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

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin—the histone-to-protamine switch.Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.     

Drosophila Centrosomin Protein is Required for Male Meiosis and Assembly of the Flagellar Axoneme

Centrosomes and microtubules play crucial roles during cell division and differentiation. Spermatogenesis is a useful system for studying centrosomal function since it involves both mitosis and meiosis, and also transformation of the centriole into the sperm basal body. Centrosomin is a protein localized to the mitotic centrosomes in Drosophila melanogaster. We have found a novel isoform of centrosomin expressed during spermatogenesis. Additionally, an anticentrosomin antibody labels both the mitotic and meiotic centrosomes as well as the basal body. Mutational analysis shows that centrosomin is required for spindle organization during meiosis and for organization of the sperm axoneme. These results suggest that centrosomin is a necessary component of the meiotic centrosomes and the spermatid basal body.     

Spermatogenic cyst and organ culture in <Emphasis Type="Italic">Drosophila pseudoobscura</Emphasis>

Spermatogenesis is a complex series of processes that involves (1) the maintenance of a renewable pool of diploid stem cells within a niche, (2) the mitotic expansion of a subpopulation of stem cells committed to the spermatogenic pathway, and (3) the differentiation of diploid cells into highly specialized, haploid spermatozoa through meiotic and post-meiotic cellular transformations. Drosophila melanogaster is a desirable model for studying spermatogenesis, as similarities exist between mammalian and fly spermatogenesis. Like mammals, flies maintain a spermatogenic stem cell niche; the steps involved in mammalian spermatogenesis are mimicked in flies, with the main difference being that fly sperm develop within cysts rather than an epithelial cell layer. Here, we report a reliable robust system for culturing whole testes and individual spermatogenic cysts obtained from mid- to late-pupal stages of Drosophila pseudoobscura. D. pseudoobscura testes can be easily distinguished in later pupal stages because of their intense red pigmentation and are easily handled because of their simple ellipsoidal morphology. Cultured cysts are comparable in length to cysts obtained from adult flies, and motility is consistently achieved in vitro. This system not only offers a method for dissecting the mechanisms involved in meiotic and post-meiotic cellular transformations, but also can be used for the study of signaling during spermatogenesis.     

Drosophila male-sterile mutation emmenthal specifically affects the mitochondrial morphogenesis

Proper mitochondrial morphogenesis is crucial for successful development of motile sperm. It was known that recessive Drosophila melanogaster mutation emm caused anomalies in the formation of a mitochondrial derivative—nebenkern and led to male sterility. Here we identified primary mutation effect and showed that emm is required for the formation and maintenance of inner mitochondrial structure starting from early spermatocytes. Abnormal mitochondria structure affects subsequent cellular processes in spermatogenesis such as meiotic cytokinesis and spermatid elongation.     

Uncoupling of gonial and spermatocyte stages by means of conditional lethal mutations in Drosophila melanogaster.

Two conditional lethals which change the developmental fate of gonial cells in male Drosophila melanogaster have been isolated. 1(1)63 arrests mitoses in gonial cysts (and other tissues) before the four division cycles have been completed, but allows meiotic divisions and normal spermatid differentiation to proceed. Gonial divisions in testes of 1(1)55, on the other hand, continue well after the usual four divisions, and as a result, cysts with hundreds of cells, many of which later degenerate, are formed. It is suggested that four gonial divisions are not a prerequisite genetically and metabolically for spermatocyte differentiation and that the activation of the spermatocyte program is not predetermined but rather requires the arrest of the gonial program. Other implications of the findings for our understanding of the genetic control of spermiogenesis are briefly discussed.     

Ultrastructure of sperm development in the plant-parasitic nematode Xiphinema theresiae

Spermatogenesis and sperm ultrastructure were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) in the longidorid Xiphinema theresiae. All germ cell stages, except spermatogonia, are present in the testes of young adult males. The nonflagellated, slightly elongated sperm displays little intraspecific variation and, although never polarized into a head and tail region, has a remarkably precise form, with a high degree of internal organization. Incipient fingerlike pseudopodia appear in the young spermatid and increase to such an extent that the adult sperm has a conspicuous “woolly” appearance. Microfilament bundles encircle the perinuclear mitochondria in the spermatid, and seem to be closely associated with the evaginated plasma membrane, especially in the spermatozoon. A large nucleus with nuclear envelope is prominent in the spermatocyte, but the envelope is absent in the young spermatid. Mitochondria are present in all germ cell stages and undergo certain morphological changes (e.g., in size and number, presence or absence of cristae), as well as changes in intracellular movements during spermatogenesis. Membranous organelles are prominent in the spermatocyte, but disappear in the older spermatid. Annulate lamellae and a residual body (i.e., cytophore) are conspicuous in the spermatocyte and spermatid, respectively; the spermatozoon clearly lacks a refringent body (i.e., acrosome).     

A new function of immunity‐related gene Zn72D in male fertility of Drosophila melanogaster

Zn72D encodes the Drosophila zinc finger protein Zn72D. It was first identified to be involved in phagocytosis and indicated to have a role in immunity. Then it was demonstrated to have a function in RNA splicing and dosage compensation in Drosophila melanogaster. In this study, we discovered a new function of Zn72D in male fertility. We showed that knockdown of Zn72D in fly testes caused an extremely low egg hatch rate. Immunofluorescence staining of Zn72D knockdown testes exhibited scattered spermatid nuclei and no actin cones or individualization complexes (ICs) during spermiogenesis, whereas the early‐stage germ cells and the spermatocytes were observed clearly. There were no mature sperms in the seminal vesicles of Zn72D knockdown fly testes, although a few sperms could be found close to the seminal vesicle. We further showed that many cytoskeleton‐related genes were significantly downregulated in fly testes due to Zn72D knockdown. Taken together these findings suggest that Zn72D may have an important function in spermatogenesis by sustaining the cytoskeleton‐based morphogenesis and individualization thus ensuring the proper formation of sperm in D. melanogaster.     

Immuno-electron microscopic localization of calmodulin and calmodulin-binding proteins in the mouse germ cells during spermatogenesis and maturation

When extracts of mouse testis were Western-blotted against a monoclonal antibody which reacts with calmodulin in the presence of Ca²⁺, all calmodulin was associated with the macromolecules of molecular weight above 50 kDa. Immuno-electron microscopy of testes using this antibody indicated that calmodulin is localized at higher density in the nucleus and cytoplasm of germ cells during the developmental phase between pachytene and round spermatid, showing the highest level just before meiotic divisions. There was no special association of calmodulin to any organelles in these cells. Extremely low levels of calmodulin occurred in spermatogonia and other testicular tissue cells. Calmodulin decreased dramatically as spermatids underwent metamorphosis, becoming detectable only at the perinuclear space of sperm heads. Further relocation to the postacrosomal region occurred during sperm transit to the cauda epididymis. Immunodetection after the calmodulin overlay on ultrathin sections revealed a sharp increase of calmodulin immunogold deposits in the nuclei of spermatids accompanying their condensation. The results indicate that some calmodulin-binding proteins, but not calmodulin itself, accumulate in the nuclei during the final steps of spermiogenesis.     

KNOCKDOWN OF ATPsyn‐b CAUSED LARVAL GROWTH DEFECT AND MALE INFERTILITY IN Drosophila

The ATPsyn‐b encoding for subunit b of ATP synthase in Drosophila melanogaster is proposed to act in ATP synthesis and phagocytosis, and has been identified as one of the sperm proteins in both Drosophila and mammals. At present, its details of functions in animal growth and spermatogenesis have not been reported. In this study, we knocked down ATPsyn‐b using Drosophila lines expressing inducible hairpin RNAi constructs and Gal4 drivers. Ubiquitous knockdown of ATPsyn‐b resulted in growth defects in larval stage as the larvae did not grow bigger than the size of normal second‐instar larvae. Knockdown in testes did not interrupt the developmental excursion to viable adult flies, however, these male adults were sterile. Analyses of testes revealed disrupted nuclear bundles during spermatogenesis and abnormal shaping in spermatid elongation. There were no mature sperm in the seminal vesicle of ATPsyn‐b knockdown male testes. These findings suggest us that ATPsyn‐b acts in growth and male fertility of Drosophila.     

Continuous spermatogenesis and the germ cell development strategy within the testis of the Jamaican Gray Anole, Anolis lineatopus

Testicular tissues from Anolis lineatopus were examined histologically to determine testicular structure, germ cell morphologies, and the germ cell development strategy employed during spermatogenesis. Anoles (N = 36) were collected from southern Jamaica from October 2004 to September 2005. Testes were extracted and fixed in Trump's fixative, dehydrated, embedded in Spurr's plastic, sectioned, and stained with basic fuchsin/toluidine blue. The testes of Jamaican Anoles were composed of seminiferous tubules lined with seminiferous epithelia, similar to birds and mammals, and were spermatogenically active during every month of the year. However, spermatogenic activity fluctuated based on morphometric data for February, May and June, and September-December. Sequential increases for these months and decreases in between months in tubular diameters and epithelial heights were due to fluctuations in number of elongating spermatids and spermiation events. Cellular associations were not observed during spermatogenesis in A. lineatopus, and three or more spermatids coincided with mitotic and meiotic cells within the seminiferous epithelium. Although the germ cell generations were layered within the seminiferous epithelium, similar to birds and mammals, the actual temporal development of germ cells and bursts of sperm release more closely resembled that reported recently for other reptilian taxa. All of these reptiles were temperate species that showed considerable seasonality in terms of testis morphology and spermatogenesis. The Jamaican Gray Anole has continuous spermatogenesis yet maintains this temporal germ cell development pattern. Thus, a lack of seasonal spermatogenesis in this anole seems to have no influence on the germ cell development strategy employed during sperm development.     

The role of Drosophila Merlin in spermatogenesis

Background

Drosophila Merlin, the homolog of the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and receptor endocytosis. Male flies carrying a Mer ³ allele, a missense mutation (Met¹⁷⁷→Ile) in the Merlin gene, are viable but sterile; however, the cause of sterility is unknown.

Results

Testis examination reveals that hemizygous Mer ³ mutant males have small seminal vesicles that contain only a few immotile sperm. By cytological and electron microscopy analyses of the Mer ³, Mer ⁴ (Gln¹⁷⁰→stop), and control testes at various stages of spermatogenesis, we show that Merlin mutations affect meiotic cytokinesis of spermatocytes, cyst polarization and nuclear shaping during spermatid elongation, and spermatid individualization. We also demonstrate that the lethality and sterility phenotype of the Mer ⁴ mutant is rescued by the introduction of a wild-type Merlin gene. Immunostaining demonstrates that the Merlin protein is redistributed to the area associated with the microtubules of the central spindle in telophase and its staining is less in the region of the contractile ring during meiotic cytokinesis. At the onion stage, Merlin is concentrated in the Nebenkern of spermatids, and this mitochondrial localization is maintained throughout sperm formation. Also, Merlin exhibits punctate staining in the acrosomal region of mature sperm.

Conclusion

Merlin mutations affect spermatogenesis at multiple stages. The Merlin protein is dynamically redistributed during meiosis of spermatocytes and is concentrated in the Nebenkern of spermatids. Our results demonstrated for the first time the mitochondrial localization of Merlin and suggest that Merlin may play a role in mitochondria formation and function during spermatogenesis.     

CHFR is important for the survival of male premeiotic germ cells

DNA damage response is required for male fertility. DNA damage repair mediates recombination between homologous chromosomes in meiotic prophase, which is essential for proper chromosome segregation during meiotic division. Interestingly, some DNA damage response proteins are also required for the survival of premeiotic germ cells, but their roles in these cells are still unclear. CHFR was recently shown to participate in DNA damage response, but it remains to be established if CHFR is required for male fertility. In this study, we characterized Chfr knockout male mice and found that around 30% of them were infertile. The onset of spermatogenesis was delayed and there was significant increase in apoptosis in premeiotic germ cells. This resulted in complete loss of germ cells in testes in 3 months and azoospermia in these mice. We further demonstrated that ATM activation was compromised in the testes of these mice. Therefore, CHFR is important for the survival of male premeiotic germ cells, which is likely through maintaining genomic stability in spermatogonial stem cells.