Males expressing AOX are defective in sperm-competition assays
In Drosophila, as in many species, females typically mate with a succession of males, storing sperm inside the female body to fertilize oocytes as they mature [14]. Sperm competition occurs by means of a substitution mechanism, in which a second male dislodges the sperm of the first male from the female’s reproductive tract and replaces it with its own gametes [14, 15]; this is apparently the motive force for the correlative evolution between the male’s giant sperm cells and the female’s spacious sperm-storage organs [16]. Newly acquired sperm competes with and usually displaces that from previous males, unless it is functionally compromised. These mechanisms can drive evolution by maximizing male reproductive success, such as by the development of larger testes, production of more abundant, more viable or longer-lasting gametes, or by modifying the response to elevated competition risk, among other characteristics [17].
To test the reproductive success of D. melanogaster males expressing AOX, we performed sperm-competition assays between flies carrying three copies of the α-tubulin-AOX construct, as described previously (3Xtub-AOX line: genotype tub-AOX
35/Y; tub-AOX
112/tub-AOX
112; tub-AOX
7/tub-AOX
7 [7]) and wild-type males of the same genetic background (w
1118). In the ‘defensive’ approach, 3Xtub-AOX males were first allowed to mate with virgin w
1118 females, which were then mated with w
1118 males in the absence of 3Xtub-AOX males. Progeny from the competing males can be distinguished by eye-colour: those from transgenic males have red eyes, whilst those from w
1118 males have white eyes. Controls consistently show that the eye-colour marker, as such, has no influence over the outcome (Additional file 1: Fig. S1).
The number of progeny originating from the sperm of 3Xtub-AOX males was decreased after the females were mated subsequently with w
1118 males (Fig. 1a, upper panel). This result is consistent with previously published data using diverse genetic backgrounds [14, 18,19,20,21] and with our own data using several other transgenes (Fig. 1b, Additional file 1: Fig. S1–S3): sperm of the first male is replaced (partially or completely) by the sperm of the second male. To implement a rigorous statistical analysis of the findings, we derived the parameter P1’ which measures the proportion of progeny sired by the first male to mate in sperm-competition assays [22]. This was similar for all lines analyzed here, although 3Xtub-AOX males sired statistically fewer offspring than some of the AOX-nonexpressor controls (Additional file 1: Table S1).
In contrast, when 3Xtub-AOX males were challenged in an ‘offensive’ approach, i.e., when the virgin w
1118 females were first crossed with w
1118 males, then with AOX-expressing males, the number of 3Xtub-AOX progeny never overcame the number of w
1118 progeny (Fig. 1a, lower panel). In fact, even after 10 days since w
1118 males were removed from the mating vials, eggs fertilized with the original male sperm were still preferentially laid over the ones fertilized with 3Xtub-AOX sperm (Fig. 1a, lower panel, vial IV). Control experiments performed with red-eyed daughterless-GAL4 (daGAL4) (Fig. 1b, lower panel) and tubulin-GeneSwitch (tubGS) and also UAS-empty
2nd and UAS-empty
3rd (Additional file 1: Fig. S1) males imply that the failure of AOX-expressing males to compete successfully in the offensive paradigm is specific to AOX, and is not a property of transgenic lines in general. The defect appeared to be AOX transgene-dose dependent. Males carrying only two copies of the tub-AOX transgene (Additional file 1: Fig. S2) appeared less impaired in the ‘offensive’ paradigm than those carrying three (Fig. 1a), whilst those with just a single copy of tub-AOX were not significantly different from control males (Additional file 1: Fig. S2). These conclusions reflect the statistical analysis of the proportion of progeny sired by the second male (P2’, [22]), wherein 3X and 2Xtub-AOX males were significantly impaired in the offensive paradigm compared to all control classes (Additional file 1: Table S2), although the differences between 3X and 2Xtub-AOX (Additional file 1: Table S2) themselves were not significant. The observed phenotypes also correlate approximately with the amount of expression of the tub-AOX transgene in the male reproductive system, observed by immunoblotting (Additional file 1: Fig. S4).
However, expressing GAL4-dependent UAS-AOX using the daGAL4 driver [6], produced no loss of sperm-competitiveness (Additional file 1: Fig. S3A, and Table S2), despite the fact that the level of AOX protein in the male reproductive system was comparable with that expressed from multiple copies of tubAOX (Additional file 1: Fig. S4). These data suggest that the effect of AOX on sperm competitiveness depends on the precise cellular context of its expression rather than simply its overall amount.
AOX-expressing males accumulate a decreased amount of mature sperm
Successful sperm competition in Drosophila depends both on the number and quality of spermatozoids and on the compatibility between the male’s seminal fluid proteins and their receptors in the female reproductive tract [17, 23,24,25]. We checked how the production of mature spermatozoids in the 3Xtub-AOX males is affected by dissecting the reproductive organs in adult males of increasing age. Spermatogenesis in D. melanogaster starts at the distal tip of the testes, taking place inside cysts. As cell differentiation proceeds, the cysts move towards the proximal end of the testes, where these organs connect to the SVs. The mature spermatozoids are then deposited in the SVs, where they are stored until mating [26]. Therefore, as adult males age, the testes tend to get thinner, because differentiation in the cysts proceeds and the fully formed spermatozoids move to the SVs, which in turn increase in size. As shown in Fig. 2 (panels a and c), the thickness of the testes of control flies decreases >50% in the first 10 days of adult life, whereas the SVs triple in size. In contrast, the testes of 3Xtub-AOX males exhibit a much less pronounced decrease in thickness (~25%), whilst at the same time their SVs remain immature. In addition, we observed an accumulation of whitish material at the proximal end of the testes of 10-day-old 3Xtub-AOX males (Fig. 2a, inset), which we investigated further (see below). The mature spermatozoids produced by these males, although few in number, did not appear to have any motility defects, as judged by visual inspection.
We were able to produce a similar phenotype using the inducible GeneSwitch (tubGS) system, in which the same α-tubulin promoter as used in the tub-AOX constructs controls the expression of a modified GAL4 that is inducible by mifepristone (RU486). This allows time-regulated expression of a GAL4-dependent AOX transgene in exactly the same tissues as in the tubAOX lines. By transferring males to food-vials containing the inducing drug on the day of eclosion, any possible developmental disturbance is avoided, whilst accurate controls can be implemented, notably flies with transgene, but lacking driver and/or drug (see Additional file 1: Fig. S5 for immunoblots indicating tight regulation of AOX protein levels using this system). Similarly as for 3Xtub-AOX males, the testes of UAS-AOX transgenic males driven by tubGS in the presence of mifepristone remained thick during the first 10 days of adult life, whilst their SVs remained small (Fig. 2b and c). These organs were as wild-type in the absence of the driver or inducing drug (Fig. 2b and c). The normal thinning of the testis over the first 10 days of adult life was also seen when both the driver and inducing drug were present, whether driving a control transgene, GFP, a catalytically inactive form of AOX, mutAOX [27] or even with no transgene at all (Additional file 1: Fig. S6A, S6C). In these cases, however, the SV remained undeveloped, but only when driver and drug were both present (Additional file 1: Fig. S6B, S6D). The whitish material near the proximal end of the testis also accumulated prominently when tubGS was used to express AOX (but not GFP or mutAOX), and again only under inducing conditions (Fig. 2b, red arrow). To confirm that this is an α-tubulin promoter-specific phenotype, we dissected the reproductive organs of 10-day-old males expressing UAS-AOX driven by daGAL4. The SVs had a normal morphology (Additional file 1: Fig. S7), and appeared to have accumulated a similar amount of mature sperm cells as controls, in agreement with the results of the sperm-competition assays (Additional file 1: Fig. S3).
Other reproductive organs were also evaluated morphologically (see Additional file 1: Fig. S8A for schematics of the reproductive system of D. melanogaster males), but no obvious alterations were observed between AOX-expressing and control males, based on visual inspection of >70 dissected flies of each genotype. Although AOX was not expressed in the accessory glands (Additional file 1: Fig. S9A), we also checked the presence of Sex Peptide in these organs by immunostaining and confocal microscopy, and again observed no difference (Additional file 1: Fig. S9B). Sex Peptide is one of the most important components of the male’s seminal fluid; it has been implicated in reproductive success in sperm-competition assays [24, 28] and its action is dependent on the compatibility with receptors in the female reproductive tract [23]. Altogether, our data indicate that AOX-expressing males are defective in sperm-competition assays due to a decreased production of mature spermatozoids.
AOX is expressed in cells of the testis and seminal vesicle sheaths
Although the SVs of AOX-expressing males present perhaps the most pronounced phenotype in our study, these are purely storage organs for mature sperm cells [26], and their morphology was not diagnostic for AOX expression (Additional file 1: Fig. S6B).
Therefore, we hypothesized that the defect in sperm production caused by AOX expression most likely affects the testes, where spermatogenesis takes place. The α-tubulin promoter has been used previously to drive transgene expression in the fly testis in both somatic cells and the germline (from the stem-cell stage to late spermatocytes, reviewed in [29]). However, using immunofluorescent confocal microscopy (Fig. 3 and Additional file 1: Fig. S10), AOX expression from the α-tubulin promoter, whether directly (Additional file 1: Fig. S10) or driven by tubGS plus mifepristone (Fig. 3), was below the level of detection in germline cells, and seen only at very low levels in some somatic cyst cells. Nevertheless, it was abundantly expressed in the somatic cells of the testis and SV sheaths. In addition, all of the immature stages of cell differentiation appeared to be present (Additional file 1: Fig. S11), judging by the characteristic changes in nuclear morphology, mitochondrial network arrangement and cellular elongation that the germ line cells go through towards the final steps in spermatogenesis [26].
At higher resolution, it was clear that not all cells in the ensheathing tissues of the testes and SVs express AOX at a high level when driven by the α-tubulin promoter (either directly, as 3Xtub-AOX or indirectly, through tubGS). Some cells highly expressing ATP5A, a complex V subunit traditionally used as a mitochondrial marker, appeared negative for AOX (Additional file 1: Fig. S12A). Using UAS-StingerGFP in combination with the tubGS driver, in order to mark the positive cells with GFP in the nucleus, we observed high expression in specific cells in the outermost sheath-cell layer (Additional file 1: Fig. S12B). The inner sheath-cell layer, formed of smooth-muscle cells with multiple small nuclei, appeared negative for AOX or GFP, in agreement with co-staining for AOX and actin (Additional file 1: Fig. S13A) in the testes of 3Xtub-AOX males. Positive cells most likely correspond with the pigment cells, which carry a single large nucleus, and are abundant around the SVs and the proximal end of the testis, where these two organs connect [30]. This is supported (Additional file 1: Fig. S13B–D) by co-staining for the empty spiracles gene product (ems), which is a marker for the pigment cells [31, 32]. Partial three-dimensional reconstruction of the microscopy images shows that the AOX and ems signals are located in the outermost cell layer of the sheath of the SV (Additional file 1: Fig. S13C) and testis (Additional file 1: Fig. S13D), whilst the underlying (smooth muscle) cells stain highly for ATP5A and actin. Note that the pigment cells are abundant in the anatomical region where the most pronounced morphological alterations were found in AOX-expressing males, marked by the accumulation of whitish material at the proximal end of the testis combined with a decreased amount of mature sperm cells in the SVs (Fig. 2a).
Mature-looking spermatozoids are lodged in the proximal end of the testis in AOX-expressing males
The proximal end of the D. melanogaster testis is also the region where the individualization process starts. This process is essential for the final stage of elongated spermatid differentiation [26], and is accomplished by an individualization complex (IC) that is assembled in the cyst region containing the spermatid nuclei and which then moves towards the tip of the tails, collecting syncitial cytoplasm and creating cystic bulges and waste bags (WBs) along the way [33, 34]. By staining the testis samples for actin and activated caspase-3, two components of the ICs, we observed no significant differences in the number of starting and established ICs in males expressing AOX driven by tubGS, but did observe an elevated number of WBs (Additional file 1: Fig. S14). Most noticeably, we observed in these males a dramatic change in the distribution of ICs throughout the organ. Starting ICs are usually present in the proximal region of the testis, but in 10 day-old males expressing AOX driven by tubGS, a significant number of them were found towards the middle region of the organ. Established ICs, which are usually found all along the testes, were concentrated at the middle and proximal end of the AOX-expressing testes. Finally, WBs appeared to be distributed in all testis regions in the AOX-expressing males, whereas in control testes they accumulated at the distal end (Additional file 1: Fig. S14C). Our observations imply that AOX expression driven by tubGS in the somatic cells of the cysts, and/or in the pigment cells of the testis sheath causes internal rearrangements in this organ, disrupting the production and delivery of mature sperm.
To investigate the nature of the whitish material found at the proximal end of the testis of AOX-expressing males, we used a transgenic construct that expresses GFP-tagged Don Juan protein (DJ-GFP) in the tail of elongated spermatids and mature spermatozoids [35, 36], enabling us to evaluate the transition between these two stages. The expression of DJ-GFP revealed a high concentration of what appeared to be individualized mature spermatozoids in the proximal end of the testis of AOX-expressing males (Fig. 4, lower panels). In control males, few of these individualized GFP-positive cells were found in this region of the testes (Fig. 4, upper panels), as expected, given that they should move to the SVs after individualization. The failure of the individualized, elongated spermatids to move into the SV and the associated disturbance in the spatial distribution of ICs could account for the accumulation of the whitish material seen by light microscopy, and is the simplest explanation for the defect seen in sperm-competition assays, although the reason why they are retained in the testis is not obvious morphologically.