Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

The testis in Drosophila males is already formed in the embryo and persists in the body cavity during larval stages. Third-instar larvae show small, round testes surrounded by a layer of future pigment cells and embedded in fat body tissue (Figure 2A). Testes in adult male flies are long, coiled tubes, which are covered by a layer of muscle cells originating from the genital disc and a pigment layer²⁴. Interestingly, larval testes contain only germ-line cells of pre-meiotic and meiotic stages until the primary spermatocyte stage, which corresponds to meiotic prophase (see also Figure 1)²³. The first cohorts of male germ cells pass through meiosis during puparium formation. At ca. 24 hr APF, post-meiotic stages with elongated flagella have already developed, and all nuclei contain histone H2AvD-RFP (Figures 1 and 2B). None of the spermatids have undergone the H-P switch as no ProtamineB-eGFP signal can be detected by fluorescence microscopy (Figure 2C). Frequently, the testes dissected at this stage contain an auto-fluorescent structure of variable size reminiscent of an agglomerate of fat body cells or one to several drops of oil (Figure 2C, arrowhead). This structure within the testis is also visible with phase-contrast microscopy. To date, we have not found any description of its nature in the literature. Testes dissected at 36 hr APF appear elongated and in some cases already begin to curl (Figure 2D). They have already attached to the tissues of the genital tract growing out from the genital imaginal disc^23,24. Furthermore, they frequently contain the first spermatids beyond the H-P switch, as indicated by the ProtamineB-eGFP signal (Figures 1 and 2D, arrow). At 48 hr APF, the testes have further elongated and clearly start curling at the proximal end. Several germ-line cysts with protamine-positive nuclei become visible (Figure 2E, arrow).

When testes dissected at 24 hr APF are kept in culture for 24 hr, they do not elongate as in the intact fly (compare Figure 2D and 2E with Figure 6A’). This is possibly due to a lack of positional and growth signals normally sent from the developing genital tract contacting the testis at its posterior tip^23,29. Furthermore, the muscle layer of the testis sheath does not develop because nascent myotubes cannot migrate from the genital disc to the testis²⁴. The testis sheath—in this situation solely the thin pigment layer—does not seem to grow sufficiently in culture to envelope the increasing number of developing germ cells within. However, germ cells grow, divide, and differentiate independently of the testis sheath. Hence, after more than 36 hr in culture, the early testes will frequently burst open, and further development is not observed. However, within a shorter time frame, it is possible to record time-lapse ex vivo live images of whole testes developing in culture, as evidenced by Figures 3A-3C (see also Supplemental video 1). A pupal testis, dissected at ca. 45 hr APF, was incubated in medium for 6 hr and imaged every 5 min. Within this time frame, several cysts of spermatids emerge from the H-P switch stage and show a bright ProtamineB-eGFP signal (Figures 3A’-3C, open arrows).

As mentioned above, male germ cells in the Drosophila testis develop as synchronized bundles of interconnected cells, named cysts¹. Figure 4A shows an overview of cysts dissected from a pupal testis at around 24 hr APF. Pre-meiotic stages, meiotic stages, early post-meiotic stages, and also cysts in the early canoe stage with elongated nuclei prior to the H-P switch can be found (Figures 1 and 4B-4E). The isolated cysts are very fragile and should be handled carefully. The two somatic cells that form the cyst’s envelope can be easily disrupted mechanically. Germ-line stem cells have the ability to survive and continue dividing after genetic ablation of the somatic cyst cells in vivo³⁰. It has been reported that in vitro, germ cells of the 16-cell stage separated from their somatic envelope further develop and differentiate¹⁹. Indeed, we noticed in our culture system that presumably late spermatocytes completed meiotic divisions with disrupted cyst cells, albeit very infrequently. Most frequently, these disrupted cysts ceased development. Following the presented protocol, intact cysts might in some cases develop ex vivo in culture medium for a maximum of 72 hr. However, we observed that a reasonable number of cysts survive up to 48 hr of incubation. Within this time frame, the cultured cysts are able to develop from the primary spermatocyte stage until after meiotic divisions as well as from the round nuclei stage with histone-based chromatin until the late canoe stage beyond the H-P switch¹². This allows live imaging of vital processes in spermatogenesis, as for example, spermatocytes entering meiotic divisions (Figure 5 and Supplemental video 2). For this experiment, the RFP-tagged histone variant H2AvD was used as chromosome marker. The primary spermatocyte stage extends for ca. 3 days in vivo³¹. Therefore, in order to record meiotic divisions, we chose cysts with spermatocytes that had visibly started chromatin condensation (Figure 5A, region 2). Meiotic division starts in spermatocytes on one side of the cyst and then follows in a wave in the remaining spermatocytes (in Figure 5A from region 2 to region 1, also see Supplemental video 2). Condensation of the chromatin soon leads to fast-moving chromosomes that are visible as bright spots (Figure 5A, region 2). After 30 min, the first spermatocytes of this region are already in telophase (Figure 5B, arrowheads). The chromosomes of the younger region 1 arrange at the metaphase plate 20 min later (Figure 5C). They complete telophase and are ready to undergo cytokinesis about 15 min later (Figure 5D). Anaphase, telophase, and cytokinesis lasted ca. 10 min in this recording (Supplemental video 2).

In mammals, as well as in Drosophila, a pronounced increase in histone H4 acetylation marks the onset of histone removal and the H-P switch during post-meiotic spermatogenesis^6,11. It has been proposed that this epigenetic modification is an essential component or even trigger of the developmental program of the H-P switch^10,32. Previously, we treated Drosophila pupal testes in culture with inhibitors targeting HATs and HDACs to influence the acetylation level of histone H4 during post-meiotic stages¹². In these pharmacological assays, we found that histone acetylation is essential for the progression of spermatogenesis from stages with histone-based chromatin to stages with protamine-based chromatin.

Figures 6 and 7 show representative results of a pharmacological assay using anacardic acid to target HATs in pupal testes. Testes at 24 hr APF were dissected from a fly strain expressing H2AvD-RFP and ProtamineB-eGFP. ProtamineB-eGFP protein was absent in these early pupal testes (Figure 6A and B). After 24 hr in culture, the control testes showed ProtamineB-eGFP signals, which indicated that the first cysts had developed beyond the H-P switch (Figure 6A’, open arrowheads). This was not the case with the testes treated with 150 µM anacardic acid (Figure 6B’). Testis squash preparations and immunofluorescence analyses offer more detailed information (Figure 7). In the untreated control, the nuclei of the relatively large primary spermatocytes can be recognized by their characteristic pattern of three regions of richly stained DNA, which correspond to the major chromosomes (4C level) of Drosophila (Figure 7A, column 1). Acetylation of histone H4 is clearly detectable at this stage (Figure 7B, column 1). The first stage after meiotic divisions is known as the Nebenkern stage and is characterized by rather large, round nuclei (see Figure 4C; not shown in Figure 7). The next stage shown in Figure 7 is identified by flagellar growth and the round shape of the germ cell nuclei, which are already much smaller than in earlier stages (Figure 7A, column 2) and show very low to nearly undetectable levels of histone H4 acetylation (Figure 7B, column 2). The round shape then elongates (Figure 7A, column 3), and the acetylation of histone H4 is again clearly detectable (Figure 7B, column 3). The spermatids then reach the canoe shape stage in which the H-P switch takes place and histones are replaced by protamines (Figures 7A–7C, columns 4 and 5). Following the canoe stage, the protaminated nuclei then further elongate into a very thin needle shape in the mature sperm (not shown here).

Testis squash preparations of testes treated with anacardic acid showed different results (Figures 7D–7F). The chromatin in germ cells treated with anacardic acid appeared more condensed than that of the untreated control (compare Figure 7D, treated and 7A, control). Immunofluorescence analyses revealed that acetylation of H4 was greatly diminished or even absent in nuclei of germ cells treated with anacardic acid (Figure 7E). It is known that highly acetylated histones are associated with regions of open chromatin, while chromatin regions that lack histone acetylation frequently show a repressive structure³³. Therefore, it is not surprising that treatment with anacardic acid influenced the chromatin structure of the treated germ cell nuclei. Importantly, no protamine-positive nuclei of late canoe stage or beyond were identified in treated testes (Figure 7F). Thus, our data are consistent with the idea that HAT activity is essential for spermiogenesis to proceed from histone-based chromatin to protamine-based chromatin stages.

Figure 1. Overview of Drosophila spermatogenesis. The left panel displays the sequence of stages in germ-cell development from a stem cell to individualized sperm. Schematic drawings show the characteristic nuclear morphology of the germ cells. One spermatogonium divides to produce 16 interconnected spermatocytes in one cyst. During this stage most of the transcripts needed for post-meiotic development are produced, translationally repressed, and stored. The bulk of transcriptional activity ends with the entry into the meiotic divisions. The histone-to-protamine switch in the chromatin takes place between the early and late canoe stage. Stages with a histone-based chromatin are highlighted in red; stages with a protamine-based chromatin are highlighted in green. The bars in the right panel indicate the stages of germ cells that can normally be found in the developing testis in larvae, pupae at ca. 24 and 36 hr after puparium formation (APF), and adult flies.

Figure 2. Drosophila testes at different stages of development. (A) Phase-contrast image of a slightly squashed whole-mount testis of a third-instar larva (L3). Only pre-meiotic and meiotic stages can be found. Spermatogonia are restricted to the anterior tip beneath the hub region (asterisk). The remaining testis is filled with spermatocytes (open arrow). (B) Phase-contrast image of a slightly squashed testis at ca. 24 hr after puparium formation (APF). In addition to spermatocytes (open arrow), the testis contains spermatids in the Nebenkern stage (open arrowheads), with round nuclei and spermatids in elongating stages with growing flagella (double open arrowhead). (C–E) Whole-mount pupal testes expressing H2AvD-RFP and ProtamineB-eGFP (overlays of phase-contrast and eGFP and RFP fluorescence images). (C) Pupal testis dissected at ca. 24 hr APF. Arrowhead marks auto-fluorescence of putative fat body cells. D) Pupal testis dissected at ca. 36 hr APF displays the first ProtamineB-eGFP-positive cyst (arrow). (E) Pupal testis at ca. 48 hr APF with a group of ProtamineB-eGFP-positive cysts (arrow). In all figure parts, asterisks indicate the hub region. Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 3. Live imaging of the histone-to-protamine switch in a pupal testis growing ex vivo. (A) Phase-contrast image of a pupal testis expressing H2AvD-RFP and ProtamineB-eGFP taken into culture at ca. 45 hr after puparium formation (APF). The seminal vesicle is indicated by a filled arrow. A paragonium (double arrowhead) remains attached to the testis. (A’) eGFP and RFP fluorescence of the testis shown in (A). The open arrow indicates a ProtamineB-eGFP-positive cyst. Scale bar: 100 µm. (B) After 2 hr 30 min in culture, several further cysts (open arrow) emerge from the histone-to-protamine transition. (C) After an additional ca. 3 hr in culture, i.e., a total of 5 hr 29 min in culture, a group of cysts (open arrow) with strong ProtamineB-eGFP signal is visible at the proximal end of the testis. See also Supplemental video 1. In all figure parts, asterisks indicate the hub region. Please click here to view a larger version of this figure.

Figure 4. Isolated cysts of germ cells at different stages of development. (A) Cysts dissected from a testis at ca. 24 hr after puparium formation (APF). Overlay of phase-contrast and H2AvD-RFP fluorescence (red) images. Scale bar: 100 µm. (B–E) Magnifications of single cysts of different stages. Overlay of differential interference contrast (DIC) and H2AvD-RFP fluorescence images. Scale bar: 20 µm. (B) Cyst of 16 spermatocytes. (C) Cyst of 64 round spermatids at the Nebenkern stage. The Nebenkern structure develops from fused mitochondria and is visible in DIC images next to the nucleus (open arrowhead). (D) Cyst of spermatids with round H2AvD-RFP-positive nuclei and elongating flagella (open arrowhead indicates an elongating Nebenkern structure that accompanies the growing axoneme). (E) Apical tip of a cyst of spermatids at the early canoe stage showing an elongated nuclear shape. The extensively elongated flagella are only partially visible. Please click here to view a larger version of this figure.

Figure 5. Live imaging of a single 16-spermatocyte cyst undergoing meiosis I ex vivo. Fluorescence images of a cyst in culture expressing H2AvD-RFP. Depicted are characteristic stages of meiosis I. Spermatocytes in the cyst pass through meiotic divisions in a wave-like manner. (A) Chromosome condensation starts in nuclei at one side of the cyst (2) and progresses through the cyst (direction indicated by arrow). Nuclei in the opposing region (1) represent an earlier stage of chromatin condensation, where histone-dense regions marking the three major chromosomes can still be discerned. Spermatocytes in region (2) contain fully condensed chromosomes, visible as bright fluorescent spots. (B) After 30 min, the first spermatocytes in region (2) are in telophase (open arrowheads), while chromosome condensation continues in region (1). (C) In the last spermatocytes of region (1), the chromosomes (arrowheads) are arranged at the metaphase plate 20 min later. (D) Within another 15 min, the last spermatocytes appear to have completed telophase and are now ready to undergo cytokinesis (open arrowheads). See also Supplemental video 2. Scale bar: 50 µm. Please click here to view a larger version of this figure.

Figure 6. Anacardic acid inhibits the histone-to-protamine switch in cultured pupal testes. Pupal testes expressing H2AvD-RFP and ProtamineB-eGFP and dissected at 24 hr after puparium formation (APF) were incubated for 24 hr in medium without inhibitor (control; A, A’; DMSO, dimethylsulfoxide) or in medium supplemented with 150 µM anacardic acid (B, B’). Hub regions indicated by asterisks. (A, B) No ProtamineB-eGFP-positive cysts can be observed after dissection. (A’) After 24 hr of incubation, some cysts underwent the histone-to-protamine switch and ProtamineB-eGFP-positive cysts (open arrowheads) can be observed. (B’) Testes treated with anacardic acid do not develop ProtamineB-eGFP-positive cysts. Scale bar: 100 µm. Please click here to view a larger version of this figure.

Figure 7. Anacardic acid affects histone H4 acetylation and the chromatin structure of spermatids. Squash preparations of spermatid nuclei from pupal testes (24 hr APF) expressing ProtamineB-eGFP incubated for 24 hr without inhibitor (A–C) or with 150 µM anacardic acid (D–F). DNA stained with Hoechst dye (A and D). H4 acetylation analyzed immunofluorescently (B and E). Presence of protamines monitored with the ProtamineB-eGFP signal (C and F). After treatment with anacardic acid, the chromatin appears strongly compacted (D) and the H4 acetylation signal is completely reduced in all spermatid stages (E). In addition, the anacardic-acid-treated testes show no cysts of late canoe stage or beyond and no ProtamineB-eGFP signal (F). Scale bar: 10 µm. Please click here to view a larger version of this figure.

Supplemental Video 1. Live imaging via eGFP and RFP fluorescence of the histone-to-protamine switch in a pupal testis growing ex vivo. Video of 6 hr time course imaging illustrates the histone-to-protamine transition by the increase in prevalence of ProtamineB-eGFP-positive cysts. Images were acquired every 5 min. For details, see Figure 3. (See "suppl_video_1.MOV" under Downloads).

Supplemental Video 2. Live imaging of a single 16-spermatocytes cyst undergoing meiosis I ex vivo. Time-lapse fluorescent imaging of a cyst in culture expressing H2AvD-RFP over the course of 80 min. This video depicts the characteristic stages of meiosis I. Images were acquired every 1 min. For details, see Figure 5. (See "suppl_video_2.MOV" under Downloads).