (coin by analogy) is made up of numerous spermatozoa tightly coiled upon themselves in a circle of approximately constant radius. Each coil wiggles giving the impression that it is vigorously revolving around its own axis in Situ. The sequence of events is as follows: the leaking 72 ‘.—a- .1. I u 73 mass from the seminal vesicle is made up of numerous tightly coiled spermatozoa arranged one to another as coins in a coin roll. The individual visible coils at this point separate from one another. By analogy this process might be visualized as coins in a coin roll separating from each other. The individual coils (coins by analogy) then begin to swell radially. It is now possible to see that each coil is really composed of many individual coils. These component coils may be termed sub-coils, and each sub-coil is in reality a single spermatozoon. At first, each spermatozoon is tightly coiled. HOwever, this coiling eventually becomes looser. When the empty seminal vesicle is removed, the sperm mass becomes spherical. As time passes, the diameter of the sperm mass enlarges assuming a disc-like Shape. This enlargement seems to be due to the dispersion of the coils and the concommitant separation of these coils into sub-coils (individual spermatozoa). There appears to be a more general dispersion at the periphery of the sperm mass than in the central area. The diameter of each sub-coil (spermatozoon) when first separated is approximately 0.07 mm. and enlarges to a maximum of approximately 0.09 mm. Each spermatozoon assumes a helical con- figuration. Eventually, the diameters of such helical configurations are approximately equal. The lengths of the helices vary Quite markedly. Some helices are made up of 8 gyres, while others consist of h or even 2 gyres. A spermatozoon which consists of 8 gyres corresponds to observations made by Cooper (1950). 7h It is not known whether or not the 2 and h gyre units represent a complete spermatozoon. AS previously mentioned, when the sperm mass is placed in Drosophila Ringer solution, the individual spermatozoa exhibit motility which is recognizable by a wiggling motion. This motion is detectable by a glittering of light points. If a source of illumination is set up such that the light waves strike the sperm- atozoa at an angle slightly above the horizontal, a glittering light point can be seen originating at one end of a spermatozoon and passing along the gyres of the helix. Eight light points originate at one end every second. Thus, the motility of these spermatozoa is assumed to be of a wave-like nature. In any one spermatozoon the direction of movement of light points along its length was always the same. Moreover, the frequency of this wave-like movement was the same for all spermatozoa observed. This frequency, which may be described as eight new light points per second, is the maximum motility obtained and is only exhibited by spermatozoa existing in a relatively tightly coiled helix. If a tightly coiled spermatozoon is observed in a position with the long axis of its helix parallel to the plane of the glass slide, an interesting phenomenon is detected. By focusing the microscOpe at the upper halves of the gyres, the passage of a light point can be followed in detail from half gyre to half gyre. The light point will travel in the same direction over the half gyres of spermatozoa which are oriented in the same direction relative to 75 the external source of light and the observer. In other words, when looking at a spermatozoon whose long axis, relative to the observer, lies in a north-south direction, and if the light point originates at the south end, the direction of this light point over the upper half of the gyres will be in a right-left direction. USing the model as described in the previous sentences, it was observed that an occasional spermatozoon exhibited a mixed pattern in which some gyres seem to be arranged in a left-right direction while most were orientated right-left. However as these tightly coiled helices loosen, all the unusual spermatozoa observed, exhibited only the right-left direction with reference to the model already described. Physically the mixed direction exhibited by some spermatozoa can be explained if one assumes there is a kink in the helix which reverses its direction. As the gyres of the helix loosen the kink is eliminated and the gyres of the helix will now exist in only one direction, which seems to be normal. AS described previously, when a freshly isolated sperm- atozoon is observed in DrOSOphila Ringer solution, it exists as a tightly coiled helix exhibiting a rapid wave-like motion. The helix subsequently loosens and simultaneously the frequency ‘ of this wave-like motion decreases. The periodicity of this motion eventually slows down sufficiently for the undulating motion along the length of the spermatozoon to be directly discernable. The round gyres making up the helix assume an angular shape 76 corresponding to a hexagonal- or heptagonal-like figure. At this stage the smooth wave-like motion ceases and is replaced by a spasmodic action. This motion emanates from the apex of the polygon, seemingly depressing the two upper adjacent sides and quickly snapping them back into their original position. This spasmodic motion is not uniform over the entire length of an individual spermatozoon, but rather is inter- mittent. . After this, motility in most of the spermatozoa completely ceases. Hewever some spermatozoa are seen to exhibit a Slight swelling at one vertex which passes along the side of the angled structure to an adjacent vertex. This type of movement is the last seen before the complete cessation of motility. An important difference exists between the sperm mass taken from males and the sperm ball taken from females. The spermatozoa in a sperm ball, as observed ig‘vitrg, exhibit centripetal gathering as well as centrifugal dispersion during their period of motility. The spermatozoa which make up the sperm mass are continuously dispersed during their motile period ig_gitgg and never tend to aggregate with one another. 2. Treatment with hyaluronidase. Dissection of the sperm masses was carried out in Drosophila Ringer solution containing concentrations of the enzyme hyaluronidase ranging from 2.5 TRU (Dorfman, 1955) per ml. to 100 TRU per ml. no differences of any kind were observed in the sperm masses when subjected to concentrations of the enzyme of less than 6h TRU per ml. Bbwever, when the enzyme concentration was 6h or more TRU per ml. the spermatozoa dispersed more quickly than they did in the DrOSOphila 77 Ringer solution. No other difference was detectable concerning the general appearance of the sperm mass and individual Spermatozoa. Furthermore, the mode, vigor, and duration of motility of individual spermatozoa was indistinguishable from that observed in DrOSOphila Ringer solution. After the cessation of motility the sperm cells did not disintegrate. 3. Treatment with trypsin. The effect of purified trypsin (Worthington Biochem. Corp.) upon the sperm mass was tested in concentrations ranging from 0.0065$ to 5% (w/v) in Drosophila Ringer solution. The trypsin did not affect the sperm mass in any way until it was present in a concentration of 0.15. At this concentration of trypsin, and above, the spermatozoa dispersed more quickly and the degree of dispersion was greater than previously observed in Ringer solution. In addition, the mass was less cloudy and slightly less sticky. The motility of the individual spermatozoa lasted longer, though the mode and vigor of the motility was the same as in Ringer. After motility ceased, the sperm cells did not disintegrate. B. Sperm ball from genital chamber of female. 1. In Drosophila Ringer. Sperm balls were obtained from the genital chamber of females in Drosophila Ringer solution. The females used varied in their contact time with males from a minimum of 10 minutes to a maximum of 65 minutes. None of these sperm balls was observed to liquefy any further than the stage it had already reached when first obtained, even after 3 hours at room.temperature. Consequently, most further observations were made 78 upon sperm.balls obtained from females whose contact time with males was 65 minutes. After the removal of the empty genital chamber, the diameter of the sperm ball did not change, regardless of whether or not the sperm cells were motile or immotile. No dispersion of cells was observable even.with physical agitation. The Cloudy appearance of the intercellular substance did not change over a 3 hour period at room temperature nor was disintegration of any kind observable. The liquefied portion of the . I Ham-1' I sperm ball stuck easily to a forceps or glass rod. 2. Treatment with hyaluronidase. [7 Treatment of sperm balls, derived from females whose contact time with males varied, with hyaluronidase in excess of 6% TRU'per ml. gave the following results. The sperm became less cloudy than in DrOSOphila Ringer. A slight dispersion of spermatozoa was Observable but was confined only to that area which has been previously liquefied in the genital chamber. The sperm ball seemed to be less sticky after treatment with this enzyme. The solid part of the ball appeared unchanged. In most cases the diameter of the sperm ball Showed no change during its contact with enzyme, whether or not the sperm'were motile. Motile sperm cells still exhibited the phenomenon of centri- fugal dispersion and centripetal gathering. There was no observable difference of any kind.with respect to sperm motility as compared to the activity in Drosophila Ringer. 3. Treatment with trypsin. No apparent change of the sperm ball was detectable when the concentration of the trypsin was less than 2.5%. When the enzyme 79 concentration reached 2.5% or more, the liquefied portion of sperm ball became less sticky and the spermatozoa in this area were dispersed more. The solid part of the sperm ball was not affected. when sperm balls, which came from females whose contact time with males was 65 minutes, were treated with trypsin (2.5% or more) their diameter increased. The centripetal gathering of sperm cells was not observable. The motility of the spermatozoa seemed to last slightly longer in the presence of the enzyme than in its absence. No other observable differences with regard to the Sperm cells themselves were apparent. C. Locomotion of spermatozoa. l. Spermatozoa from males. Locomotion as used herein is defined as the movement of a spermatozoon from one location to another. This kind of movement is quite different from motility which describes a motion in a fixed location. Since a spermatozoon which is immotile is incapable of locomotion, a relationship between the two kinds of movement is probable. HOwever, it must be pointed out that a motile spermatozoon does not necessarily exhibit locomotion.‘ .- When Sperm cells are observed in DrOSOphila Ringer solution they seem to disperse as previously described. Since the sperm cells in the sperm mass are embedded in a sticky, viscous material it is difficult to ascertain whether true locomotion is occuring, or whether the spermatozoa are simply being moved passively as the mass spreads out. However, it is believed that the dispersion of the spermatozoa is passive because their axes exhibit completely random orientation. 80 2. Spermatozoa from females. It is very difficult to determine whether or not the centripetal gathering and centrifugal dispersion of spermatozoa in sperm balls involves true locomotion. However, the location of each spermatozoon is constantly changing and its pathcway is regular, unlike Brownian movement. Furthermore, this change of location was exhibited only by motile sperm. A fresh piece of virgin female oviduct or fresh non-fertilized eggs placed in the vicinity of a sperm ball suspended in DrosOphila Ringer solution never attracted individual spermatozoa. 3. Attraction of sperm by oviduct fragments and eggs. When a fresh piece of oviduct was placed near a sperm mass few of the spermatozoa were attracted to it. Fresh, non-fertilized eggs obtained from.the oviducts of virgin females, had the same effect. The number of spermatozoa attracted in‘both cases was low, but the incidence was higher with the oviduct. Moreover the oviduct seemed to exert its attraction over a greater distance than the egg. Attraction of spermatozoa to both of these structures did not occur in every test. In both cases the movement of the spermatozoa is believed to be true locomotion. The axes of the individual sperm‘were orientated in the same direction.while the movement proceeded. The vigor of motility of locomoting spermatozoa did not change as they approached the oviduct or the egg. Each spermatozoon followed the shortest route to either of the structures. The maximum.distance over which a spermatozoon may be attracted to an egg is estimated at 0.02 mm. It takes approximately 30 seconds 81 for the sperm cells to travel this distance. More spermatozoa are attracted to the ends of the egg than to its middle. As a spermatozoon moves toward the egg the axis of its helical structure is orientated perpendicular to the surface of the egg. At the moment of contact between a spermatozoon and the surface of the egg, a sudden increase in the vigor of the spermatozoon is discernible. Immediately after contact, the spermatozoon recoils from the wall of the 31 egg and resumes its previous rate of motility. It may then approach the egg again, making contact at a different location, and exhibiting increased motility once again. Most spermatozoa approach an egg repeatedly in this fashion. The attraction of spermatozoa by oviduct fragments and un- fertilized eggs was enhanced by extracts of male flies. These extracts were prepared by homogenizing ly0philized males of the YA stock in DrosOphila Ringer solution (2%‘by dry weight of flies). The homogenate was centrifuged at 3500 xg for 30 minutes, and the supernatant dialyzed against water. The non-dialyzable fraction was lyophilized. Immediately before each experiment this lyophilized fraction was restored to its original volume with Drosophila Ringer solution, centrifuged at 3500 xg for 30 minutes, and the supernate used in the experiment. Sperm.masses were obtained by dissection of seminal vesicles in the extract, and the spermatozoa were dispersed by physical agitation. When unfertilized eggs or oviduct fragments were placed in such suspensions, spermatozoa were attracted in larger numbers and over greater distances than in Drosophila Ringer solution. Oviduct still exhibited 82 greater attraction than eggs. The vigor of sperm motility was not changed, but the duration of motility was markedly increased. h. Possible in 13332 fertilization. In one experiment of the sort described in the preceeding section, an instance of actual fertilization was observed. The opportunity to repeat this observation has not presented itself, but since no such case has been reported in the literature the details are recorded here. An unfertilized egg, obtained from the oviduct of a virgin Oregon R female, was placed in a suspension of sperm prepared in above male extracts in DrOSOphila Ringer solution from the seminal vesicle of an Oregon R male. The egg attracted a few sperm to its surface, as described above. One spermatozoon was observed to contact the anterior portion of the egg in the region of the microPyle. Instead of recoiling, as did the other spermatozoa, it remained attached to the egg where it exhibited unusually vigorous and prolonged motility. This motility ceased suddenly, rather than gradually as described in section IV, 1, and the spermatozoon was never released from the surface of the egg. The egg was transferred to an individual vial of DrOSOphila medium. A single larva and, subsequently, a single pups were observed in the vial at times apprOpriate to the develOpmental rate at the temperature of incubation (25°C). A normal male emerged from the pupal case, indistinguishable phenotypically from males of the Oregon R stock. V. EXPERIMENTAL MODIFICATION OF SPERMATOZOAL MOTILITY A. Treatment with immune globulin 1. Materials and methods. Antisera were obtained by the immunization of rabbits with extracts of X/X (Oregon R females), X/Y (YA males), and.ii/Y (YA female) flies. These antisera, in point of fact, were the same as those used in experiments described in Part I of this dissertation, and details are given there. Globulin fractions from thesesera were prepared by the following technique: to the sera an equal volume of a saturated ammonium sulfate solution was added. The precipitate was collected by centrifugation (3,200 xg, 30 minutes), resuspended in a 50% saturated ammonium sulfate solution, and recentrifuged. This procedure was repeated twice. The precipitate was then resuspended in a 0.85% saline solution and dialysed against distilled water for 21+ hours in the cold (5°C). The dialysate was quick-frozen and lyophilized. The lyOphilized globulin fraction was solubilized in Drosophila Ringer solution at a concentration of h$ (w/v). Sperm masses were prepared from ,X/Y (YA) virgin males of various ages. A seminal vesicle was placed on a depression slide con- taining the globulin solution. The sperm mass was removed from the vesicle and the motility of the spermatozoa at the periphery of the mass was observed. 83 8h 2. Results. The time recorded for immobilization of spermatozoa was that time at which half of the sperm in the peripheral area of the sperm mass were immotile. In no case was immobilization a consequence of agglutination. Table 3 and Figure 5 summarize the observations. It is obvious from the data that anti X/Y and anti fi/Y sera prolong motility as compared to normal sera (unimmunized) and anti X/X sera in all age groups. Furthermore, it is evident that the age of the fly from which the sperm mass has been taken, in some cases, has a profound effect upon the length of motility. The prolongation of motility constituted an unexpected result and will be discussed below. B. Treatment with fly extracts l. Hhterials and methods Extracts of X/Y (YA male) flies were prepared by the procedure described in Section IV, C, 3. Dialysis was performed by immersing a bag containing 5 ml. of extract in 30 ml. of distilled water (constant agitation, at hrs., 5°C). During the 2h hour period, four 30 ml. volumes of distilled water were used. At the end of this period the contents of the bag constituted the non-dialyzable fraction and the four 30 ml. volumes of surrounding fluid constituted the dialyzable fraction. Whole extract, as well as these two fractions, was used in the following experiments. Sperm masses were collected from.the seminal vesicles of six- day old virgin males (YA) and suspended in the apprOpriate fractions. The duration of motility was recorded as that time at which half of the sperm in the peripheral area of the sperm mass were immotile. Table 3. Time required for immobilization by immune globulin of spermatozoa from sperm mass. 85 Age Time4(seconds) required for 50% immobilization gile Anti-x/Y Anti-iii: Anti-x/x Normal Control * (days) v SD N v SD N v SD N v SD N v SD N 1 2178 35h 6 21.0 2.6 6 1h.5 2.h 6 2 1h28 11k 6 1776 8h 6 10.3 0.8 6 3 1650 96 6 1500 11h 7 18.6 2.7 6 10.0 1.3 6 38h6 38h 6 h 1890 186 6 1728 210 6 29.8 0.8 6 17.8 0.8 6 5 1986 102 6 2226 20k 6 12.1 0.9 6 16.3 1.0 6 6‘ . 26h6 26k 6 22.5 2.9 7 17.3 1.6 6 3750 38A 6 7 3096 156 9 3030 60 6 no.8 h.7 6 23.3 2.8 6 8 19.5 2.3 6 h.6 0.6 6 9 3528 378 7 3300 216 8 5h.8 2.8 9 52.8 1.5 8 5670 396 6 10 332k 3A8 5 3582 168 6 35.3 h.7 6 25.0 1.3 6 . 11 3876 29k 6 73966 276 6 28.8 5.h 6 30.8 h.7 6 12 A506 210 6 3978 378 6 19.6 3.3 6 17.6 2.5 6 35h6 h26 6 13 h518 huh 6 5136 258 6 31.6 3.3 6 25.0 2.7 7 1h #698 20h 6 716A A32 7 hh.1 3.h 6 25.0. 2.2 6 15 2388 96 6 h776 96 6 3h.1 3.0 6 27.5 2.1 6 3h86 306 6 V : Arithmetic mean SD : Standard deviation N : No. of experiments *Sperm suspended in Drosophila Ringer solution ill llllllallll 'ail‘l’li I In! l.|‘a I) I‘ll; TIME REQUIRED FOR 50% IMMOBILIZATION (SECONDS) I04 :03 I0' '00i l " ”’3 I \ ’./ 2’" r:- x CONTROL (DROSOPHILA amass) ... O ANTI X/Y GLOBULIN _ o ANTI x—X/Y GLOBULIN \ ‘.\ A ANTI xxx GLOBULIN b A NORMAL GLOBULIN P l 1 l J L l l 1 I l l l J 3 4 5 6 7 8 9|O ll I2 I3 I4 l5 AGE OF MALES (DAYS) FIGURE 5. VIABILITY OF SPERM MASS TREATED WITH VARIOUS GLOBULINS Table A. Determination of time needed for immobilization of spermatozoa from sperm masses in the presence of fly extract fractions. Fraction tested Time (minutes) required for 50% immobilization V SD N Whole extract 0.6 0.068 6 Dialyzable fraction 0.33 O.h6 6 Non-dialyzable fraction 167.6 2h.5 6 V = Arithmetic mean SD : Standard deviation N : No. of eXperiments 88 2. Results Table A reveals that the non-dialysable fraction supports motility for a much longer period of time than either of the other two fractions tested. VI. AGGLUTINATION 0F SPERMATOZOA BY NORMAL AND IMMUNE GLOBULIN. 1. Materials and methods. Spermatozoa from 2h-day old virgin males (YA) were suspended in Drosophila Ringer solution and physically agitated to separate individual spermatozoa. Globulin fractions (obtained as described in Section v, A) from normal, anti X/Y, anti x/x and anti ii/Y sera were added, individually in equal volumes, to these suspensions and incubated at 37.5 00 for 20 minutes. After this incubation the spermatozoa were observed. 2. Results Table 5 shows that all the g10bulins tested were capable of agglutinating the sperm cells. The effect, therefore, cannot be attributed to specific antibodies. Table 5. Agglutination of spermatozoa by normal and immune globulin. Globulin Presence (+) or absence (-) tested of agglutination Anti XXY + (YA) Anti XX 9 (Oregon R) Anti XY (YA ) a- Normal s Drosophila Ringer - 89 VII . DISCUSS ION A. In 1122 Observations Spermiogensis has been described in detail by Cooper (1950) and Miller (1950). They both reported that the spermatids and sperm- atozoa occur in compact bundles in the sperm tube. These observations have been confirmed in the present study. huller (1950) reports that the sperm are not vibratile in the I' testis and seminal vesicle of normal males. This is in opposition to the work reported by Shen (1932), and Hadorn and Stern (1938) who state g I that the sperm are motile in the seminal vesicle and in the testis. Both of these opposing viewpoints can be reconciled by the present study. It was found in normal males that the extended sperm bundles present in the regions of the distal portion and basal gyre II of the testis are not motile, while the convoluted sperm in the testis are motile. A unique observation in this work is that the sperm bundle has an outer membrane, and that there are structures resembling sperm bundles which contain no sperm. One possible interpretation to account for this membrane is that it is the membrane of a single, giant cell.within which a whole group of 20 to 100 sperm can form. This leads to the position that a single spermatid gives rise to many sperm cells. However, in view of COOper's (1950) detailed observations of all the stages of the trans- formation of the spermatid into a spermatozoon, this interpretation seems unlikely. Another view, more consistent with the available literature, is that the membrane is laid down around each cluster of sperm cells by an excretory activity of the "nutritive cells" or perhaps the cells of 90 91 the testis sheath. The structures which contain only hyaline fluid and not sperm may be sperm bundles in which the cells have degenerated. Their absence in gyre I may be attributable to resorption in the apical portion of the testis and gyre II. The observations on X/O males are consistent with this interpretation. The presence of a few sperm bundles in region A of newly emerged X/O males, suggests that some sperm are formed prior to eclosion, but that these are abnormal (non-motile and never convoluted). ft The presence of structures containing hyaline fluid in the upper portion of the testis and gyre II may be attributable to degeneration of the sperm I’ which they formerly contained. Indeed, in some of these structures, I sperm exhibiting unclear demarcation can be seen, this may represent an intermediate stage in the degenerative process. Recently Brosseau (1960) has investigated the genital tract of X/O males. His work supported the fact that meiosis seems to be completely normal as do the early stages of spermiOgenesis. However, he reports that the vasa efferentia were usually devoid of sperm. Brosseau feels the breakdown occurs at the time of sperm elongation and matur- ation. These observations have been confirmed in the present study. In view of the findings made herein on both X/Y and X/O males, it seems advantageous to suggest a classification for developing spermatozoa using a number of parameters. This classification is presented in Table 6, and schematically in Figure 6. Males not possessing a complete Y chromosome are only able to reach Stage 1 of spermiogenesis. 11 r- .. Anon» .. 03.50.3800 3.3..“ 00000pr HH 0.03 Homes... -9333 m... on 830.3 03 sued Hanna 8 macaw e m - anodes: seed smooths H on“ Hanna. 3 c w w r w 03588.00 madman scanned Hope:— 3 003m 3 f 8 .m .m o m u madman HH 0% H0000... I . . .m 0009300 Enema 000003 003.30 H330 a. 0m3m r“ H003 00000 m n 3635 . w 0 90.09.50 shone 0930>noo HH 0&8 Human m 0m3m m x003 000.33 .m a 00.002 an Enema 3.3.. 9500 HH 0.63 H0009 .... 3333 -H ER Ream m omaam f 0 0330: met 30.3w? 030.3000 w a .3035 50mm 339600 HH 0.03 demon : owepm 8 m abandon P . e 30.335 03883 30305 .m a .2033 500m 003 2500 Head-Em a. swepm .m 6030985090 0.39.85 1! . agape t 003085000 6.30 05 0.300390 0030.50 003003000 and“! 35:85 no 0:308 fine .62: «mean no 0096 0030004 no 093m 69G 03¢ .500 show 0H! «0 0033008330 00 030330.30 » 0n» «0 000.30 05 00.0 £33850 N 0039000 0 5.; 009300000 0003.8509— uo 0000090 .8 03000.3 05 om Sung 93 Figure 6. Schematic Representation of Developing Sperm. /,Stage 2 ———-+ Stage 3 44% Stage h —>mature sperm Stage l\‘Stage 1d ———————~> Stage 2d 0; resorption \ l Degeneration B. I_n_ vim observations. 1. Non-experimental observations. It has been observed that motility of spermatozoa in_!it£g is a function of the compactness of the helical structure of the sperm- atozoa. As the gyres of the helix of a spermatozoon separate,there is a simultaneous decrease in motility. This loss of motility is a gradual process directly prOportional to the compactness of the individual gyres. In order to study locomotion of spermatozoa in vitgg, the spenn cells must be separated from the rest of the sperm mass. Of the various attempts made to obtain this end chemically, none were successful. The separation of individual spermatozoa occurred only when the sperm mass was physically agitated. 2. Experimental observations. The apparent fertilization of an egg cell by a spermatozoon 1n_vit£2_has been cited. This observation offers vast possibilities as a tool for further studies. Deoxyribonucleic acid (DNA) from Drosophila melanogaster has been isolated and characterized by Mead (1960). With this in mind, the possibility of transformation experiments in Drosophila are feasible. As suggested by Dr. Allen 8. Fox, perhaps by carrying out 9h in vitro fertilization in the presence of pure Drosophila DNA, the DNA might be carried in the fertilized egg effecting an observable transfor- mation. Up to this time transformation experiments have been carried out only with bacteria. Drosophila is a metazoan whose genetic con- stitution has been studied extensively. Hence, if transformation experiments could be effected in Drosophila the Opportunity would be afforded for correlating the chemistry of the hereditary material with its morphological manifestations. C. Experimental immobilization Henle (1938) found that anti-bull sperm serum agglutinated bull sperm and completely inhibited their motility. These experiments were repeated by Tyler (l9h6) using sea-urchin sperm. His results are compatible with those of Henle. Extensive work on the immobilization of Paramecium by homolOgous antibody has been carried out by Beale, Sonneborn et al. (195%). Their work also supports the previous obser- vations that homologous antibody causes the cessation of motility. Generally, the cessation of motility in the presence of homologous antibody has been attributed to the reaction of the surface antigens of the organism with its antibody. This union of antigen and antibody is believed to physically hinder motility. When the motility of DrOSOphila spermatozoa from the seminal vesicle of the male are studied in the presence of various immune globulins, the results are quite unexpected. The duration of motility appear- ed essentially unaffected in the presence of homolOgous antibody (Anti-X/Y). 9S Mbreover, a similar result is also observed with anti-EE/Y globulin. When the sperm are tested in the presence of anti-X/X globulin and/or un- immunized rabbit globulin, the duration of motility is much shorter. In view of these results, immobilization of Drosophila sperm in sera must be attributable to causes other than those involved in a specific antigen- antibody interaction. That other factors might be involved has been demonstrated by Iorenz and Tyler (1951). These workers showed that trace concentrations of certain metals shorten the duration of sea-urchin sperm motility. This adverse effect of the metals can be overcome by the addition of protein, which is believed to chelate them (Tyler and Rothschild 1951). lorenz and Tyler point out that many different kinds of proteins are capable of functioning in this capacity and that their effect of overcoming the adverse effect of metal on the sperm motility is non-specific. A possible interpretation of the results obtained herein must take into consideration several factors. The restoration of the duration of motility, compared to the control, takes place only when the Y chromosome is present in the immunizing flies regardless of their sex. This fact suggests that this effect on the duration of motility is not non-specific. Since motility is greatly shortened in the presence of normal globulin as well as anti-X/X globulin, this, on the contrary, suggests a non—specific effect. In other words, something seems to be present in rabbit globulin which is capable of suppressing the duration of motility. When the globulin from.anti-X/Y or anti-EXVY sera is used, this suppression is overcome. Therefore, it might be assumed that a 96 specific antigen-antibody reaction attributable to antibody to'a Y-chromo- somal product, occurs at the surface of a sperm cell and prevents the endogenous incorporation of a "motility inhibitor". As Tyler (l9h6) has shown using sea-urchin sperm, if the antibody is univalent, the duration of motility is unaffected even though it has reacted with the surface antigens. This work suggests that the specific antibody elicited by the presence of the Y chromosome might also be univalent. VIII. SUMMARY 1. Mbthods are described for observing the development of spermatozoa in the male genital organs. 2. It was found that the presence of a complete Y chromosome was necessary for the formation of motile mature sperm. Males that are X/O, X/YL, and X/YS fail to produce mature sperm. In X/O flies spermiogenesis is arrested at a stage designated "Stage 1". In X/Y males, motility is exhibited in the testis and seminal vesicle. 3. Methods are described for studying spermatozoa deposited in the k female genital organs utilizing time-scaled observations. h. The sperm mass from the seminal vesicle of males was studied in 23352 with respect to motility and locomotion in DrOSOphila Ringer solution and in the presence of hyaluronidase and trypsin. 5. Locomotion of spermatozoa from the sperm mass was initiated only in the presence of a portion of female oviduct or unfertilized egg.‘ 6. A possible case of an in 31332 fertilization is described and its ramifications discussed. 7. The sperm ball obtained from the genital chamber of impregnated females was also studied in Drosophila Ringer solution with respect to motility and locomotion. The effect of hyaluronidase and trypsin upon the sperm ball.was observed. 8. Sperm cells from normal X/Y males were treated with the globulin fractions of anti-X/Y, anti-fin, anti-X/X and non-immunized sera. 97 98 9. A possible interpretation of the results in the immobilization experiments is discussed. 10. Male fly extracts were prepared and the motility of spermatozoa in both the non-dialyzable and dialyzable fractions were studied. 11. It was observed that the non-dialysable fraction of X/Y extracts prolonged motility. l2. Agglutination tests of spermatozoa were carried out in normal, anti-X/Y, anti-SEX/Y, and anti-X/X globulins. All globulins produced agglutination. IX. REFERENCES Beale, G. H., 195%. The antigens, in "The Genetics of Paramecium aurelia" (G. H. Beale), pp. 77-123. Cambridge Univ. Press, Cambridge, England” Bodenstein, D., l9h6. Investigation on the locus of action of DDT in flies. Biol. Bull. wood's Hble, 90:1h8-ls7. Brosseau, G. E. Jr., 1960. Genetic analysis of the male fertility factors on the Y chromosome of Drosophila melanogaster. Genetics, #52257-27h. COOper, K. W}, 1950. Normal spermatogenesis in Drosophila, in "Biology of Drosophila" (M. Demerec, ed.), pp. 1-56. Wiley, new Ybrk. Dorfman, A., 1955. Mucopolysaccharidases, in "Methods in Enzymology" (s. P. Colowick and N. 0. Kaplan, eds.) Vol. 1, pp. 166-173. Academic Press, wa York. Ephrussi, B. and G. w. Beadle, 1936. A technique of transplantation for Drosophila. Am. Nat., 70:218-255. Hadorn, E. and C. Stern, 1938. The determination of sterility in Drosophila males without a complete Y chromosome. Am. Nat., 72:h2-52. Henle,‘w., G. Henle and L. A Chambers, 1938. Studies on the anti- genic structure of some mammalian spermatozoa. J. Exp. Med., ‘58:335-352§ . 'Kaufmann, B. P., 1933. Interchange between X and Y chromosomes in attached X: females of Drosophila melanogaster. Proc. Nat. Acad. Sci., 19:830-838. Lorenz, F. W. and A. Tyler, 1951. Extension of motile life span of spermatozoa of the domestic fowl by amino acids and proteins. Proc. Soc. Exp. Biol. & med., 78:57-62. 99 100 Mead, C. G , 1960. Isolation and characterization of the deoxyribo- nucleic acids of Drosophila melanogaster. Thesis for Ph. D. Michigan State university. Miller, A., 1950. The internal anatomy and histology of the imago of Drosophila melanogaster, in "Biology of Drosophila" (M. Demerec, ed.), pp. h20-53l. Wiley, New York. Miller, D. D. l9hl. "Genetics and the Origin of Species" (Th. Dobzhansky), p. 271. Columbia Univ. Press, New York. Oliver, C. P., and R. C. Anderson, l9h3. Infertility due to sperm non— motility in a female of Drosophila melanogaster. Genetics, 28:85 (Abstr.). Oliver, C. P. and R. C. Anderson, 19h5. The effects of remating on the fecundity of an infertile mutant female. Amer. Nat., 79:89—9h. Shen, T. H., 1932. Zytologische Uhtersuchungen fiber Sterilitat beim Mannchen von Drosophila melanogaster und beim Fl - Mannchen der Kreuzung zwischen D, simulanereibchen und D, melanogaster- Mannehen. z. Zellforsch., 15:5u7-580. Stern, G., 1929. Uhtersuchungen fiber Aberrationen des Y-Chromosoms von Drosophila melanogaster. Z. indukt. Abstamm.- u. Vererbungslehre, 51:253-353- TY1er, A., 19h6. loss of fertilizing power of sea-urchin and urechis sperm treated with univalent antibodies vs. antifertilizin. Proc. Soc. Exp. Biol. & Med., 62:197-199. Tyler, A and L. Rothschild, 1951. Metabolism of sea-urchin spermatozoa and induced anaerobic motility in solutions of amino acids. Proc. Soc. Exp. Biol. & med., 76:52-58. V 9’ IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII MinimumumuWMHIIH 1 0 N O U