Hidden Effects of X Chromosome Introgressions on Spermatogenesis in Drosophila simulans x D. mauritiana Hybrids Unveiled by Interactions Among Minor Genetic Factors

Corresponding author: Horacio F. Naveira, Departamento de Bioloxía Celular e Molecular, Facultade de Ciencias, Universidade de A Coruña, Campus da Zapateira s/n, 15071 A Coruña, Spain., horaci{at}udc.es (E-mail).

Communicating editor: C.-I WU

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

One of the most frequent outcomes of interspecific hybridizations in Drosophila is hybrid male sterility. Genetic dissection of this reproductive barrier has revealed that the number of responsible factors is very high and that these factors are frequently engaged in complex epistatic interactions. Traditionally, research strategies have been based on contrasting introgressions of chromosome segments that produce male sterility with those that allow fertility. Few studies have investigated the phenotypes associated with the boundary between fertility and sterility. In this study, we cointrogressed three different X chromosome segments from Drosophila mauritiana into D. simulans. Hybrid males with these three segments are usually fertile, by conventional fertility assays. However, their spermatogenesis shows a significant slowdown, most manifest at lower temperatures. Each of the three introgressed segments retards the arrival of sperm to the seminal vesicles. Other small disturbances in spermatogenesis are evident, which altogether lead to an overall reduction in the amount of motile sperm in their seminal vesicles. These results suggest that a delay in the timing of spermatogenesis, which might be brought about by the cumulative action of many different factors of minor segment, may be the primary cause of hybrid male sterility.

HYBRID male sterility factors in Drosophila apparently have evolved more rapidly than either female sterility or inviability determinants (WU 1992 ; HOLLOCHER and WU 1996 ; TRUE et al. 1996 ). However, we still do not know what these factors really are. The preceding statement is actually based on the study of introgressions of chromosome segments, not of single genes. Although our knowledge on this matter has considerably increased in the last few years, and despite the formidable power of DNA technology, there are no published accounts of the molecular characterization of any hybrid sterility gene thus far. But some of these factors should be clonable in the foreseeable future (e.g., Odysseus, PEREZ and WU 1995 ). In our opinion, the reason for the difficulty in obtaining this kind of result lies in the finding that multilocus weak allele interactions are probably the most common cause of hybrid male sterility in Drosophila (NAVEIRA and FONTDEVILA 1986 , NAVEIRA and FONTDEVILA 1991 ; NAVEIRA 1992 ; CABOT et al. 1994 ; PALOPOLI and WU 1994 ; WU and PALOPOLI 1994 ; PEREZ and WU 1995 ; DAVIS and WU 1996 ; NAVEIRA and MASIDE 1998 ). Thus, sterility is usually brought about by the cointrogression in an intact chromosome segment of an indeterminate number of linked interacting factors. When an introgressed segment that produces sterility is split by recombination into smaller segments, some of them retain partial or full sterility effects, but, if the dissection is continued, sterility finally vanishes: one gene is simply not enough.

Fertility testing of introgressions is usually carried out by observing the ability to leave offspring after single or mass matings, or simply by checking for the presence of motile sperm in seminal vesicles. Higher resolution of introgression effects would be possible through progeny counts (JOHNSON et al. 1993 ), as well as through the analysis of spermatogenesis. Thus, for this second approximation, in sterile hybrids between Drosophila buzzatii and D. koepferae, several kinds of abnormalities in spermatogenesis have been described, which apparently result from the cumulative action of minor genetic factors (NAVEIRA and FONTDEVILA 1991 ). In hybrids between D. mauritiana and D. simulans, larger perturbations of spermatogenesis as introgression is increased are well documented (PEREZ et al. 1993 ; CABOT et al. 1994 ; DAVIS and WU 1996 ), although backcross introgressive hybrids usually display a more severe phenotype than F₁ males (WU et al. 1993 ). However, there is little information on the defects associated with the transition from fertility to sterility. "Semisterile" males in buzzatii x koepferae hybrids and "subfertile" and "quasi-sterile" males in mauritiana x simulans hybrids have in common a reduced ability to produce motile sperm and leave offspring, but no apparent abnormality in their spermatogenesis has been found except for an accumulation of coiled sperm bundles at the entrance to the seminal vesicles. It is our intention with this article to fill in this gap and show the subtle disturbances that introgression often brings about in the spermatogenesis of fertile and subfertile hybrid males.

We have introgressed three X-linked regions from D. mauritiana into D. simulans. Hybrid males bearing just a single one of these regions, any pairwise combination, or even the full set of the three of them are able to leave offspring, although the amount of motile sperm is severely reduced in some of the genotypic classes. Their spermatogenesis is considerably slowed down, but cytological abnormalities are not very conspicuous: (1) a different shape in the nucleolus of mature primary spermatocytes; (2) a higher than normal number of nuclei in the tail region of elongated cysts; and (3) an accumulation of degenerating coiled sperm bundles, with scattered sperm nuclei, at the basal region of the testis. The delay in the timing of spermatogenesis may be the primary effect of introgressions, which later gives rise to all the observed cytological abnormalities and eventually to a reduced production of motile sperm. This effect is increased by the combined action of different introgressed factors. It is our hypothesis that complete sterility results from pushing this time lag too far, after the introgression of a "sufficient" number of factors (polygenic combination) scattered throughout the genome.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and mutants:
A D. simulans strain carrying the X-linked mutations y, w, v, m, and f (y, yellow, 1B; w, white, 3C; v, vermilion, 10A; m, miniature, 10E; f, forked, 15F) was constructed from existing mutant stocks. Females from this strain were crossed with males from a wild-type strain of D. mauritiana, and F₁ hybrid females were backcrossed to the D. simulans strain (F₁ hybrid males are always sterile). Individual backcrosses were repeated during several (at least 10) generations in three different directions by selecting just one of these three markers: y⁺, m⁺, or f⁺. Males bearing any of these markers are introgressive hybrids. At the beginning of the introgressive hybridization most of these males are sterile, but after several backcross generations fertile males can be easily obtained, after the elimination of the major part of the D. mauritiana genome (COYNE and CHARLESWORTH 1989 ). Some of these fertile males were individually used to establish fixed hybrid stocks for each of the three visible markers (strains of types A, B, and C in Figure 1), following a simple crossing scheme (Figure 2). Each of these strains contained an X chromosome region of undetermined length from D. mauritiana, produced by crossing over and marked by the pertinent wild-type allele on an otherwise D. simulans genetic background. The intact chromosome regions surrounding each marker apparently harbor minor factors of hybrid male sterility, which may give rise to sterile males when recombined into the same genome (NAVEIRA 1992 ). Strains of type A (marked by y⁺) were crossed with strains of type C (marked by f⁺), so that the fertility of hybrid males incorporating both parental introgressions could be finally assessed (Figure 2). Those combinations of y⁺ and f⁺ introgressions that still allowed hybrid male fertility were selected and used to obtain fixed hybrid strains incorporating both markers and the corresponding intact X chromosome segments from D. mauritiana (strains of type A+C in Figure 2). Finally, in an analogous way, a segment marked by m⁺ was added to the genotype, thus giving rise to the fixed hybrid stock y⁺ m⁺ f ⁺ (H in Figure 1) that we used in our experiments.

View larger version (21K): In this window In a new window Download PPT slide   Figure 1. Crossing protocol to generate the fixed hybrid stock (H) used in the experiments described in this article. Three kinds of X chromosome segments (marked by alleles y+, m+, and f+) were separately introgressed from D. mauritiana into D. simulans. Rectangles, chromosomes; large rectangles, sex pair (the Y ended by a hook); small rectangles, autosomes (first and last steps); A, B, and C, collections of fixed hybrid strains with single-introgressed segments produced as shown in Figure 2. Strain H incorporates three introgressed segments from different regions of the X chromosome, after appropriate crosses involving strains of type A and C (see Figure 2) and B.

View larger version (23K): In this window In a new window Download PPT slide   Figure 2. Detailed crossing protocol to generate fixed hybrid strains of type A and A plus C in Figure 1. Strains of type A represent a collection of introgressions of the distal part of the X chromosome from D. mauritiana, marked with y+, that allow hybrid male fertility. Similar protocols were applied to obtain strain collections of types B and C (Figure 1). Strains of type A plus C result from recombining introgressed segments from A (y+) and C (f+) into the same chromosome (then marked both with y+ and f+), as an intermediate step toward the final obtention of strain H (Figure 1). Wild-type alleles always mark chromosome segments from D. mauritiana. Fertile hybrid males that initiate the crossing protocol are shown in Figure 1. The stage denoted "establishment" consists of single-brother-sister matings for five consecutive generations, so that the stock can safely be considered genetically homogeneous.

The D. simulans m and v strains were kindly supplied by J. Coyne. All the other mutant strains of this species were provided by the Mid-American Drosophila Stock Center (Bowling Green, OH). The D. mauritiana wild-type stock was supplied by J. F. McDonald from a collection by J. David (CNRS, France). All crosses were carried out using instant Drosophila medium, formula 4-24 from Carolina Biological Supply Company, at room temperature (21–25°), unless otherwise specified.

DNA markers:
Approximate lengths of intact chromosome segments cointrogressed with y⁺, m⁺, and f⁺ were determined on the basis of the species-specific DNA sequence of PCR products from the loci armadillo (arm, placed in cytological interval 2B15), RNA polymeraseII-215-kD subunit (RpII215, 10C2-5), gastrulation defective (gd, 11A7-8), rudimentary (r, 15A), beta Spectrin (ßSpec, 16C1-4), and Shaker (Sh, 16F). DNA from individual flies was extracted according to protocol 48 of ASHBURNER 1989 . From each sample of genomic DNA, a single PCR amplification was performed. Primers for RpII215 and gd were the same as those in DAVIS and WU 1996 . Primer sequences for the other loci were as follows: (forward and reverse) arm, 5' TAA ACG AGC GTG CTG GTC GGT CG 3' (beginning at nucleotide 1311) and 5' TGG TTC GAT TCT GGG CTG GCA TG 3' (beginning at nucleotide 1840); r, 5' GCA GCA AAG AGA GAC GTT CGC 3' (beginning at nucleotide 2939) and 5' GGT AAC AGT CCG TAG AGG CCA 3' (beginning at nucleotide 3787); ßSpec, 5' AAA CCG TAG ATC CCC AGC CA 3' (beginning at nucleotide 110) and 5' GAG GGA AGC ATT GCC ATC GA 3' (beginning at nucleotide 551); Sh, 5' CCA AAG TGA AGG CCG AGG AGG TAC 3' (beginning at nucleotide 3608) and 5' GAG ATT CCT GGG ACT GCC GCA ACG 3' (beginning at nucleotide 4092). All the nucleotide positions of the 5' oligonucleotide base correspond to published D. melanogaster sequences (GenBank accession numbers X54468, M37783, M92288, and X58188, respectively). Expected PCR product sizes were 529 (arm), 442 (RpII215), 244 (gd), 848 (r), 441 (ßSpec), and 484 bp (Sh). Primer annealing temperature was 55° for all reactions, except for gd (50°). DNA fragments amplified from the two parental species and from the fixed hybrid strain y⁺ m⁺ f⁺ were cloned in pGEM-T (Promega, Madison, WI). For each strain, the sequences of both strands were determined by the dideoxynucleotide method (SANGER et al. 1977 ) with the AutoRead sequencing kit (Pharmacia, Piscataway, NJ) on a ALF express-automated DNA sequencer (Pharmacia) from universal vector primers. Sequence analysis and alignment of the different gene regions were performed using the CLUSTAL V program (HIGGINS et al. 1992 ).

Assays of male fertility:
The fertility of hybrid males and controls was determined by dissection. Flies were cultured at 23° during at least one generation, and emerging males were aged for at least 3 days at this same temperature before the assay. Testes and vasa deferentia were dissected in 0.9% NaCl saline solution, and the seminal vesicles were checked for the presence of motile sperm. Males were classified as fertile if any motile sperm were observed. Average time for sperm to be released into 50% of the seminal vesicles (t₅₀) and 95% confidence intervals was estimated by probit regression analysis after log transformation of the time from ecdysis (in hours), using SPSS 7.5. One generation of flies was raised at the corresponding temperature (either 18° or 21°), and the emerging males were aged at this temperature before the assay, which was carried out for age intervals of 6–24 hr (males grown at 18°) or 6–12 hr (21°), by the same technique described above.

Spermatogenesis:
The analysis of spermatogenesis was performed by three different methods. Live premeiotic and postmeiotic stages, up to the beginning of spermatid cyst elongation, were characterized under phase contrast optics, after dissection of the testes in saline solution and very gentle squashing. To determine the number of cysts in either preindividualization, individualization, or spiralization stages (Figure 3), testes were dissected in saline solution, transferred to 45% acetic acid for 10 sec and then to a drop of lactic acetic orcein (60:40), gently squashed in the same solution, and examined immediately under phase contrast. This same technique was applied to count spermatid nuclei in the head region of the cysts. In contrast, counts of nuclei in the tail region were obtained after staining with DAPI (4',6-daimidino-2-phenylindole dichloride) and observation with a fluorescence microscope: testes were dissected in saline solution and their sheaths removed to allow the cysts to spread in the fluid; the tissue was air-dried, immediately fixed in a drop of alcohol (70%), air-dried again, and stained in the dark with a drop of DAPI (0.5 µg/ml in 0.18 M Tris-HCl, pH 7.5); when completely dried, the slides were mounted in fluormount and stored in the dark at 4°.

View larger version (178K): In this window In a new window Download PPT slide   Figure 3. Head regions of three cysts, one of them with individualized spermatid nuclei (arrow), the other two with elongated but still nonindividualized spermatids (arrowheads). Orcein staining under phase contrast.

Microscopy:
Live and orcein-stained slides were examined under phase contrast with an Olympus photomicroscope (Olympus, Lake Success, NY), objective UPlanFl 0.75 Ph2 x40. DAPI-stained slides were examined with a Nikon photomicroscope with epifluorescence equipment (Nikon, Melville, NY). A high-pressure mercury lamp DC 100 W USHIO was used for illumination, with Nikon filter combination EX330~380, DM400, BA420, and objective CF Fluor 0.85 x40. Photographs were taken on either Agfapan 25 Professional or Kodak Gold 100 film.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have introduced three marker alleles of the X chromosome from D. mauritiana into D. simulans by repeated backcrossing. The presence of each of these markers actually indicates the cointrogression of a linked intact chromosome segment from D. mauritiana, containing an indeterminate number of other genes from this species, which may or may not affect hybrid male fertility. Among the several independent lines thus obtained, we have selected three in which hybrid male fertility is apparently not affected (strains of type A, B, and C in Figure 1). As shown in Table 1, sterility frequencies at 23° in these hybrid males were very low and similar to those of pure D. simulans (0.4–0.8%). When the three different introgressed segments were recombined into the same genome to build strain H (Figure 1 and Figure 2), sterility frequencies increased slightly to 4.6% (² = 8.33, P = 0.004). Hybrid males of strain H are thus usually fertile. However, DAPI staining of their spermatid cysts revealed a large difference in D. simulans controls (Table 2, ² = 182.5, P < 0.5%), showing that many unelongated spermatid nuclei are displaced from their normal position in the head region of the cysts. Thus, 58% of the cysts in these hybrids contained at least 11 nuclei (over a maximum of 64) lagging in the tail region, compared to only 1% in D. simulans. Or, shown in a different way, whereas nearly 52% of the cysts of D. simulans had all the nuclei properly positioned in the head region, not a single cyst of this kind was found in the hybrids, which 90% of the time had only between 30 and 60 nuclei in this region (Table 3). No conspicuous differences in size were apparent among nuclei, in either head or tail regions. Orcein staining of relatively undisturbed elongating or fully elongated cysts shows the progressive enrichment in individualization and spiralization stages within the testis, as flies develop (Table 4). So, in young pupae of D. simulans, 73% of the cysts contained spermatids that had not begun to individualize, in 26%, individualization had already started, and in only 1%, spermatid spiralization was taking place. These numbers changed to 40, 45, and 15%, respectively, in recently emerged adults (< 6 hr since ecdysis). Hybrids show a similar pattern, but are considerably delayed in developmental time. So, the contents of the testes of recently emerged hybrid adults strongly resemble those of D. simulans young pupae (² = 1.8, P = 0.407). Correspondingly, the vast majority (94%) of the cysts from young hybrid pupae contained spermatids that had not even begun to individualize, and it was in adults 18–36 hr old that the expected increase in individualization and spiralization stages was observed. More evidence of this apparent delay in the timing of hybrid spermatogenesis is provided by the analysis of live cells in the testes of third-instar larvae and prepupae (Table 5). Up to 27% of the testes from D. simulans larvae contained at least a few elongating cysts, but in only 2% of the testes of hybrid larvae had spermatogenesis arrived thus far; in fact, the most advanced stages reached by spermatogenesis in the majority (48%) of hybrid larvae were primary spermatocytes, while all the testes inspected in D. simulans contained several cysts that had already left meiosis behind. In hybrid prepupae, although elongating cysts are frequently observed (13%), 31% of the testes had not gone beyond premeiotic or meiotic stages.

All the observations described above were carried out at 23° on hybrids from strain H, which bears three different introgressed X chromosome segments. To investigate the contribution of each of these segments to the apparent slowdown of spermatogenesis, we recorded the time taken for sperm to be released into the seminal vesicles of different kinds of recombinant males, which contained just one or two of the D. mauritiana chromosome markers, as well as control males from strain H, D. simulans ywvmf, and D. mauritiana wild type. Flies were raised and aged at 18° to enhance the differences among genotypic classes (Table 6). As expected, males bearing the three chromosome markers (strain H) become fertile considerably later than D. simulans or D. mauritiana, which are not significantly different. However, the lag in the arrival of sperm to the seminal vesicles shows a clear interaction with temperature: the time for 50% of the males to become fertile (t₅₀) is nearly nine times greater for strain H than for D. simulans at 18°, but barely two times greater when the temperature is raised to 21°. All the introgressed segments retard the spermatogenesis at 18°, with the one marked by m⁺ making the largest contribution, but their effects are not independent. So, combinations m⁺f⁺ and y⁺f⁺ produce the same effect as single markers, whereas y⁺m⁺ produces an even greater slowdown than the cointrogression of the three segments. No dissection of the effects of the different markers has been carried out at 23°. At this temperature, all the D. simulans males 0–6 hr old had motile sperm. In contrast, no sperm were observed in males 0–6 hr old from stock H, but all the males examined in the interval 30–48 hr already had them.

As far as cytological disturbances in hybrid spermatogenesis are concerned, only two were noted in strain H, in addition to the misplacement and incomplete elongation of spermatid nuclei in the tail region of the cysts. First, abnormal nucleoli, with characteristic extrusions, were observed in all the mature primary spermatocytes (Figure 4). Second, there was an accumulation of degenerating coiled sperm bundles in the basal testicular region, just before the constriction that marks the entry to the seminal vesicle, which generally contained only a few motile sperm. The coiling stage is apparently normal at first, with sperm nuclei lying closely associated at the center of the coiled tails (Figure 5A), but then these nuclei are scattered (Figure 5B), and cysts finally degenerate.

View larger version (140K): In this window In a new window Download PPT slide   Figure 4. (A) Mature primary spermatocytes of hybrids (B) and D. simulans controls. In hybrids, the nucleolus, or bodies attached to it, display abnormal shapes (arrows). Live material under phase contrast.

View larger version (140K): In this window In a new window Download PPT slide   Figure 5. Coiling stages of spermiogenesis in hybrids. (A) Cyst in a relatively apical position within the testis, showing closely associated spermatid nuclei (arrow) in its head region. (B) Another cyst of the same male, localized near the basal constriction that marks the entrance to the seminal vesicle, showing the scattering of spermatid nuclei (arrowheads). Orcein staining under phase contrast.

All these disturbances in males from strain H lead to a reduction in their amount of motile sperm, but generally not so much as to prevent them from leaving at least some offspring. This is what DAVIS and WU 1996 designate as subfertility (a property of introgressions that cause a significant reduction, or even complete absence, of motile sperm, while producing at least some males that prove to be fertile by mating). Approximately 5% of males from strain H fail to produce motile sperm and are thus sterile (Table 1). However, no difference in spermatogenesis was observed between them and their fertile siblings. These exceptional cases of complete sterility within strain H may result simply from the whole blockage of the release of sperm into seminal vesicles.

To find out approximate lengths of introgressed segments from D. mauritiana in strain H, a high-resolution mapping experiment was undertaken. Six genes were examined both in the parental stocks of D. simulans and D. mauritiana and in the resulting hybrid strain H. The diagnostic features of the sequences obtained are outlined in Figure 6, where D. melanogaster is also included as a reference. Twelve nucleotide differences were found between D. mauritiana and D. simulans in the analyzed region of armadillo, 7 were found in RpII215, 10 in gastrulation defective, 1 in beta Spectrin, and 10 in Shaker. As regards rudimentary, despite several attempts with different DNA samples at annealing temperatures from 55° to 50°, the PCR product from D. mauritiana could not be observed in our routine agarose gels, whereas an abundant product was always obtained from D. simulans. Only after reduction of the annealing temperature to 48°, was a faint band of the expected size also observed in D. mauritiana. In this case, then, the origin of DNA (either simulans or mauritiana) in the hybrid strain was inferred simply by the presence or absence of a PCR product at the restrictive annealing temperatures. With the aid of these DNA markers and the information from visible mutants that we already had, we could establish approximate bounds for each of the three introgressed segments from D. mauritiana into the X chromosome of D. simulans. Our results are shown in Figure 7. The segment marked by yellow (y⁺) ends before reaching armadillo (i.e., it extends from the telomere to an undetermined point before 2B in the polytene map). The intact segment marked by miniature (m⁺) begins somewhere between vermilion (10A) and RpII215 (10C) and ends before gastrulation defective (11A). Finally, the segment marked by forked (f⁺) initiates after rudimentary (15A) and ends after beta Spectrin (16C), but before reaching Shaker (16F).

View larger version (45K): In this window In a new window Download PPT slide   Figure 6. Nucleotide differences among D. melanogaster, D. simulans, and D. mauritiana. First four rows show the base position relative to the aligned sequence of D. melanogaster obtained from GenBank; s, synonymous substitution in an exon; r, amino acid replacement substitution; l, length polymorphism in an exon; n, any kind of change within a noncoding region. Length polymorphism is indicated by an asterisk in sequences shortened relative to others.

View larger version (11K): In this window In a new window Download PPT slide   Figure 7. High-resolution mapping of D. mauritiana introgressions in hybrid strain H. Thick bars, introgressed segments from D. mauritiana; thin line, D. simulans material. The dotted line is used when the two adjacent markers exhibit different species patterns, so that the boundary of the introgressed segment must lie somewhere between them. Thick arrowheads show maximum limits of each introgressed segment. Both visible and DNA markers are shown above the top line, while their cytological locations are shown below it. Distances between markers within each of the three regions are drawn to scale.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

There is increasing evidence that the building blocks of hybrid male sterility are not single genes capable of causing sterility by themselves in a foreign background, but interacting gene sets made out of minor factors whose individual introgression has virtually no effect on male fertility (for reviews see WU and PALOPOLI 1994 ; MASIDE and NAVEIRA 1996 ; NAVEIRA and MASIDE 1998 ).

The results presented in this article indicate that the immediate effect of an introgression is, apparently, a simple delay in the timing of spermatogenesis that is enhanced when different introgressions are recombined into the same genome to give rise to subfertile males. No marked cytological disturbances were observed. Counts of spermatid nuclei per cyst and the similar sizes of these nuclei indicate that the four successive mitotic divisions of spermatogonia and the two meiotic divisions ordinarily take place. The somewhat abnormal shape of the nucleolus, or bodies attached to it, detected in all the mature primary spermatocytes of males from strain H, might indicate problems in the assembly of ribosomal particles and thus a reduction in overall translation rate. The abnormal abdomen syndrome of D. mercatorum, produced by a decrease in functional rDNA, actually causes delayed sexual maturation in males and yet no change in egg-to-adult development time (HOLLOCHER and TEMPLETON 1994 ). However, no bobbed-like phenotype was ever observed in the hybrids from strain H, and, therefore, if such a decline in translation rate actually occurs, it must be restricted to the germ line. All the morphogenetic events of spermiogenesis are apparently not disturbed in the hybrids, except perhaps the last one (entrapment). The accumulation of degenerating coiled cysts at the basal testicular region of subfertile males suggests an impairment of the entrapment stage, which would lead to the reduced amount of motile sperm observed in the seminal vesicles. It is conceivable that an occasional reinforcement of this barrier to sperm release may lead to the complete sterility observed in a few hybrids.

All our results show a considerable heterogeneity: within the same testis, between both testes of the same male, within hybrids of the same genotype raised at the same temperature, among different temperatures, and among different genotypes. In spite of this diversity, significant effects were detected.

First, the subfertility associated with some introgressions is extremely cold sensitive, so that it would be unwise to carry out sterility tests at temperatures below 21°. In this respect, introgressions seem to intensify the effects of temperature on the control cultures of D. simulans. In this species, it takes about 20 hr at 18° for 50% of the males to have motile sperm in their seminal vesicles, but newly eclosed males at 23° already have them. Apparently, the slowdown of developmental processes produced by raising the flies at lower temperatures affects the germ line more than the soma. This difference is greatly exacerbated in the hybrids.

Second, not all the introgressed segments produce the same effect. This was best shown at 18°. The segment marked by m⁺ brings about the largest delay in spermatogenesis, and f⁺ the smallest one. Besides, the three segments are engaged in complex interactions. Whereas the combination y⁺m⁺ retards spermatogenesis considerably more than expected from an independent additive contribution of each segment, the incorporation of f⁺ seems to alleviate this effect very moderately.

It is important to realize that the different introgressions do not exhibit qualitatively distinct effects on spermatogenesis. The differences among them are apparently a simple question of degree. On the other hand, they do not display conspicuous phenes, but a perturbation in the timing of spermatogenesis stages. However, consecutive morphogenetic events are controlled and executed by independent programs (LIFSCHYTZ 1987 ), so that at least a slight asynchrony should be expected in the hybrids. The introgressions studied in this article lead to nothing so evident as the precocious mitochondrial aggregation observed in some male sterile mutants (LIFSCHYTZ and HAREVEN 1977 ), but probably to minor differences in the pace of independent events, sufficient to produce the observed cytological abnormalities. It can be hypothesized that all three chromosome segments contain loci involved in a germ-line-specific timer, which should be operating before the onset of meiosis, because primary spermatocytes are undoubtedly affected. In principle there are two possibilities. It may be that the products of introgressed genes are involved in message degradation of a specific gene, analogous to the regulation of string in early Drosophila development (YASUDA and SCHUBIGER 1992 ). Or perhaps it is a system that depends on the titration of some material (e.g., transcriptional inhibitors) by DNA, similar to what might control midblastula transition in Xenopus (KIRSCHNER et al. 1985 ). In this last model, introgressed chromosome segments would have inferior titration efficiency compared to the recipient species' homologs they substitute, and a heterogeneous slowdown of independent sequential events would result. One way or another, when taken beyond a certain limit, the heterogeneity of this delay could finally bring about the complete sterility of hybrids and all the concomitant cytological disturbances. The very high number and overspread distribution of minor sterility factors (NAVEIRA and MASIDE 1998 ; WU et al. 1996 ), the similar nature of their effects (NAVEIRA and FONTDEVILA 1991 ), and their pervasive epistasis (NAVEIRA and FONTDEVILA 1991 ; PALOPOLI and WU 1994 ; PEREZ and WU 1995 ) seem to favor the second possibility.

The three X chromosome regions introgressed from D. mauritiana into D. simulans that we studied in this article had been the subject of previous analysis. First, COYNE and CHARLESWORTH 1989 suggested that each of these regions contained a single-sterility factor with complete penetrance (major gene). Later, NAVEIRA 1992 demonstrated that two of these regions contained minor interacting factors with incomplete penetrance. Finally, high-resolution mapping experiments by WU and co-workers led to the conclusion that no major genes of sterility (genes sufficient by themselves to cause sterility) were actually included in these chromosome regions and that a complex web of epistasis underlay this hybrid-restricted character (for review see PALOPOLI and WU 1994 ). Notwithstanding this general pattern, several factors of relatively strong effect were reported. First, for the distal part of the X chromosome, marked by yellow, inclusion of interval 4C–5E in the appropriate introgressed tract usually brings about sterility (CABOT et al. 1994 ). According to our high-resolution mapping, that cytological interval is not included in the segment actually introgressed in our hybrids (it goes from 1A to no farther than 2B), so that it cannot be responsible for the effects described here. Second, another strong factor was located in 11A, proximal to miniature, whereas a weak effect was traced to the region between vermilion and miniature (DAVIS and WU 1996 ). Of these two factors, probably only the weak one has been introgressed in our hybrids (the intact segment from mauritiana extends from 10A/10C to no farther than the proximal part of 11A). Finally, a large effect was mapped to the proximal region of forked, to interval 16C1–16D5, which harbors the Odysseus gene (PEREZ and WU 1995 ), while no factors were found distal to this marker up to 12B (PEREZ et al. 1993 ). In our case, it is quite possible that the intact segment from D. mauritiana marked by f⁺ includes Odysseus, because the introgression extends to at least the most proximal part of cytological interval 16C. Were it so, then we should conclude that this gene makes only a very minor contribution to the disturbances described in this article. It is the segment marked by m⁺ and, above all, its interaction with the segment marked by y⁺ that brings about the highest delay in hybrid spermatogenesis. In our view, this discrepancy may be simply a consequence of the different combinations of epistatic factors involved. WU and co-workers investigated the effect of cointrogressing factors linked to relatively long intact segments at either side of each visible marker. Thus, to get full sterility, Odysseus needs to be introgressed together with other genes in the interval 12B–15F (PEREZ and WU 1995 ). In contrast, we have produced a combination of rather short, intact segments, lying far apart on the X chromosome. Apparently, the effect of a given factor, such as Odysseus, is extremely dependent upon the gene set cointrogressed with it.

	ACKNOWLEDGMENTS

We are particularly grateful to E. Hauschteck-Jungen, from the University of Zürich, for her hospitality and valuable assistance with DAPI staining. We thank J. Coyne and J. F. McDonald for providing fly stocks and N. Johnson and an anonymous referee for their very helpful suggestions to improve the original manuscript. This work was supported by grants from Ministerio de Educación y Ciencia (PB92-0386) and Xunta de Galicia (XUGA 10305B95), Spain. X.M. and J.B. were supported by fellowships from Xunta de Galicia and Universidade da Coruña.

Manuscript received October 16, 1997; Accepted for publication June 29, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ASHBURNER, M., 1989 Protocol 48: preparation of DNA from single flies, pp. 108–109 in Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

BAINBRIDGE, S. P. and M. BOWNES, 1981  Staging the metamorphosis of Drosophila melanogaster. J. Embryol. Exp. Morphol. 66:57-80[Medline].

CABOT, E. L., A. W. DAVIS, N. A. JOHNSON, and C.-I. WU, 1994  Genetics of reproductive isolation in the Drosophila simulans clade: complex epistasis underlying hybrid male sterility. Genetics 137:175-189[Abstract].

COYNE, J. A. and B. CHARLESWORTH, 1989  Genetic analysis of X-linked sterility in hybrids between three sibling species of Drosophila. Heredity 62:97-106.

DAVIS, A. W. and C.-I. WU, 1996  The broom of the sorcerer's apprentice: the fine structure of a chromosomal region causing reproductive isolation between two sibling species of Drosophila. Genetics 143:1287-1298[Abstract].

FULLER, M. T., 1993 Spermatogenesis, pp. 71–147 in The Development of Drosophila melanogaster, edited by M. BATE and A. MARTINEZ-ARIAS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

HIGGINS, D. G., A. J. BLEASBY, and R. FUCHS, 1992  CLUSTAL V: improved software for multiple sequence alignment. Comput. Appl. Biosci. 8:189-191[Abstract/Free Full Text].

HOLLOCHER, H. and A. R. TEMPLETON, 1994  The molecular through ecological genetics of abnormal abdomen in Drosophila mercatorum. VI. The non-neutrality of the Y chromosome rDNA polymorphism. Genetics 136:1373-1384[Abstract].

HOLLOCHER, H. and C.-I. WU, 1996  The genetics of reproductive isolation in the Drosophila simulans clade: X versus autosomal effects and male versus female effects. Genetics 143:1243-1255[Abstract].

JOHNSON, N. A., H. HOLLOCHER, E. NOONBURG, and C.-I. WU, 1993  The effects of interspecific Y chromosome replacements on hybrid sterility within the Drosophila simulans clade. Genetics 135:443-453[Abstract].

KIRSCHNER, M., J. NEWPORT, and J. GERHART, 1985  The timing of early developmental events in Xenopus. Trends Genet. 1:41-47.

LIFSCHYTZ, E., 1987  The developmental program of spermiogenesis in Drosophila: a genetic analysis. Int. Rev. Cytol. 109:211-256[Medline].

LIFSCHYTZ, E. and D. HAREVEN, 1977  Gene expression and the control of spermatid morphogenesis in Drosophila melanogaster. Dev. Biol. 58:276-294[Medline].

MASIDE, X. and H. NAVEIRA, 1996  A polygenic basis of hybrid sterility may give rise to spurious localizations of major sterility factors. Heredity 77:488-492.

NAVEIRA, H., 1992  Location of X-linked polygenic effects causing sterility in male hybrids of Drosophila simulans and D. mauritiana. Heredity 68:211-217.

NAVEIRA, H. and A. FONTDEVILA, 1986  The evolutionary history of Drosophila buzzatii. XII. The genetic basis of sterility in hybrids between D. buzzatii and its sibling D. serido from Argentina. Genetics 144:841-857.

NAVEIRA, H. and A. FONTDEVILA, 1991  The evolutionary history of Drosophila buzzatii. XXI. Cumulative action of multiple sterility factors on spermatogenesis in hybrids of D. buzzatii and D. koepferae. Heredity 67:57-72.

NAVEIRA, H., and X. MASIDE, 1998 The genetics of hybrid male sterility in Drosophila, pp. 330–338 in Endless Forms: Species and Speciation, edited by D. J. HOWARD and S. H. BERLOCHER. Oxford University Press, NY.

PALOPOLI, M. F. and C.-I. WU, 1994  Genetics of hybrid male sterility between Drosophila sibling species: a complex web of epistasis is revealed in interspecific studies. Genetics 138:329-341[Abstract].

PEREZ, D. E. and C.-I. WU, 1995  Further characterization of the Odysseus locus of hybrid sterility in Drosophila: one gene is not enough. Genetics 140:201-206[Abstract].

PEREZ, D. E., C.-I. WU, N. A. JOHNSON, and M.-L. WU, 1993  Genetics of reproductive isolation in the Drosophila simulans clade: DNA marker-assisted mapping and characterization of a hybrid male sterility gene, Odysseus (Ods). Genetics 134:261-275[Abstract].

SANGER, F., S. NICKLEN, and A. R. COULSON, 1977  DNA sequencing with chain terminating inhibitors. Proc. Nat. Acad. Sci. USA 74:5463-5467[Abstract/Free Full Text].

TRUE, J. R., B. S. WEIR, and C. C. LAURIE, 1996  A genome-wide survey of hybrid incompatibility factors by the introgression of marked segments of Drosophila mauritiana chromosomes into Drosophila simulans. Genetics 142:819-837[Abstract].

WU, C.-I., 1992  A note on Haldane's rule: hybrid inviability vs. hybrid sterility. Evolution 46:1584-1587.

WU, C.-I. and M. F. PALOPOLI, 1994  Genetics of postmating reproductive isolation in animals. Annu. Rev. Genet. 28:283-308[Medline].

WU, C.-I., D. E. PEREZ, A. W. DAVIS, N. A. JOHNSON, E. L. CABOT et al., 1993 Molecular genetic studies of postmating reproductive isolation in Drosophila, pp. 191–203 in Mechanisms of Molecular Evolution, edited by N. TAKAHATA and A. G. CLARK. Sinauer Associates, Sunderland, MA.

WU, C.-I., N. A. JOHNSON, and M. F. PALOPOLI, 1996  Haldane's rule and its legacy: why are there so many sterile males? TREE 11:281-284.

YASUDA, G. K. and G. SCHUBIGER, 1992  Temporal regulation in the early embryo: is MBT too good to be true? Trends Genet. 8:124-127[Medline].

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