Genetic Analysis of a Y-Chromosome Region That Induces Triplosterile Phenotypes and Is Essential for Spermatid Individualization in Drosophila Melanogaster

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Genetic Analysis of a Y-Chromosome Region That Induces Triplosterile Phenotypes and Is Essential for Spermatid Individualization in Drosophila melanogaster

Benjamin Timakov and Ping Zhang Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-2131 Manuscript received October 21, 1999 Accepted for publication January 24, 2000

ABSTRACT Mb of DNA but has only 40ف The heterochromatic Y chromosome of Drosophila melanogaster contains six loci mutable to male sterility. Region h1-h9 on YL, which carries the kl-3 and kl-5 loci, induces male sterility when present in three copies. We show that three separate segments within the region are responsible for the triplosterility and have an additive effect on male fertility. The triplosterile males displayed pleiotropic defects, beginning at early postmeiotic stages. However, the triplosterility was unaf- fected by kl-3 or kl-5 alleles. These data suggest that region h1-h9 is complex and may contain novel functions in addition to those of the previously identified kl-3 and kl-5 loci. The kl-3 and kl-5 mutations as well as deficiencies within region h1-h9 result in loss of the spermatid axonemal outer dynein arms. Examination using fluorescent probes showed that males deficient for h1-h3 or h4-h9 displayed a postmeiotic lesion with disrupted individualization complexes scattered along the spermatid bundle. In contrast, the kl-3 and kl-5 mutations had no effect on spermatid individualization despite the defect in the axonemes. These results demonstrate that region h1-h9 carries genetically separable functions: one required for spermatid individualization and the other essential for assembling the axonemal dynein arms.

HE Y chromosome of Drosophila melanogaster is an Despite complete sterility, X/0 males have fully grown Tunusual component in the fruit fly genome. It is testes that contain developing germ-line cells, including composed entirely of heterochromatin that contains re- mature primary spermatocytes (Lindsley and Toku- petitive DNA sequences, replicates during late S phase, yasu 1980). Moreover, X/0 males are able to support and remains condensed throughout the cell cycle the development of transplanted X/Y polar cells into (Gatti and Pimpinelli 1992). Although it is involved fertile sperm (Marsh and Wieschaus 1978), demon- in neither sex determination nor viability, the Y chro- strating that essential functions of the Y-linked fertility mosome is required for male fertility. Thus, Drosophila genes are restricted to the germ-line cells. X/0 males have a normal appearance, but are com- Several studies show that X/0 males and males carrying pletely sterile (Bridges 1916). Its large physical size of large deficiencies of the Y chromosome display numer- -of a normal male genome ous abnormalities in spermiogenesis, apparently re %12ف Mb accounts for 40ف but only six genes essential for spermatogenesis have sulting from degradation of defective spermatids pro- been identified by saturation mutagenesis (Kennison duced at early stages of spermatogenesis (Kiefer 1966, 1981; Hazelrigg et al. 1982; Gatti and Pimpinelli 1973). By comparing primary spermatocytes in X/Y and 1983). In a direction from the telomere on the Y long X/0 males, ultrastructural studies revealed differences in arm (YL) to the telomere on the Y short arm (YS), the a number of structural elements, such as a quantitative fertility genes are designated as kl-5, kl-3, kl-2, kl-1 (on reduction of reticular and tubular elements in the nuclei YL), and ks-1 and ks-2 (on YS). The existence of kl-4, of X/0 males (Meyer et al. 1961; Lifschytz and Meyer tentatively identified by Brosseau (1960), has not been 1977). However, Hardy et al. (1981) observed the nu- confirmed (Kennison 1981). In addition to the fertility clear components in primary spermatocytes of both X/ genes, several types of middle repetitive elements are Y and X/0 males and speculated that these nuclear ele- present on the Y chromosome, including the bb locus ments are present, but dispersed, in X/0 nuclei. of rDNA genes, the Su(Ste) locus that interacts with the Genetic analysis using segmental aneuploidy for the X-linked Ste locus, and two ABO loci that interact with Y chromosome has revealed that two YL regions con- a specific euchromatic gene, Abnormal oocyte (see Gatti taining kl-3 and kl-5 have peculiar properties. First, elec- and Pimpinelli 1992). tron microscopic examination (Hardy et al. 1981) shows that males deficient for either gene lost the outer dynein arms in the axoneme of the sperm tails. This Corresponding author: Ping Zhang, Department of Molecular and Cell Biology, U-2131, University of Connecticut, 354 Mansfield Rd., defect in the sperm tails was seen in mutations of kl-3 Storrs, CT 06269-2131. E-mail: [email protected] and kl-5 induced by P-element insertions (Zhang and

Genetics 155: 179–189 (May 2000) 180 B. Timakov and P. Zhang

Stankiewicz 1998). In addition, deficiency for kl-3 or The following stocks with X·Y reciprocal translocations were kl-5 is correlated with the absence of a high-molecu- obtained from the Bloomington Stock Center: T(1;Y)V24, T(1;Y)W27, T(1;Y)P7, T(1;Y)W19, and T(1;Y)V8. A stock car- lar-weight polypeptide with electrophoretic mobility sim- rying the FM7a, nod7 chromosome was also obtained from the ilar to that of Chlamydomonas dynein heavy chains Bloomington Stock Center. Four Y chromosomes carrying (Goldstein et al. 1982). The possibility that the fertility the kl-328, kl-361, kl-3104b, and kl-516 alleles have been described genes may encode dynein heavy chains is supported in previously (Zhang and Stankiewicz 1998). a recent molecular analysis showing that the predicted T(1;Y) chromosomes: Male-fertile reciprocal translocations product of a DNA sequence within the kl-5 locus is a between X and Y were employed in this study (Kennison 1981). Each translocation carries a breakpoint within Y and dynein ␤-heavy chain polypeptide (Gepner and Hays another in X heterochromatin, as shown in Figure 1. To 1993). better describe the separable components of the transloca- Second, in addition to the postmeiotic defect in the tions, nomenclature adopted by FlyBase (1999) is used sperm tails, males deficient for kl-3 and kl-5 display ab- throughout this article. A translocation segregant of a translo- normalities in the developing germ-line cells. Deficiency cation, Ts, is named using its telomeres as landmarks. For for kl-5 results in failure to develop aggregates of tubuli example, Ts(1Rt;YLt)V24 has the 1R (X right arm) telomere and the YL (Y long) telomere. and deficiency for kl-3 results in the absence of reticu- Rearranged Y chromosomes carrying duplicated YL materi- lar materials in the nuclei of primary spermatocytes als: Two rearranged Y chromosomes, Y2 and Y146, were gener- (Hardy et al. 1981). In addition, the YL regions con- ated in a genetic screen to recover Y chromosomes that containing kl-3 and kl-5 are specifically associated with the tain duplications of the Y long arm. The screen was carried out presence of two nuclear giant lampbrush loop-like struc- in K. Golic’s lab. Rearrangements were induced by irradiating C(1)RM, y v bb/BSYyϩ females with 3000 Rad of ␥ rays within tures in the primary spermatocytes that resemble those 12 hr of eclosion, aging for 1–2 days, and mass mating to X·YS, of lampbrush chromosomes in amphibian oocytes y Sxl v g f/yϩY males. Interchanges between YL of one chroma- (Meyer 1963; Hess and Meyer 1963; Bonaccorsi et al. tid and YS of its sister generated derivatives duplicated for the 1988). It has been proposed that these peculiar nuclear terminus of YL and lacking that of YS. These were recovered as X·YS, y Sxl v g f/YL·YL, BS BS (two doses of BS and loss of structures play roles other than encoding proteins and ϩ are essential for spermatogenesis (Hennig 1985, 1993; y ). Both Y2 and Y146 carry male-sterile mutations due to deletions on the short arms. Genetic complementation tests Bonaccorsi et al. 1988; Gatti and Pimpinelli 1992). on male fertility show that Y2 was complemented by Thus, the kl-3 and kl-5 regions on YL appear to have Ts(1Rt;YSt)V8, yϩ ks-2ϩ and, thus, lost only the ks-2 locus. On dual functions to encode proteins of the outer dynein the other hand, Y146 was complemented by Ts(1Rt;YSt)W19, arm and to organize nuclear structures of the primary yϩ ks-1ϩ ks-2ϩ but not Ts(1Rt;YSt)V8, yϩ ks-2ϩ, indicating that spermatocytes. Y146 lost at least the ks-1 locus. By coupling with cytological Ј Finally, although an extra Y chromosome poses no examination using 4 ,6-diamidino-2-phenylindole (DAPI) staining and N banding (Gatti and Pimpinelli 1983), the problem for X/Y/Y males or X/X/Y females, males with orders of the rearranged chromosomes were shown as follows: three copies of the Y chromosome lack motile sperm h1-h24|h3-h1 for Y2 (a duplication of h1-h3 and a deletion of and are sterile (Morgan et al. 1934; Cooper 1956). h25) and h1-h21|h4-h1 for Y146 (a duplication of h1-h4 and Since X/Y/Y/YS males are fertile, the dominant triplo- a deletion of h22-h25). sterility of X/Y/Y/Y males results from the hyperploidy Fertility tests and examination of postmeiotic defects in segmental aneuploidy for the Y chromosome: To test the effect of YL (Williamson and Meidinger 1979). Further ge- of various Y segments on male fertility, several types of duplica- netic mapping data (Kennison 1981) show that the kl-3 tions and deficiencies of the Y chromosome were constructed. region is responsible for the triplosterile effect of YL. To generate the segmental aneuploidy for Y duplications, an Here we report our analysis on the genetic complexity X chromosome balancer derivative, FM7a, nod7, was employed. of two YL regions containing the kl-3 and kl-5 genes. In addition to the multiple inversions carried on FM7a, which 31d 8 a Of Our results show that the hyperploidy of either the kl-3 are marked by y sc w v B (Lindsley and Zimm 1992), FM7a, nod7 carries two additional rearrangements (Zhang and region or the kl-5 region interferes with spermiogenesis, Hawley 1990). One is a reciprocal translocation between indicating that the factors responsible for the triplosteri- FM7a and chromosome 3, T(1;3)17A;92A, which induces lity are dispersed on YL. The analysis further suggests X-chromosome primary nondisjunction in females of FM7a, ,In FM7a, nod7/X/Y females .%30ف that neither the kl-3 nor the kl-5 locus is involved in the nod7/X at a frequency of triplosterility. Moreover, our studies reveal that sets of X-chromosome secondary nondisjunction is elevated to 60– postmeiotic defects are associated with deficiencies 70%. To measure fertility, 1- to 3-day-old males were mated individually to five 3- to 5-day-old y/y females in vials and their within the kl-3 and kl-5 regions, but absent in males progeny were counted until the 18th day of the crosses. carrying ms(Y) mutations induced by P-element inser- Specific details of the sterility tests done for various Y-chro- tions. mosome combinations are as follows:

1. y/yϩY/YL·YL, BS BS (Y2 or Y146) males: FM7a nod7/y fe- ϩ MATERIALS AND METHODS males were crossed to y/y Y males to produce primary exceptional FM7a, nod7/y/yϩ Y daughters. These females Drosophila strains: Flies were cultured on standard corn- were crossed to X·YS, y Sxl v g f/YL·YL, BS BS males and meal and agar medium at 25Њ. Unless stated otherwise, strains their y/yϩY/YL·YL, BS BS sons were tested for fertility and and mutations are as described in Lindsley and Zimm (1992). examined for defects in spermatogenesis. Y Long Arm and Spermatogenesis 181

2. w/YL·YL, BS BS/ms(Y) males: FM7a, nod7/w females were the presence of three copies of the Y chromosome in- S S crossed to X·YS, y Sxlvgf/YL·YL, B B males. Their FM7a, duces dominant male sterility (Cooper 1956) and YL nod7/w/YL·YL, BS BS daughters were crossed to X·Y, y/ms(Y) males and the resulting w/YL·YL, BS BS/ms(Y) males were is responsible for the triplosterile phenotypes (William- tested for fertility. son and Meidinger 1979). 3. Ts(1Lt;YSt),yϩ/YL·YL, BS BS males: C(1)DX, y f/YL·YL, BS BS To investigate whether the YL materials duplicated females were crossed to T(1;Y) males, and their onto the short arms of the Y2 (h1-h3) and Y146 chromo- ϩ Ts(1Lt;YSt),y /YL·YL, BS BS sons were tested for fertility. ϩ ϩ somes (h1-h4) affect male fertility, y/y Y/Y146 and 4. Ts(1Lt;YSt),y /0 males: C(1)RM, y v/0 females were crossed ϩ to T(1;Y) males. The resulting Ts(1Lt;YSt),yϩ/0 sons that y/y Y/Y2 males were produced via X-chromosome non- 7 carried a terminal deletion from the YL telomere were disjunction in FM7a, nod /y females (materials and examined for defects in spermatogenesis. methods). As shown in Table 1, none of the y/yϩY/ 5. Synthetic deficiencies: Males carrying internal deletions of Y146 males (N ϭ 70) were fertile, as assayed by individu- YL were synthesized by combining two different T(1;Y)sas ally mating to females. In similar fertility tests, none of described in Kennison (1981) and Hardy et al. (1981). the y/yϩY/Y2 males (N ϭ 60) produced progeny. Thus, Examination of mutant phenotypes by phase-contrast mi- dominant male sterility was induced by combining ei- croscopy: Testes of 1- to 3-day-old males were dissected out ther Y2 or Y146 with a normal Y chromosome. Since in PBS and transferred to a drop of PBS on a glass slide. The neither X·YS/Y2 nor X·YS/Y146 males displayed sig- testes were torn open, squashed under the weight of a coverslip (Kemphues et al. 1980), and examined by phase-contrast mi- nificantly lower fertility than the controls (Table 1), ϩ croscopy for defects in primary and secondary spermatocytes, the dominant sterility observed for y/y Y/Y2 and and spermatid tails. This method was also used to observe y/yϩY/Y146 males most likely resulted from the pres- needle-shaped crystals associated with the Ste phenotype in ence of three copies of the h1-h3 region and the h1-h4 primary spermatocytes. region. Staining of testes with rhodamine-conjugated phalloidin and DAPI: Testes were removed from 20 1- to 3-day-old males in The effects of the kl-3 and kl-5 loci on the triplosteril- PBS and fixed for 10 min in a rotating 1.5-ml Eppendorf tube ity: The kl-3 and kl-5 loci (Figure 1) are located within with 1 ml 6% formaldehyde in phosphate buffer (16 mm the duplicated YL segments on the rearranged Y chro- KH2PO4/K2HPO4, pH 6.8, 75 mm KCl, 25 mm NaCl, 4 mm mosomes, i.e., h1-h3 on Y2 and h1-h4 on Y146. Thus, MgCl2). Tissues were washed twice for 5 min each with PBS Y2 and Y146 presumably carry two copies of the kl-5ϩ and stained for 20 min with rhodamine-conjugated phalloidin ϩ at 8 U/ml in PBS (Molecular Probes, Eugene, OR). Tissues allele and Y146 possibly carries two copies of the kl-3 ϩ were then washed twice for 5 min each in PBS and stained allele. To address whether three copies of the kl-5 or for 10 min with DAPI (0.5 ␮g/ml in PBS). After staining, kl-3ϩ alleles cause male sterility in y/yϩY/Y2 and y/yϩY/ individual testes were transferred into 10 ␮l DAPI staining Y146 males, we employed four Y chromosomes that solution on a coverslip. Under a dissection microscope, the carry ms(Y) mutations in kl-3 and kl-5. These alleles, all sheath was peeled away with a glass scalpel, which was made by pulling a micropipette (1- to 5-␮l Micro-Pipet; Fisher Scientific, of which are induced by P-element insertions (Zhang Pittsburgh) over a flame, cooling, and breaking to produce an and Stankiewicz 1998), are as follows: kl-328, kl-361, kl- extremely sharp end. The samples were mounted in ProLong 3104b, and kl-516. Fertility tests show that males of the antifade (Molecular Probes) and viewed with an Olympus following genotypes were sterile: yw/Y2/kl-328, yw/Y2/ Provis microscope equipped with filters to observe epifloures- kl-361, yw/Y2/kl-3104b, and yw/Y2/kl-516 (Table 1). In cence illumination. Images from the DAPI and rhodamine channels were captured individually by using a cooled CCD similar tests, no progeny were produced from males of digital camera (SPOT; Diagnostic Instruments Inc.) and pro- yw/Y146/kl-328, yw/Y146/kl-361, yw/Y146/kl-3104b, and cessed by using Adobe Photoshop software (Adobe Systems yw/Y146/kl-516 (Table 1). The results show that the Inc.) on an Apple computer. The above procedure was per- yw/Y2/ms(Y) and yw/Y146/ms(Y) males were sterile, formed at least three times for each mutant genotype. All despite removing a functional copy of the kl-3ϩ allele reactions were performed at room temperature. or the kl-5ϩ allele. We conclude that three doses of the functional fertility genes are not required for inducing the triplosterility and thus the kl-3 and kl-5 loci are not RESULTS involved in causing the triplosterile phenotypes. Two rearranged Y chromosomes impaired male fertil- The triplosterile phenotypes and the Y long arm: ity when combined with a regular Y chromosome: In D. To map the YL region responsible for the triplosterile melanogaster, an extra Y chromosome in either males or phenotypes, we used a set of three reciprocal transloca- females causes no adverse effect. Results from fertility tion between the X and Y chromosomes, T(1;Y)V24, tests (Table 1) showed that males carrying an extra Y T(1;Y)W27, and T(1;Y)P7 (Figure 1). As shown in Table 1, short arm (X·YS/yϩY) and males carrying an entire extra both Ts(1Lt;YSt)P7,yϩ/Y146 and Ts(1Lt;YSt)W27,yϩ/Y146 Y chromosome (X·Y/Y) produced 111.1 progeny/male males produced large numbers of progeny (88.7 and 98.8 and 122.7 progeny/male, respectively, similar to males progeny/male, respectively), comparable to wild-type X/ carrying a regular Y chromosome (y/Y; 98.5 progeny/ Y males (96.6–120.0 progeny/male). Thus, combining male), a marked Y chromosome (y/yϩY; 120.0 progeny/ the Y146 chromosome with a Y fragment from h10 to male), or a Y chromosome that is attached to an X h25 had little, if any, effect on male fertility. In contrast, chromosome (X·Y/0; 96.4 progeny/male). However, a significant reduction of male fertility was present in 182 B. Timakov and P. Zhang

TABLE 1 The effect of region h1-h4 and ms(Y) alleles on the triplosterile phenotype

Total % with Genotype Description Progeny/male males progeny Control y/Y 1 copy of h1-h4 98.5 Ϯ 20.2 17 100 y/yϩY 1 copy of h1-h4 120.0 Ϯ 21.8 22 100 X·Y, y/0 1 copy of h1-h4 96.4 Ϯ 14.0 20 100 X·YS/yϩYa 1 copy of h1-h4 111.1 Ϯ 18.0 20 100 X·Y/Y 2 copies of h1-h4 122.7 Ϯ 24.1 20 100 X·YS/Y2 2 copies of h1-h3 118.3 Ϯ 18.6 20 100 X·YS/Y146 2 copies of h1-h4 88.6 Ϯ 17.7 20 100 Triplosterile y/yϩY/Y2 3 copies of h1-h3 0 60 0 y/yϩY/Y146a 3 copies of h1-h4 0 70 0 Triplosterile with ms(Y)kl-3 or kl-5 yw/Y2/ms(Y)16 3 copies of h1-h3 and 2 copies of kl-5ϩ 0200 yw/Y2/ms(Y)28 3 copies of h1-h3 and 2 copies of kl-3ϩ 0200 yw/Y2/ms(Y)61 3 copies of h1-h3 and 2 copies of kl-3ϩ 0200 yw/Y2/ms(Y)104b 3 copies of h1-h3 and 2 copies of kl-3ϩ 0200 yw/Y146/ms(Y)16 3 copies of h1-h4 and 2 copies of kl-5ϩ 0200 yw/Y146/ms(Y)28 3 copies of h1-h4 and 2 copies of kl-3ϩ 0200 yw/Y146/ms(Y)61 3 copies of h1-h4 and 2 copies of kl-3ϩ 0200 yw/Y146/ms(Y)104b 3 copies of h1-h4 and 2 copies of kl-3ϩ 0200 Mapping with Ts Ts(1Lt;YSt)P7,yϩ/Y2 2 copies of h1-h3 101.3 Ϯ 34.6 39 95 Ts(1Lt;YSt)W27,yϩ/Y2 2 copies of h1-h3 90.1 Ϯ 41.9 59 86 Ts(1Lt;YSt)V24,yϩ/Y2 2 copies of h1-h3 107.7 Ϯ 46.0 71 94 Ts(1Lt;YSt)P7,yϩ/Y146 2 copies of h1-h4 88.7 Ϯ 24.6 20 100 Ts(1Lt;YSt)W27,yϩ/Y146 2 copies of h1-h4 98.8 Ϯ 41.9 60 98 Ts(1Lt;YSt)V24,yϩ/Y146 2 copies of h1-h3 and 3 copies of h4 29.9 Ϯ 32.2 67 81 a Two sibling genotypes produced from a genetic cross, as described in materials and methods.

Ts(1Lt;YSt)V24,yϩ/Y146 males, which produced only 90.1, and 107.7 progeny/male, respectively; see Table 1). 29.9 progeny/male (Table 1). The Ts(1Lt;YSt)V24,yϩ Thus, a combination of Y2 and an YL fragment from segregant carries the entire Y chromosome except for h4 to h25 has no effect on male fertility, consistent with region h1-h3 (Figure 1). The sharp decline in male the observation that the duplication on Y2 is limited to fertility is manifested more dramatically at the fertility h1-h3. Moreover, the results argue that an additional level of the individual males. As plotted in Figure 2, the YL region, namely the h1-h3 region, plays an important vast majority of individual males of X/yϩY; Ts(1Lt; role in inducing triplosterile phenotypes, since y/yϩY/ YSt)P7,yϩ/Y146; and Ts(1Lt;YSt)PW27,yϩ/Y146 pro- Y2 and y/yϩY/Y146 males were completely sterile (Table duced large numbers of progeny. For example, 95% 1 and Figure 2). of y/yϩY; 65% of Ts(1Lt;YSt)P7,yϩ/Y146; and 70% of Postmeiotic defects associated with triplosterility: In Ts(1Lt;YSt)PW27,yϩ/Y146 males produced Ͼ80 progeny spermatogenesis of D. melanogaster (reviewed in Lind- male. However, only 10% of Ts(1Lt;YSt)V24,yϩ/Y146 sley and Tokuyasu 1980; Fuller 1993), a germ-line males produced Ͼ80 progeny/male, whereas most of stem cell undergoes four rounds of mitotic divisions, them either failed to produce any progeny (19%) or giving rise to 16 primary spermatocytes surrounded by 2 produced very small numbers, e.g., Յ40 progeny/male somatic cyst cells. After the meiotic divisions, 64 haploid (46%). The data suggest that an addition of the h4-h9 round spermatids are produced in a cyst and undergo region, which is carried in the Ts(1Lt;YSt)V24,yϩ chro- extensive syncytial cellular differentiation, including nu- mosome, into males carrying the Y146 chromosome clear condensation, spermatid elongation, individual- greatly reduced male fertility. This result agrees with a ization, and coiling. By using a ﬂuorescent probe, rhoda- previous observation that three copies of the h4-h9 re- mine-conjugated phalloidin, which binds to F-actin, gion cause male fertility problems (Kennison 1981). Fabrizio et al. (1998) have shown that F-actin is a major In contrast, males of the following genotypes pro- component of the individualization complex (IC). ICs duced large numbers of offspring: Ts(1Lt;YSt)P7,yϩ/Y2; are formed at the aligned nuclear heads of spermatid Ts(1Lt;YSt)W27,yϩ/Y2; and Ts(1Lt;YSt)V24,yϩ/Y2 (101.3, bundles, traverse along the length of the tails, and re- Y Long Arm and Spermatogenesis 183

Figure 1.—T(1;Y) chromosomes used to study the effects of various Y regions on male fertility. The top ﬁgure illustrates banding patterns from h1 to h25 of the Y chromosome of D. melanogaster as determined by Gatti and Pimpinelli (1983). The Y-linked fertility loci (kl-5, kl-3, kl-2, and kl-1 on the long arm; ks-1 and ks-2 on the short arm) and their approximate locations (thick bars) are shown above the Y chromosome. Schematic drawings of six reciprocal X·Y translocations are adapted from Hardy et al. (1981). The breakpoints of the translocations are located in h4 for T(1;Y)V24; h10 for T(1;Y)W27; h11 for T(1;Y)P7; h20-h21 for T(1;Y)V8; and h24 for T(1;Y)W19 (Kennison 1981; Gatti and Pimpinelli 1983; Hardy et al. 1984; Bonaccorsi et al. 1988). Open boxes, Y-chromosomal materials; stippled boxes, X-heterochromatin; thin lines, X euchromatin; open circles, Y centromeres; solid circles, X centromeres. Ts(1Rt;YLt)W27,yϩ carries an additional reciprocal translocation between the X, Y, and third chromosomes (Hardy et al. 1984) but acts like the other Ts(1;Y)s in this article. solve the syncytial spermatids of a cyst into 64 cells with (approximately one-fourth) are located in positions individual membranes (Tokuyasu et al. 1972). Exami- away from the aligned nuclear heads, indicating that nation using a combination of two ﬂuorescent probes, they have traveled along the tails. DAPI and rhodamine-conjugated phalloidin, shows that When testes of either X/yϩY/Y146 or X/yϩY/Y2 were the individualization complexes in wild type are often examined, departure from normal development was associated with spermatid nuclear heads (Fabrizio et seen at early postmeiotic stages of spermatid differentia- al. 1998; Figure 3, A and B). A small fraction of ICs tion (Figure 3, C–E; Table 2). Before nuclear elonga-

Figure 2.—Changes in the distribution of individual male fertility in wild type and in segmental aneuploids carrying various Y segments. Sharp decline in individual male fertility was shown in males carrying Y146 and Ts(1Lt;YSt)V24,yϩ or yϩY. In the fertility tests, 1- to 3-day-old males of various genotypes were individually mated to five females in vials, and the progeny were scored (materials and methods). The number indicated on the x-axis is the highest number of offspring in that class, except the last that shows individuals produced Ͼ80 offspring. 184 B. Timakov and P. Zhang tion, round spermatid nuclei began to fall apart in a In semisterile males of Ts(1Lt;YSt)V24,yϩ/Y146, which large number of spermatid bundles, resulting in singu- contain three copies of the h4 region, postmeiotic de- lar nuclear heads that were dispersed throughout the fects were similar to but much less severe than in the tails (Figure 3C). In the more extreme case, accounting triplosterile males of y/yϩY/Y2 and y/yϩY/Y146. For -of the triplosterile males, the testes contained example, spermatid bundles with scattered singular nu %10ف for exclusively round spermatids scattered throughout the clear heads were seen frequently (Figure 3, F and G), length of the tails. Although it appears that nuclear but accounted for only a small proportion of spermatid elongation in some of the scattered nuclei continued bundles. In contrast to those of the triplosterile males, despite their earlier dissociation with each other, the some ICs in Ts(1Lt;YSt)V24,yϩ/Y146 were located away dispersed nuclei were devoid of phalloidin staining, in- from the nuclear bundles, apparently resulting from dicating that F-actin was unable to assemble around the caudal movement along the tails (Figure 3, F and G). scattered nuclei. In addition, many of the spermatid Consistent with the observation that ICs traversed along bundles with scattered nuclei were much smaller in spermatid tails of the semisterile mutants, we observed diameter than normal ones, suggesting that some of the individualized spermatids in the basal region of the tes- spermatid tails failed to develop (Figure 3C). However, tes and mature sperm in the seminal vesicles (data not there were other spermatid bundles that displayed shown). aligned nuclear heads associated with ICs, as in wild Postmeiotic defects associated with deficiencies type (Figure 3, D and E). The number of such bundles within the h1-h10 region: Males deficient for region h1- with normal-looking ICs varied among males, ranging h3 (a kl-5 deletion) or h4-h9 (a kl-3 deletion) lost the to 40% of the total spermatid cysts. The ICs outer dynein arms in the spermatid bundles (Hardy et 10ف from were apparently arrested at the elongated nuclear al. 1981). kl-3 or kl-5 mutations induced by P-element heads, since all the observed ICs were localized with the insertions caused the same axonemal defect (Zhang aligned nuclear heads. and Stankiewicz 1998). We have further examined postmeiotic defects associated with various deficiencies within the h1-h10 region by staining testes with the fluorescent probes DAPI and rhodamine-conjugated phalloidin. The analysis shows that a group of three deficiencies

Figure 3.—Wild-type and triplosterile testes stained with rhodamine-conjugated phalloidin and DAPI. (A, D, and F) Images from phalloidin staining (red). (B, C, E, and G) Com- posite images from phalloidin staining (red) and DAPI staining (blue). Images of the spermatid bundles are generally oriented from the apical tip (AP) of the testes (left) to the basal region (right). (A and B) Wild-type testes. Condensed nuclear heads of 64 haploid cells in a cyst were stained with DAPI. During spermatogenesis, the nuclear bundles advance toward the basal region of the testes (right), while the spermatid tails grow in the opposite direction (left; Lindsley and Tokuyasu 1980). Most of the aligned nuclear heads (triangles) are associated with ICs (stained with phalloidin, red). As the ICs travel along the length of the tails (arrows), they grow in volume (compare the sizes of ICs from left to right) by collecting cytoplasmic materials between the spermatid tails (Lindsley and Tokuyasu 1980; Fabrizio et al. 1998). Approx- imately one-fourth of the ICs in a testis have traversed caudally along the spermatid tails and mature ICs are stained strongly by phalloidin and are visible readily through the sheath of intact testes (data not shown). (C–E) y/yϩY/Y146 testes. The majority of spermatid bundles display individual nuclei scattered throughout the tail length (arrowheads). Some of the spermatid tails were probably absent in the cysts, since the bundles were smaller in diameter than normal ones. A small number of spermatid bundles display ICs located at the aligned nuclear heads (triangles). (F and G) Semisterile testes of Ts(1Lt;YSt)V24,yϩ/Y146. Isolated nuclei (arrowheads) were frequently seen along the length of the tails. Some ICs (arrows) were located along the tails in positions away from the nuclear heads. Bars, 20 ␮m. Y Long Arm and Spermatogenesis 185

TABLE 2 Postmeiotic defects in segmental aneuploids for the Y chromosome and ms(Y) mutations

Genotype Description Early postmeiotic defect X/yϩY/Y146 Triplosterile X/yϩY/Y2 Triplosterile Defect before individualization Ts(1Lt;YSt)P7,yϩ/0 Df h1-h10 Ts(1Lt;YSt)P7,yϩ/ Df h4-h10 Ts(1Rt;YLt)V24,BS Individualization defect Ts(1Lt;YSt)V24,yϩ/0 Df h1-h3 Ts(1Lt;YSt)W27,yϩ/0 Df h1-h9 Ts(1Lt;YSt)W27,yϩ/ Df h4-h9a Ts(1Rt;YLt)V24,BS Defect after individualization X/kl-328 kl-3, P inducedb X/kl-361 kl-3, P inducedb X/kl-3104b kl-3, P inducedb X/kl-516 kl-5, P inducedb Ts(1Lt;YSt)P7,yϩ/ kl-2 c Ts(1Rt;YLt)W27,BS a The deficiency causes lost or greatly reduced axonemal outer dynein arms (Hardy et al. 1981). b Figure 4.—Mutant testes of h1-h10 deletions stained with Mutations are induced by P-element insertions and display rhodamine-conjugated phalloidin and DAPI. Tissues were earlier postmeiotic defects, i.e., lost or greatly reduced axo- stained either with phalloidin (red) in A, C, E, and G or with nemal outer dynein arms (Zhang and Stankiewicz 1998). c phalloidin and DAPI (blue) in B, D, F, and H. Arrowheads A synthetic deficiency of the h10 region. Ultrastructural indicate disrupted ICs associated with singular elongated nu- analysis shows that the sperm axonemes are normal (Hardy clear heads that were scattered along the tails (A–F). Arrows et al. 1984). indicate intact ICs located at the aligned nuclear heads (G and H). Images of the spermatid bundles are oriented from the apical tip of the testes (left) to the basal region (right). in h1-h9 induced postmeiotic defects with disrupted ICs (A and B) Ts(1Lt;YSt)V24,yϩ/0 (deficiency from h1 to h3). (C ϩ (Table 2 and Figure 4, A–F). These include deletions and D) Ts(1Lt;YSt)W27,y /0 (deficiency from h1 to h9). (E ϩ S of h1-h3, h4-h9, and h1-h9. The mutant testes contained and F) Ts(1Lt;YSt)W27,y /Ts(1Rt;YLt)V24,B (synthetic deficiency from h4 to h9). (G and H) Ts(1Lt;YSt)P7,yϩ/ large numbers of spermatid bundles that displayed a Ts(1Rt;YLt)V24,BS (deficiency from h4 to h10). Bars, 10 ␮m. phenotype similar to that of the triplosterile mutations, i.e., scattered nuclei throughout the spermatid bundles. However, unlike the triplosterile mutations in which We have also extended our examination into the ad- round nuclei were dispersed (Figure 3C), the scattered jacent region h10, which contains the kl-2 locus (Fig- singular nuclei in the deficiencies were needle shaped ure 1). In males carrying a synthetic deficiency for the (Figure 4B). Furthermore, while a small number of the h10 region, Ts(1Lt;YSt)P7,yϩ/Ts(1Rt;YLt)W27,BS, nor- ICs were located with aligned nuclear bundles, as in mal sperm axonemes are present in ultrastructural stud- wild type, most of the ICs of the mutants were disrupted ies (Hardy et al. 1984). By staining with DAPI and rho- in spermatid bundles (Figure 4, A–F). The dispersed damine-conjugated phalloidin, we show that ICs from ICs were fragmented and distributed along elongated this deficiency were formed at the elongated nuclear spermatid bundles. They were cone shaped, with the bundles and traversed caudally along the spermatid blunt end pointing apically and the pointed end di- tails, as in wild type (Table 2), suggesting the sperma- rected basally. The isolated sperm heads often appeared tids were individualized. Phase-contrast examination to trail the IC fragments. These rhodamine-staining showed that the mutant produced a large number of cones are the individual investment cones formed individualized spermatids in the basal region of the tes- around each individualizing spermatid (Fabrizio et al. tes. However, the defective spermatids were degraded 1998). These cones normally migrate together in a com- before entering the seminal vesicles. pact mass (Figure 3, A and B). However, in these mu- Interestingly, two larger deficiencies, Ts(1Lt;YSt)P7, tants the mass dispersed into single cones, each with its yϩ/0 (deficient for h1-h10) and Ts(1Lt;YSt)P7,yϩ/ own sperm nucleus. Ts(1Rt;YLt)V24,BS (deficient for h4-h10), exhibited a 186 B. Timakov and P. Zhang

Figure 5.—Defective sperm from kl-3 and kl-5 mutations. Images of live squashed testes were captured with a phase-contrast microscope. (A) Wild-type testes. Motile sperm display smooth and coiled morphology of the individualized tails. (B and C) Testes from X/kl-516 mutant, (D) X/kl-328, (E) X/kl-361, and (F) X/kl-3104b show irregular- ities of the spermatid tails. In X/kl-516 testes, spermatid bundles with numerous bulges distributed along the tails were frequently observed (arrows in C). X/ ms(Y) males were produced from crosses between X/X females and X·Y/ms(Y) males. Bars, 20 ␮m. striking defect at the individualization process. In the DISCUSSION mutant testes, there were no individualized spermatids Hyperploidy of YL and the triplosterility: Males with and nearly every IC was associated with a nuclear bundle three doses of region h4-h9 are sterile (Kennison 1981), (Figure 4, G and H; Table 2), indicating that the IC was but motile sperm are present in the seminal vesicles arrested at the nuclear head. In addition, the nuclear where usually only mature sperm are stored (Hardy et heads of the deficiencies appear to be slightly less con- al. 1981). However, three doses of YL cause male sterility densed than those of the wild type. Although we occa- without motile sperm (Williamson and Meidinger sionally observed ICs that were located along the sper- 1979). The observation that hyperploidies of region h4- -IC per h9 and YL induce different phenotypes raises the ques 0.5ف) matid tails, away from the nuclear bundles male), none were located near the apical tip of the tion that genetic information on YL, in addition to h4- testes (i.e., at the opposite end of the nuclear bundles). h9, is involved in the triplosterile effect. Our analysis Thus, the mutants were unable to produce individual- shows that separable regions on YL are responsible for ized spermatids, which was confirmed by phase-contrast the triplosterility. Region h4-h9 can be further divided microscopy (data not shown). into two segments on the basis of effects on male fertility. Spermatid individualization and ms(Y) mutations in Since three copies of region h4 induce only semisterility h1-h9: In contrast to the individualization defects associ- (Table 1), region h5-h9 must play a critical role in caus- ated with the h1-h10 deficiencies, as shown above, the ing the complete triplosterility of males carrying three individualization process in males carrying kl-3 or kl-5 doses of region h4-h9. mutations appeared to be normal. By staining with DAPI Although a previous survey (Kennison 1981) has and rhodamine-conjugated phalloidin, normal-looking shown that h4-h9 was the only YL region that causes ICs were seen in the mutant testes of X/kl-328, X/kl- male sterility with three doses, our analysis has revealed 361, X/kl-3104b, and X/kl-516 (Table 2). Some ICs were that y/yϩY/Y2 males carrying three copies of region h1- localized with aligned nuclear bundles, whereas others h3 were sterile (Table 1). We have further determined were located along the tails, away from the nuclear bun- that two additional genotypes with three copies of the dles. The results suggest that the ICs were able to tra- h1-h3 region, X·YS/Y2/Ts(1Rt;YLt)V24,BS and X·YS/ verse caudally along the tails to produce individualized Y146/Ts(1Rt;YLt)V24,BS, were also sterile (data not spermatids. The hypothesis was confirmed in experi- shown). The reasons why the previous study failed to ments using phase-contrast microscopy, which showed observe the effect of the h1-h3 region on the triplosteril- the presence of individualized spermatids in the mutant ity remain unknown. It is possible that the Y-chromo- testes. Although we found no obvious abnormality in some derivatives used in these studies behaved differ- the individualization process by using the fluorescent ently: (1) Y2 and Y146 used in this study were derived probes, the individualized spermatids displayed abnor- directly from BSYyϩ, whereas Kennison (1981) em- mal morphology (Figure 5). The defective spermatids ployed a highly derived wϩY2 chromosome (Lindsley degenerated before reaching the seminal vesicle where and Zimm 1992); and (2) we used Ts(1Rt;YLt)V24,BS mature sperm were stored. carrying the h1-h3 region. Kennison (1981) employed Y Long Arm and Spermatogenesis 187 at least two different T(1;Y)’s, in addition to wϩY2, to specifically associated with mutations in the fertility generate three copies of the h1-h3 region, although the genes. All four alleles of kl-3 and kl-5 induce male sterility Y derivatives used in the experiments were not specified. with characteristic phenotypes, i.e., lost or greatly re- Differential effect on male fertility has been described duced outer dynein arms of the sperm tail (Zhang and for some Y chromosomes, which induced male sterility Stankiewicz 1998). Nonetheless, the individualization with two doses (Grell 1969). process continues to produce individualized spermatids Consistent with the observation that dispersed factors (Figure 5 and Table 2). In contrast, males deficient for on YL cause the triplosterility, genetic complementation regions h1-h3, h4-h9, and h1-h9 exhibit defects in the analysis indicates that the kl-3 and kl-5 genes do not individualization process (Table 2). The disrupted indi- affect the triplosterility. By using four Y chromosomes vidualization complexes display cone-shaped IC compo- carrying ms(Y) mutations in kl-3 and kl-5, we have shown nents with the elongated nuclei that are scattered that X/Y2/ms(Y) or X/Y146/ms(Y) males, which carry throughout the length of the tail (Figure 4). three copies of the h1-h3 or h1-h4 region but only two We propose that region h1-h9 harbors two types of doses of the functional kl-3ϩ or kl-5ϩ loci, are sterile separable functions. One is encoded by the kl-3 and (Table 1). kl-5 fertility genes that are mutable to male sterility with Defects associated with the hyperploidy of YL: The loss of the axonemal outer dynein arms. The other func- most striking phenotype characteristic of the triploster- tion is revealed by the individualization defects associ- ile testes is the presence of numerous round nuclei ated with deficiencies within the h1-h9 region. An alter- caudally dispersed along the length of the sperm tails native explanation to the phenotypic difference between (Figure 3C). Staining with DAPI and rhodamine-conju- the deficiencies and the ms(Y) mutations is that the gated phalloidin reveals that F-actin fails to accumulate ms(Y) mutations are not null alleles. Although we cannot around the dispersed nuclei, although some of the nu- rule it out, this possibility is very unlikely since the two clei display elongating morphology, indicating that nu- functions of organizing the outer dynein arms and sper- clear elongation is initiated but incomplete. In addition, matid individualization appear to be distinct. a small number of ICs are formed but are always located Relationship between the triplosterility and the defi- with aligned nuclear heads, suggesting that the ICs are ciencies within h1-h9: The effects of the triplosterility stalled. appeared at early postmeiotic stages, whereas lesions of The pleiotropic defects associated with the triplosteri- the h1-h9 deficiencies were not seen until the individual- lity may be caused indirectly by an earlier lesion in ization process (Table 2). Despite the phenotypic differ- spermatogenesis. Since genetic control in spermatogen- ence, the triplosterility shares several similar features esis is not a linear cascade of dependent steps, early with deficiencies in h1-h9. First, in both cases, region lesions in the male germ-line cells often do not block h1-h9 can be divided into segments that play similar late stages of spermatogenesis (Lifschytz 1987; Fuller roles, as judged by fertility in the triplosterility or defects 1993). It is also possible that the triplication of the in the individualization process. Second, the pheno- h1-h10 region disrupts multiple developmental steps types associated with the triplosterility and the h1-h9 during spermatogenesis. Unlike most mutations that deficiencies seem to be independent of the fertility fac- arise by altering gene structures, the triplosterile muta- tors kl-3 and kl-5, which are located in this region. Fi- tion does not impair genetic function on YL. Rather, nally, segments within h1-h9 may exert cumulative effect the triplosterility results from hyperploidy of YL that on both the triplosterility and spermatid individualiza- may interfere with several genetic functions in spermio- tion. For example, three doses of region h4 induce genesis. semisterility (Table 1), but three doses of region h4-h9 Relationship between deficiencies and ms(Y) muta- induce complete sterility (Kennison 1981). A similar tions: The repetitive nature, absence of meiotic recom- cumulative effect has been indicated by the h1-h10 defi- bination, and absence of polytene banding have made ciency, which displays largely arrested ICs at the aligned it very difficult to elucidate functions of the Y chromo- nuclear bundles. This defect occurs at an earlier stage some in spermatogenesis. Studies on functional proper- of spermiogenesis than that of the h1-h9 or h10 defi- ties of the YL region containing kl-3 and kl-5 have been ciencies (Table 2). The analysis indicates that the ge- carried out mostly by examining defects associated with netic factors responsible for both the triplosterility and large chromosome rearrangements such as deficiencies, spermatid individualization are dispersed on YL and are X·Y translocations, and Y-autosome translocations functionally redundant, suggesting the involvement of (Hardy et al. 1981; Kennison 1981; Goldstein et al. heterochromatic repetitive sequences. 1982; Bonaccorsi et al. 1988; Bonaccorsi and Lohe The possible functions of the YL region: In the Y 1991). The results obtained from these studies likely chromosome of D. melanogaster, nine satellite sequences of the DNA %80ف represent a composite of functions of the fertility genes and the rDNA repeats account for and the corresponding genomic regions. Here we em- sequences (Appels and Peacock 1978; Lohe et al. ployed ms(Y) mutations of kl-3 and kl-5, which are in- 1993). In addition to several types of middle repetitive duced by single P-element insertions, to reveal defects elements related to transposable elements (Pimpinelli 188 B. Timakov and P. Zhang et al. 1995; Zhang and Spradling 1995), the vast major- some (Elgin 1996; Platero et al. 1998; Wallrath ity of the DNA sequence within the h1-h9 region is 1998). These results provide an important avenue for composed of six satellite repeats of 5- to 7-bp (Bonac- investigating how the heterochromatic h1-h9 region corsi and Lohe 1991; Lohe et al. 1993). plays essential roles in spermatid differentiation. The heterochromatic regions of Drosophila chromo- We are grateful to Kent Golic for the rearranged Y chromosomes somes behave as suppressors of position effect variega- and technical advice. We thank Christopher Bazinet for technical tion (PEV), a genetic phenomenon in which a euchro- advice on staining with the fluorescent probes. We also thank the matic gene is relocated to heterochromatin and is Bloomington Drosophila Stock Center for providing stocks. Finally, inactivated by the juxtaposed heterochromatin (Spof- we thank Linda Strausbaugh for critical comments on the manuscript. This work was supported by a grant from the University of Connecticut ford 1976; Elgin 1996). The heterochromatic Y chro- Research Foundation. mosome acts as a strong PEV suppressor (Spofford 1976; Henikoff 1992; Lloyd et al. 1997). Moreover, various segments along the length of the Y chromosome LITERATURE CITED are known to suppress PEV in an additive manner, such that the degree of PEV suppression is directly related Appels, R., and W. J. Peacock, 1978 The arrangement and evolution of highly repeated (satellite) DNA sequences with special to the amount of the Y materials (Dimitri and Pisano reference to Drosophila. Int. Rev. Cytol. 8 (Suppl.): 69–126. 1989). Although there are additive effects on triplosteri- Bonaccorsi, S., and A. 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