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

Corresponding author: Ping Zhang, Department of Molecular and Cell Biology, U-2131, University of Connecticut, 354 Mansfield Rd., Storrs, CT 06269-2131., ping{at}uconnvm.uconn.edu (E-mail)

Communicating editor: S. HENIKOFF

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The heterochromatic Y chromosome of Drosophila melanogaster contains 40 Mb of DNA but has only 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 unaffected 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.

THE Y chromosome of Drosophila melanogaster is an unusual component in the fruit fly genome. It is composed entirely of heterochromatin that contains repetitive DNA sequences, replicates during late S phase, and remains condensed throughout the cell cycle (GATTI and PIMPINELLI 1992 ). Although it is involved in neither sex determination nor viability, the Y chromosome is required for male fertility. Thus, Drosophila X/0 males have a normal appearance, but are completely sterile (BRIDGES 1916 ). Its large physical size of 40 Mb accounts for 12% of a normal male genome but only six genes essential for spermatogenesis have been identified by saturation mutagenesis (KENNISON 1981 ; HAZELRIGG et al. 1982 ; GATTI and PIMPINELLI 1983 ). In a direction from the telomere on the Y long arm (YL) to the telomere on the Y short arm (YS), the fertility genes are designated as kl-5, kl-3, kl-2, kl-1 (on YL), and ks-1 and ks-2 (on YS). The existence of kl-4, tentatively identified by BROSSEAU 1960 , has not been confirmed (KENNISON 1981 ). In addition to the fertility genes, several types of middle repetitive elements are present on the Y chromosome, including the bb locus of rDNA genes, the Su(Ste) locus that interacts with the X-linked Ste locus, and two ABO loci that interact with a specific euchromatic gene, Abnormal oocyte (see GATTI and PIMPINELLI 1992 ).

Despite complete sterility, X/0 males have fully grown testes that contain developing germ-line cells, including mature primary spermatocytes (LINDSLEY and TOKUYASU 1980 ). Moreover, X/0 males are able to support the development of transplanted X/Y polar cells into fertile sperm (MARSH and WIESCHAUS 1978 ), demonstrating that essential functions of the Y-linked fertility genes are restricted to the germ-line cells.

Several studies show that X/0 males and males carrying large deficiencies of the Y chromosome display numerous abnormalities in spermiogenesis, apparently resulting from degradation of defective spermatids produced at early stages of spermatogenesis (KIEFER 1966 , KIEFER 1973 ). By comparing primary spermatocytes in X/Y and X/0 males, ultrastructural studies revealed differences in a number of structural elements, such as a quantitative reduction of reticular and tubular elements in the nuclei of X/0 males (MEYER et al. 1961 ; LIFSCHYTZ and MEYER 1977 ). However, HARDY et al. 1981 observed the nuclear components in primary spermatocytes of both X/Y and X/0 males and speculated that these nuclear elements are present, but dispersed, in X/0 nuclei.

Genetic analysis using segmental aneuploidy for the Y chromosome has revealed that two YL regions containing kl-3 and kl-5 have peculiar properties. First, electron 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 defect in the sperm tails was seen in mutations of kl-3 and kl-5 induced by P-element insertions (ZHANG and STANKIEWICZ 1998 ). In addition, deficiency for kl-3 or kl-5 is correlated with the absence of a high-molecular-weight polypeptide with electrophoretic mobility similar to that of Chlamydomonas dynein heavy chains (GOLDSTEIN et al. 1982 ). The possibility that the fertility genes may encode dynein heavy chains is supported in a recent molecular analysis showing that the predicted product of a DNA sequence within the kl-5 locus is a dynein ß-heavy chain polypeptide (GEPNER and HAYS 1993 ).

Second, in addition to the postmeiotic defect in the sperm tails, males deficient for kl-3 and kl-5 display abnormalities in the developing germ-line cells. Deficiency for kl-5 results in failure to develop aggregates of tubuli and deficiency for kl-3 results in the absence of reticular materials in the nuclei of primary spermatocytes (HARDY et al. 1981 ). In addition, the YL regions containing kl-3 and kl-5 are specifically associated with the presence of two nuclear giant lampbrush loop-like structures in the primary spermatocytes that resemble those of lampbrush chromosomes in amphibian oocytes (MEYER 1963 ; HESS and MEYER 1963 ; BONACCORSI et al. 1988 ). It has been proposed that these peculiar nuclear structures play roles other than encoding proteins and are essential for spermatogenesis (HENNIG 1985 , HENNIG 1993 ; BONACCORSI et al. 1988 ; GATTI and PIMPINELLI 1992 ). Thus, the kl-3 and kl-5 regions on YL appear to have dual functions to encode proteins of the outer dynein arm and to organize nuclear structures of the primary spermatocytes.

Finally, although an extra Y chromosome poses no problem for X/Y/Y males or X/X/Y females, males with three copies of the Y chromosome lack motile sperm and are sterile (MORGAN et al. 1934 ; COOPER 1956 ). Since X/Y/Y/YS males are fertile, the dominant triplosterility of X/Y/Y/Y males results from the hyperploidy of YL (WILLIAMSON and MEIDINGER 1979 ). Further genetic mapping data (KENNISON 1981 ) show that the kl-3 region is responsible for the triplosterile effect of YL. Here we report our analysis on the genetic complexity of two YL regions containing the kl-3 and kl-5 genes. Our results show that the hyperploidy of either the kl-3 region or the kl-5 region interferes with spermiogenesis, indicating that the factors responsible for the triplosterility are dispersed on YL. The analysis further suggests that neither the kl-3 nor the kl-5 locus is involved in the triplosterility. Moreover, our studies reveal that sets of postmeiotic defects are associated with deficiencies within the kl-3 and kl-5 regions, but absent in males carrying ms(Y) mutations induced by P-element insertions.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila strains:
Flies were cultured on standard corn-meal and agar medium at 25°. Unless stated otherwise, strains and mutations are as described in LINDSLEY and ZIMM 1992 . The following stocks with X·Y reciprocal translocations were 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 carrying the FM7a, nod⁷ chromosome was also obtained from the Bloomington Stock Center. Four Y chromosomes carrying the kl-3²⁸, kl-3⁶¹, kl-3^104b, and kl-5¹⁶ alleles have been described previously (ZHANG and STANKIEWICZ 1998 ).

T(1;Y) chromosomes:
Male-fertile reciprocal translocations between X and Y were employed in this study (KENNISON 1981 ). Each translocation carries a breakpoint within Y and another in X heterochromatin, as shown in Fig 1. To better describe the separable components of the translocations, nomenclature adopted by FLYBASE 1999 is used throughout this article. A translocation segregant of a translocation, Ts, is named using its telomeres as landmarks. For example, Ts(1Rt;YLt)V24 has the 1R (X right arm) telomere and the YL (Y long) telomere.

View larger version (23K): In this window In a new window Download PPT slide   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,y+ 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.

Rearranged Y chromosomes carrying duplicated YL materials:
Two rearranged Y chromosomes, Y2 and Y146, were generated in a genetic screen to recover Y chromosomes that contain duplications of the Y long arm. The screen was carried out in K. Golic's lab. Rearrangements were induced by irradiating C(1)RM, y v bb/B^SYy⁺ females with 3000 Rad of rays within 12 hr of eclosion, aging for 1–2 days, and mass mating to X·YS, y Sxl v g f/y⁺Y males. Interchanges between YL of one chromatid and YS of its sister generated derivatives duplicated for the terminus of YL and lacking that of YS. These were recovered as X·YS, y Sxl v g f/YL·YL, B^S B^S (two doses of B^S and loss of y⁺). Both Y2 and Y146 carry male-sterile mutations due to deletions on the short arms. Genetic complementation tests on male fertility show that Y2 was complemented by Ts(1Rt;YSt)V8, y⁺ ks-2⁺ and, thus, lost only the ks-2 locus. On the other hand, Y146 was complemented by Ts(1Rt;YSt)W19, y⁺ ks-1⁺ ks-2⁺ but not Ts(1Rt;YSt)V8, y⁺ ks-2⁺, indicating that Y146 lost at least the ks-1 locus. By coupling with cytological examination using 4',6-diamidino-2-phenylindole (DAPI) staining and N banding (GATTI and PIMPINELLI 1983 ), the orders of the rearranged chromosomes were shown as follows: h1-h24|h3-h1 for Y2 (a duplication of h1-h3 and a deletion of h25) and h1-h21|h4-h1 for Y146 (a duplication of h1-h4 and a deletion of h22-h25).

Fertility tests and examination of postmeiotic defects in segmental aneuploidy for the Y chromosome:
To test the effect of various Y segments on male fertility, several types of duplications and deficiencies of the Y chromosome were constructed. To generate the segmental aneuploidy for Y duplications, an X chromosome balancer derivative, FM7a, nod⁷, was employed. In addition to the multiple inversions carried on FM7a, which are marked by y^31d sc⁸ w^a v^Of B (LINDSLEY and ZIMM 1992 ), FM7a, nod⁷ carries two additional rearrangements (ZHANG and HAWLEY 1990 ). One is a reciprocal translocation between FM7a and chromosome 3, T(1;3)17A;92A, which induces X-chromosome primary nondisjunction in females of FM7a, nod⁷/X at a frequency of 30%. In FM7a, nod⁷/X/Y females,X-chromosome secondary nondisjunction is elevated to 60–70%. 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 progeny were counted until the 18th day of the crosses.

Specific details of the sterility tests done for various Y-chromosome combinations are as follows:

1. y/y⁺Y/YL·YL, B^S B^S (Y2 or Y146) males: FM7a nod⁷/y females were crossed to y/y⁺ Y males to produce primary exceptional FM7a, nod⁷/y/y⁺ Y daughters. These females were crossed to X·YS, y Sxl v g f/YL·YL, B^S B^S males and their y/y⁺Y/YL·YL, B^S B^S sons were tested for fertility and examined for defects in spermatogenesis.

2. w/YL·YL, B^S B^S/ms(Y) males: FM7a, nod⁷/w females were crossed to X·YS, y Sxl v g f/YL·YL, B^S B^S males. Their FM7a, nod⁷/w/YL·YL, B^S B^S daughters were crossed to X·Y, y/ms(Y) males and the resulting w/YL·YL, B^S B^S/ms(Y) males were tested for fertility.

3. Ts(1Lt;YSt),y⁺/YL·YL, B^S B^S males: C(1)DX, y f/YL·YL, B^S B^S females were crossed to T(1;Y) males, and their Ts(1Lt;YSt),y⁺/YL·YL, B^S B^S sons were tested for fertility.

4. Ts(1Lt;YSt),y⁺/0 males: C(1)RM, y v/0 females were crossed to T(1;Y) males. The resulting Ts(1Lt;YSt),y⁺/0 sons that carried a terminal deletion from the YL telomere were examined for defects in spermatogenesis.

5. Synthetic deficiencies: Males carrying internal deletions of YL were synthesized by combining two different T(1;Y)s as described in KENNISON 1981 and HARDY et al. 1981 .

Examination of mutant phenotypes by phase-contrast microscopy:
Testes of 1- to 3-day-old males were dissected out in PBS and transferred to a drop of PBS on a glass slide. The testes were torn open, squashed under the weight of a coverslip (KEMPHUES et al. 1980 ), and examined by phase-contrast microscopy for defects in primary and secondary spermatocytes, and spermatid tails. This method was also used to observe needle-shaped crystals associated with the Ste phenotype in primary spermatocytes.

Staining of testes with rhodamine-conjugated phalloidin and DAPI:
Testes were removed from 20 1- to 3-day-old males in PBS and fixed for 10 min in a rotating 1.5-ml Eppendorf tube with 1 ml 6% formaldehyde in phosphate buffer (16 mM KH₂PO₄/K₂HPO₄, pH 6.8, 75 mM KCl, 25 mM NaCl, 4 mM MgCl₂). Tissues were washed twice for 5 min each with PBS and stained for 20 min with rhodamine-conjugated phalloidin at 8 U/ml in PBS (Molecular Probes, Eugene, OR). Tissues were then washed twice for 5 min each in PBS and stained for 10 min with DAPI (0.5 µg/ml in PBS). After staining, individual testes were transferred into 10 µl DAPI staining solution on a coverslip. Under a dissection microscope, the sheath was peeled away with a glass scalpel, which was made by pulling a micropipette (1- to 5-µl Micro-Pipet; Fisher Scientific, Pittsburgh) over a flame, cooling, and breaking to produce an extremely sharp end. The samples were mounted in ProLong antifade (Molecular Probes) and viewed with an Olympus Provis microscope equipped with filters to observe epiflourescence illumination. Images from the DAPI and rhodamine channels were captured individually by using a cooled CCD digital camera (SPOT; Diagnostic Instruments Inc.) and processed by using Adobe Photoshop software (Adobe Systems Inc.) on an Apple computer. The above procedure was performed at least three times for each mutant genotype. All reactions were performed at room temperature.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Two rearranged Y chromosomes impaired male fertility when combined with a regular Y chromosome:
In D. melanogaster, an extra Y chromosome in either males or females causes no adverse effect. Results from fertility tests (Table 1) showed that males carrying an extra Y short arm (X·YS/y⁺Y) and males carrying an entire extra Y chromosome (X·Y/Y) produced 111.1 progeny/male and 122.7 progeny/male, respectively, similar to males carrying a regular Y chromosome (y/Y; 98.5 progeny/male), a marked Y chromosome (y/y⁺Y; 120.0 progeny/male), or a Y chromosome that is attached to an X chromosome (X·Y/0; 96.4 progeny/male). However, the presence of three copies of the Y chromosome induces dominant male sterility (COOPER 1956 ) and YL is responsible for the triplosterile phenotypes (WILLIAMSON and MEIDINGER 1979 ).

To investigate whether the YL materials duplicated onto the short arms of the Y2 (h1-h3) and Y146 chromosomes (h1-h4) affect male fertility, y/y⁺Y/Y146 and y/y⁺Y/Y2 males were produced via X-chromosome nondisjunction in FM7a, nod⁷/y females (MATERIALS AND METHODS). As shown in Table 1, none of the y/y⁺Y/Y146 males (N = 70) were fertile, as assayed by individually mating to females. In similar fertility tests, none of the y/y⁺Y/Y2 males (N = 60) produced progeny. Thus, dominant male sterility was induced by combining either Y2 or Y146 with a normal Y chromosome. Since neither X·YS/Y2 nor X·YS/Y146 males displayed significantly lower fertility than the controls (Table 1), the dominant sterility observed for y/y⁺Y/Y2 and y/y⁺Y/Y146 males most likely resulted from the presence of three copies of the h1-h3 region and the h1-h4 region.

The effects of the kl-3 and kl-5 loci on the triplosterility:
The kl-3 and kl-5 loci (Fig 1) are located within the duplicated YL segments on the rearranged Y chromosomes, i.e., h1-h3 on Y2 and h1-h4 on Y146. Thus, Y2 and Y146 presumably carry two copies of the kl-5⁺ allele and Y146 possibly carries two copies of the kl-3⁺ allele. To address whether three copies of the kl-5⁺ or kl-3⁺ alleles cause male sterility in y/y⁺Y/Y2 and y/y⁺Y/Y146 males, we employed four Y chromosomes that carry ms(Y) mutations in kl-3 and kl-5. These alleles, all of which are induced by P-element insertions (ZHANG and STANKIEWICZ 1998 ), are as follows: kl-3²⁸, kl-3⁶¹, kl-3^104b, and kl-5¹⁶. Fertility tests show that males of the following genotypes were sterile: y w/Y2/kl-3²⁸, y w/Y2/kl-3⁶¹, y w/Y2/kl-3^104b, and y w/Y2/kl-5¹⁶ (Table 1). In similar tests, no progeny were produced from males of y w/Y146/kl-3²⁸, y w/Y146/kl-3⁶¹, y w/Y146/kl-3^104b, and y w/Y146/kl-5¹⁶ (Table 1). The results show that the y w/Y2/ms(Y) and y w/Y146/ms(Y) males were sterile, despite removing a functional copy of the kl-3⁺ allele or the kl-5⁺ 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 involved in causing the triplosterile phenotypes.

The triplosterile phenotypes and the Y long arm:
To map the YL region responsible for the triplosterile phenotypes, we used a set of three reciprocal transloca-tion between the X and Y chromosomes, T(1;Y)V24, T(1;Y)W27, and T(1;Y)P7 (Fig 1). As shown in Table 1, both Ts(1Lt;YSt)P7,y⁺/Y146 and Ts(1Lt;YSt)W27,y⁺/Y146 males produced large numbers of progeny (88.7 and 98.8 progeny/male, respectively), comparable to wild-type X/Y males (96.6–120.0 progeny/male). Thus, combining the Y146 chromosome with a Y fragment from h10 to h25 had little, if any, effect on male fertility. In contrast, a significant reduction of male fertility was present in Ts(1Lt;YSt)V24,y⁺/Y146 males, which produced only 29.9 progeny/male (Table 1). The Ts(1Lt;YSt)V24,y⁺ segregant carries the entire Y chromosome except for region h1-h3 (Fig 1). The sharp decline in male fertility is manifested more dramatically at the fertility level of the individual males. As plotted in Fig 2, the vast majority of individual males of X/y⁺Y; Ts(1Lt;YSt)P7,y⁺/Y146; and Ts(1Lt;YSt)PW27,y⁺/Y146 produced large numbers of progeny. For example, 95% of y/y⁺Y; 65% of Ts(1Lt;YSt)P7,y⁺/Y146; and 70% of Ts(1Lt;YSt)PW27,y⁺/Y146 males produced >80 progeny male. However, only 10% of Ts(1Lt;YSt)V24,y⁺/Y146 males produced >80 progeny/male, whereas most of them either failed to produce any progeny (19%) or produced very small numbers, e.g., 40 progeny/male (46%). The data suggest that an addition of the h4-h9 region, which is carried in the Ts(1Lt;YSt)V24,y⁺ chromosome, into males carrying the Y146 chromosome greatly reduced male fertility. This result agrees with a previous observation that three copies of the h4-h9 region cause male fertility problems (KENNISON 1981 ).

View larger version (22K): In this window In a new window Download PPT slide   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,y+ or y+Y. 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 >80 offspring.

In contrast, males of the following genotypes produced large numbers of offspring: Ts(1Lt;YSt)P7,y⁺/Y2; Ts(1Lt;YSt)W27,y⁺/Y2; and Ts(1Lt;YSt)V24,y⁺/Y2 (101.3, 90.1, and 107.7 progeny/male, respectively; see Table 1). Thus, a combination of Y2 and an YL fragment from h4 to h25 has no effect on male fertility, consistent with the observation that the duplication on Y2 is limited to h1-h3. Moreover, the results argue that an additional YL region, namely the h1-h3 region, plays an important role in inducing triplosterile phenotypes, since y/y⁺Y/Y2 and y/y⁺Y/Y146 males were completely sterile (Table 1 and Fig 2).

Postmeiotic defects associated with triplosterility:
In spermatogenesis of D. melanogaster (reviewed in LINDSLEY and TOKUYASU 1980 ; FULLER 1993 ), a germ-line stem cell undergoes four rounds of mitotic divisions, giving rise to 16 primary spermatocytes surrounded by 2 somatic cyst cells. After the meiotic divisions, 64 haploid round spermatids are produced in a cyst and undergo extensive syncytial cellular differentiation, including nuclear condensation, spermatid elongation, individualization, and coiling. By using a fluorescent probe, rhodamine-conjugated phalloidin, which binds to F-ACTIN, FABRIZIO et al. 1998 have shown that F-actin is a major component of the individualization complex (IC). ICs are formed at the aligned nuclear heads of spermatid bundles, traverse along the length of the tails, and resolve the syncytial spermatids of a cyst into 64 cells with individual membranes (TOKUYASU et al. 1972 ). Examination using a combination of two fluorescent probes, DAPI and rhodamine-conjugated phalloidin, shows that the individualization complexes in wild type are often associated with spermatid nuclear heads (FABRIZIO et al. 1998 ; Fig 3A and Fig B). A small fraction of ICs (approximately one-fourth) are located in positions away from the aligned nuclear heads, indicating that they have traveled along the tails.

View larger version (36K): In this window In a new window Download PPT slide   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) Composite 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 ). Approximately 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/y+Y/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,y+/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 µm.

When testes of either X/y⁺Y/Y146 or X/y⁺Y/Y2 were examined, departure from normal development was seen at early postmeiotic stages of spermatid differentiation (Fig 3, C–E; Table 2). Before nuclear elongation, round spermatid nuclei began to fall apart in a large number of spermatid bundles, resulting in singular nuclear heads that were dispersed throughout the tails (Fig 3C). In the more extreme case, accounting for 10% of the triplosterile males, the testes contained exclusively round spermatids scattered throughout the length of the tails. Although it appears that nuclear elongation in some of the scattered nuclei continued despite their earlier dissociation with each other, the dispersed nuclei were devoid of phalloidin staining, indicating that F-actin was unable to assemble around the scattered nuclei. In addition, many of the spermatid bundles with scattered nuclei were much smaller in diameter than normal ones, suggesting that some of the spermatid tails failed to develop (Fig 3C). However, there were other spermatid bundles that displayed aligned nuclear heads associated with ICs, as in wild type (Fig 3D and Fig E). The number of such bundles with normal-looking ICs varied among males, ranging from 10 to 40% of the total spermatid cysts. The ICs were apparently arrested at the elongated nuclear heads, since all the observed ICs were localized with the aligned nuclear heads.

In semisterile males of Ts(1Lt;YSt)V24,y⁺/Y146, which contain three copies of the h4 region, postmeiotic defects were similar to but much less severe than in the triplosterile males of y/y⁺Y/Y2 and y/y⁺Y/Y146. For example, spermatid bundles with scattered singular nuclear heads were seen frequently (Fig 3F and Fig G), but accounted for only a small proportion of spermatid bundles. In contrast to those of the triplosterile males, some ICs in Ts(1Lt;YSt)V24,y⁺/Y146 were located away from the nuclear bundles, apparently resulting from caudal movement along the tails (Fig 3F and Fig G). Consistent with the observation that ICs traversed along spermatid tails of the semisterile mutants, we observed individualized spermatids in the basal region of the testes and mature sperm in the seminal vesicles (data not shown).

Postmeiotic defects associated with deficiencies within the h1-h10 region:
Males deficient for region h1-h3 (a kl-5 deletion) or h4-h9 (a kl-3 deletion) lost the outer dynein arms in the spermatid bundles (HARDY et al. 1981 ). kl-3 or kl-5 mutations induced by P-element insertions caused the same axonemal defect (ZHANG 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 in h1-h9 induced postmeiotic defects with disrupted ICs (Table 2 and Fig 4, A–F). These include deletions of h1-h3, h4-h9, and h1-h9. The mutant testes contained large numbers of spermatid bundles that displayed a phenotype similar to that of the triplosterile mutations, i.e., scattered nuclei throughout the spermatid bundles. However, unlike the triplosterile mutations in which round nuclei were dispersed (Fig 3C), the scattered singular nuclei in the deficiencies were needle shaped (Fig 4B). Furthermore, while a small number of the ICs were located with aligned nuclear bundles, as in wild type, most of the ICs of the mutants were disrupted in spermatid bundles (Fig 4, A–F). The dispersed ICs were fragmented and distributed along elongated spermatid bundles. They were cone shaped, with the blunt end pointing apically and the pointed end directed basally. The isolated sperm heads often appeared to trail the IC fragments. These rhodamine-staining cones are the individual investment cones formed around each individualizing spermatid (FABRIZIO et al. 1998 ). These cones normally migrate together in a compact mass (Fig 3A and Fig B). However, in these mutants the mass dispersed into single cones, each with its own sperm nucleus.

View larger version (34K): In this window In a new window Download PPT slide   Figure 4. Mutant testes of h1-h10 deletions stained with rhodamine-conjugated phalloidin and DAPI. Tissues were stained either with phalloidin (red) in A, C, E, and G or with phalloidin and DAPI (blue) in B, D, F, and H. Arrowheads indicate disrupted ICs associated with singular elongated nuclear heads that were scattered along the tails (A–F). Arrows 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). (A and B) Ts(1Lt;YSt)V24,y+/0 (deficiency from h1 to h3). (C and D) Ts(1Lt;YSt)W27,y+/0 (deficiency from h1 to h9). (E and F) Ts(1Lt;YSt)W27,y+/Ts(1Rt;YLt)V24,BS (synthetic deficiency from h4 to h9). (G and H) Ts(1Lt;YSt)P7,y+/Ts(1Rt;YLt)V24,BS (deficiency from h4 to h10). Bars, 10 µm.

We have also extended our examination into the adjacent region h10, which contains the kl-2 locus (Fig 1). In males carrying a synthetic deficiency for the h10 region, Ts(1Lt;YSt)P7,y⁺/Ts(1Rt;YLt)W27,B^S, normal sperm axonemes are present in ultrastructural studies (HARDY et al. 1984 ). By staining with DAPI and rhodamine-conjugated phalloidin, we show that ICs from this deficiency were formed at the elongated nuclear bundles and traversed caudally along the spermatid tails, as in wild type (Table 2), suggesting the spermatids were individualized. Phase-contrast examination showed that the mutant produced a large number of individualized spermatids in the basal region of the testes. However, the defective spermatids were degraded before entering the seminal vesicles.

Interestingly, two larger deficiencies, Ts(1Lt;YSt)P7,y⁺/0 (deficient for h1-h10) and Ts(1Lt;YSt)P7,y⁺/Ts(1Rt;YLt)V24,B^S (deficient for h4-h10), exhibited a striking defect at the individualization process. In the mutant testes, there were no individualized spermatids and nearly every IC was associated with a nuclear bundle (Fig 4G and Fig H; Table 2), indicating that the IC was arrested at the nuclear head. In addition, the nuclear heads of the deficiencies appear to be slightly less condensed than those of the wild type. Although we occasionally observed ICs that were located along the spermatid tails, away from the nuclear bundles (0.5 IC per male), none were located near the apical tip of the testes (i.e., at the opposite end of the nuclear bundles). Thus, the mutants were unable to produce individualized spermatids, which was confirmed by phase-contrast microscopy (data not shown).

Spermatid individualization and ms(Y) mutations in h1-h9:
In contrast to the individualization defects associated with the h1-h10 deficiencies, as shown above, the individualization process in males carrying kl-3 or kl-5 mutations appeared to be normal. By staining with DAPI and rhodamine-conjugated phalloidin, normal-looking ICs were seen in the mutant testes of X/kl-3²⁸, X/kl-3⁶¹, X/kl-3^104b, and X/kl-5¹⁶ (Table 2). Some ICs were localized with aligned nuclear bundles, whereas others were located along the tails, away from the nuclear bundles. The results suggest that the ICs were able to traverse caudally along the tails to produce individualized spermatids. The hypothesis was confirmed in experiments using phase-contrast microscopy, which showed the presence of individualized spermatids in the mutant testes. Although we found no obvious abnormality in the individualization process by using the fluorescent probes, the individualized spermatids displayed abnormal morphology (Fig 5). The defective spermatids degenerated before reaching the seminal vesicle where mature sperm were stored.

View larger version (124K): In this window In a new window Download PPT slide   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 irregularities 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 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Hyperploidy of YL and the triplosterility:
Males with three doses of region h4-h9 are sterile (KENNISON 1981 ), but motile sperm are present in the seminal vesicles where usually only mature sperm are stored (HARDY et al. 1981 ). However, three doses of YL cause male sterility without motile sperm (WILLIAMSON and MEIDINGER 1979 ). The observation that hyperploidies of region h4-h9 and YL induce different phenotypes raises the question that genetic information on YL, in addition to h4-h9, is involved in the triplosterile effect. Our analysis shows that separable regions on YL are responsible for the triplosterility. Region h4-h9 can be further divided into two segments on the basis of effects on male fertility. Since three copies of region h4 induce only semisterility (Table 1), region h5-h9 must play a critical role in causing the complete triplosterility of males carrying three doses of region h4-h9.

Although a previous survey (KENNISON 1981 ) has shown that h4-h9 was the only YL region that causes male sterility with three doses, our analysis has revealed that y/y⁺Y/Y2 males carrying three copies of region h1-h3 were sterile (Table 1). We have further determined that two additional genotypes with three copies of the h1-h3 region, X·Y^S/Y2/Ts(1Rt;YLt)V24,B^S and X·Y^S/Y146/Ts(1Rt;YLt)V24,B^S, were also sterile (data not shown). The reasons why the previous study failed to observe the effect of the h1-h3 region on the triplosterility remain unknown. It is possible that the Y-chromosome derivatives used in these studies behaved differently: (1) Y2 and Y146 used in this study were derived directly from B^SYy⁺, whereas KENNISON 1981 employed a highly derived w⁺Y2 chromosome (LINDSLEY and ZIMM 1992 ); and (2) we used Ts(1Rt;YLt)V24,B^S carrying the h1-h3 region. KENNISON 1981 employed at least two different T(1;Y)'s, in addition to w⁺Y2, to generate three copies of the h1-h3 region, although the Y derivatives used in the experiments were not specified. Differential effect on male fertility has been described for some Y chromosomes, which induced male sterility with two doses (GRELL 1969 ).

Consistent with the observation that dispersed factors on YL cause the triplosterility, genetic complementation analysis indicates that the kl-3 and kl-5 genes do not affect the triplosterility. By using four Y chromosomes carrying ms(Y) mutations in kl-3 and kl-5, we have shown that X/Y2/ms(Y) or X/Y146/ms(Y) males, which carry three copies of the h1-h3 or h1-h4 region but only two doses of the functional kl-3⁺ or kl-5⁺ loci, are sterile (Table 1).

Defects associated with the hyperploidy of YL:
The most striking phenotype characteristic of the triplosterile testes is the presence of numerous round nuclei caudally dispersed along the length of the sperm tails (Fig 3C). Staining with DAPI and rhodamine-conjugated phalloidin reveals that F-actin fails to accumulate around the dispersed nuclei, although some of the nuclei display elongating morphology, indicating that nuclear elongation is initiated but incomplete. In addition, a small number of ICs are formed but are always located with aligned nuclear heads, suggesting that the ICs are stalled.

The pleiotropic defects associated with the triplosterility may be caused indirectly by an earlier lesion in spermatogenesis. Since genetic control in spermatogenesis is not a linear cascade of dependent steps, early lesions in the male germ-line cells often do not block late stages of spermatogenesis (LIFSCHYTZ 1987 ; FULLER 1993 ). It is also possible that the triplication of the h1-h10 region disrupts multiple developmental steps during spermatogenesis. Unlike most mutations that arise by altering gene structures, the triplosterile mutation does not impair genetic function on YL. Rather, the triplosterility results from hyperploidy of YL that may interfere with several genetic functions in spermiogenesis.

Relationship between deficiencies and ms(Y) mutations:
The repetitive nature, absence of meiotic recombination, and absence of polytene banding have made it very difficult to elucidate functions of the Y chromosome in spermatogenesis. Studies on functional properties of the YL region containing kl-3 and kl-5 have been carried out mostly by examining defects associated with large chromosome rearrangements such as deficiencies, X·Y translocations, and Y-autosome translocations (HARDY et al. 1981 ; KENNISON 1981 ; GOLDSTEIN et al. 1982 ; BONACCORSI et al. 1988 ; BONACCORSI and LOHE 1991 ). The results obtained from these studies likely represent a composite of functions of the fertility genes and the corresponding genomic regions. Here we employed ms(Y) mutations of kl-3 and kl-5, which are induced by single P-element insertions, to reveal defects specifically associated with mutations in the fertility genes. All four alleles of kl-3 and kl-5 induce male sterility with characteristic phenotypes, i.e., lost or greatly reduced outer dynein arms of the sperm tail (ZHANG and STANKIEWICZ 1998 ). Nonetheless, the individualization process continues to produce individualized spermatids (Fig 5 and Table 2). In contrast, males deficient for regions h1-h3, h4-h9, and h1-h9 exhibit defects in the individualization process (Table 2). The disrupted individualization complexes display cone-shaped IC components with the elongated nuclei that are scattered throughout the length of the tail (Fig 4).

We propose that region h1-h9 harbors two types of separable functions. One is encoded by the kl-3 and kl-5 fertility genes that are mutable to male sterility with loss of the axonemal outer dynein arms. The other function is revealed by the individualization defects associated with deficiencies within the h1-h9 region. An alternative explanation to the phenotypic difference between the deficiencies and the ms(Y) mutations is that the ms(Y) mutations are not null alleles. Although we cannot rule it out, this possibility is very unlikely since the two functions of organizing the outer dynein arms and spermatid individualization appear to be distinct.

Relationship between the triplosterility and the deficiencies within h1-h9:
The effects of the triplosterility appeared at early postmeiotic stages, whereas lesions of the h1-h9 deficiencies were not seen until the individualization process (Table 2). Despite the phenotypic difference, the triplosterility shares several similar features with deficiencies in h1-h9. First, in both cases, region h1-h9 can be divided into segments that play similar roles, as judged by fertility in the triplosterility or defects in the individualization process. Second, the phenotypes associated with the triplosterility and the h1-h9 deficiencies seem to be independent of the fertility factors kl-3 and kl-5, which are located in this region. Finally, segments within h1-h9 may exert cumulative effect on both the triplosterility and spermatid individualization. For example, three doses of region h4 induce semisterility (Table 1), but three doses of region h4-h9 induce complete sterility (KENNISON 1981 ). A similar cumulative effect has been indicated by the h1-h10 deficiency, which displays largely arrested ICs at the aligned nuclear bundles. This defect occurs at an earlier stage of spermiogenesis than that of the h1-h9 or h10 deficiencies (Table 2). The analysis indicates that the genetic factors responsible for both the triplosterility and spermatid individualization are dispersed on YL and are functionally redundant, suggesting the involvement of heterochromatic repetitive sequences.

The possible functions of the YL region:
In the Y chromosome of D. melanogaster, nine satellite sequences and the rDNA repeats account for 80% of the DNA sequences (APPELS and PEACOCK 1978 ; LOHE et al. 1993 ). In addition to several types of middle repetitive elements related to transposable elements (PIMPINELLI et al. 1995 ; ZHANG and SPRADLING 1995 ), the vast majority of the DNA sequence within the h1-h9 region is composed of six satellite repeats of 5- to 7-bp (BONACCORSI and LOHE 1991 ; LOHE et al. 1993 ).

The heterochromatic regions of Drosophila chromosomes behave as suppressors of position effect variegation (PEV), a genetic phenomenon in which a euchromatic gene is relocated to heterochromatin and is inactivated by the juxtaposed heterochromatin (SPOFFORD 1976 ; ELGIN 1996 ). The heterochromatic Y chromosome acts as a strong PEV suppressor (SPOFFORD 1976 ; HENIKOFF 1992 ; LLOYD et al. 1997 ). Moreover, various segments along the length of the Y chromosome are known to suppress PEV in an additive manner, such that the degree of PEV suppression is directly related to the amount of the Y materials (DIMITRI and PISANO 1989 ). Although there are additive effects on triplosterility, the finding that the region responsible for the triplosterile lesions is delimited to h1-h9 argues that the genetic complexity of h1-h9 is independent of PEV, which is not limited to any particular region of the Y chromosome.

There has been extensive discussion over the potential roles of the lampbrush loop-like structures that are present in the primary spermatocyte nuclei of >50 Drosophila species (HACKSTEIN 1987 ). It has been shown that the kl-3 and kl-5 regions are correlated with the presence of two giant lampbrush loop-like structures in primary spermatocytes and that the kl-3 loop binds a non-Y-encoded and testis-specific protein (BONACCORSI et al. 1988 ; PISANO et al. 1993 ). Three satellite elements in the region, AAGAC, AAGAG, and AATAA, are abundantly transcribed in primary spermatocytes and remain on the loops where they are synthesized (BONACCORSI et al. 1990 ; GATTI and PIMPINELLI 1992 ). The unusual properties of the loops, combined with the noncoding nature of the satellite sequences, have led to theories that the peculiar nuclear structures in primary spermatocytes have a protein-binding role (HENNIG 1985 , HENNIG 1993 ; BONACCORSI et al. 1988 ; GATTI and PIMPINELLI 1992 ). Accordingly, the loops, which are in essence subnuclear organelles, would act as protein-binding sites for post-translational processing of some proteins important in spermatogenesis.

The proposed roles of the nuclear loops are largely undefined. Several blocks of satellite sequences in the h1-h9 region are known binding sites for a growing number of proteins that remodel chromatin structure and regulate gene expression (CSINK and HENIKOFF 1998 ). The Trithorax-like protein binds to AAGAA and AAGAGAG simple repeats of region h1-h9 on the mitotic Y chromosome (PLATERO et al. 1998 ), whereas the ORC2 protein binds to the AT-rich repeats of region h1-h9 (PAK et al. 1998 ). The interaction of the proteins with satellite DNA may help to recruit other proteins onto the Y chromosome, such as HP1, another component of chromatin structures found on the Y chromosome (ELGIN 1996 ; PLATERO et al. 1998 ; WALLRATH 1998 ). These results provide an important avenue for investigating how the heterochromatic h1-h9 region plays essential roles in spermatid differentiation.

	ACKNOWLEDGMENTS

We are grateful to Kent Golic for the rearranged Y chromosomes and technical advice. We thank Christopher Bazinet for technical advice on staining with the fluorescent probes. We also thank the Bloomington Drosophila Stock Center for providing stocks. Finally, we thank Linda Strausbaugh for critical comments on the manuscript. This work was supported by a grant from the University of Connecticut Research Foundation.

Manuscript received October 21, 1999; Accepted for publication January 24, 2000.

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