CG9920 is necessary for mitochondrial morphogenesis and individualization during spermatogenesis in Drosophila melanogaster.

Drosophila melanogaster is an ideal model organism for investigating spermatogenesis due to its powerful genetics, conserved genes and visible morphology of germ cells during sperm production. Our previous work revealed that ocnus (ocn) knockdown resulted in male sterility, and CG9920 was identified as a significantly downregulated protein in fly abdomen after ocn knockdown, suggesting a role of CG9920 in male reproduction. In this study, we found that CG9920 was highly expressed in fly testes. CG9920 knockdown in fly testes caused male infertility with no mature sperms in seminal vesicles. Immunofluorescence staining showed that depletion of CG9920 resulted in scattered spermatid nuclear bundles, fewer elongation cones that did not migrate to the anterior region of the testis, and almost no individualization complexes. Transmission electron microscopy revealed that CG9920 knockdown severely disrupted mitochondrial morphogenesis during spermatogenesis. Notably, we found that CG9920 might not directly interact with Ocn, but rather was inhibited by STAT92E, which itself was indirectly affected by Ocn. We propose a possible novel pathway essential for spermatogenesis in D. melanogaster, whereby Ocn indirectly induces CG9920 expression, potentially counteracting its inhibition by the JAK-STAT signaling pathway.

Quantitative proteomics and phosphoproteomics reveal insights into mechanisms of ocnus function in Drosophila testis development.

BACKGROUND: Testis is the only organ supporting sperm production and with the largest number of proteins and tissue-specific proteins in animals. In our previous studies, we have found that knockdown of ocnus (ocn), a testis-specific gene, resulted in much smaller testis with no germ cells in Drosophila melanogaster. However, the molecular consequences of ocn knockdown in fly testes are unknown. RESULTS: In this study, through iTRAQ quantitative proteomics sequencing, 606 proteins were identified from fly abdomens as having a significant and at least a 1.5-fold change in expression after ocn knockdown in fly testes, of which 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs), apart from those proteins involved in spermatogenesis, the others extensively affected biological processes of generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. Re-analyses of the transcriptome revealed 150 differential expressed genes (DEGs) appeared in the DEPs, and their changing trends in expressions after ocn knockdown were consistent. Many common down-regulated DEGs and DEPs were testis-specific or highly expressed in the testis of D. melanogaster. Quantitative RT-PCR (qRT-PCR) confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated after ocn knockdown in fly testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated phosphorylated proteins were also identified (13 phosphoproteins appeared in both up- and down-regulated groups due to having multiple phosphorylation sites). In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways. CONCLUSIONS: Given the drastic effect of the ocn knockdown on tissue development and testis cells composition, the differences in protein abundance in the ocn knockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation of ocn. Nevertheless, our results suggest that the expression of ocn is essential for Drosophila testis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation. These DEPs and DEPPs identified may provide significant candidate set for future studies on the mechanism of male reproduction of animals, including humans.

Cytochrome c1-like is required for mitochondrial morphogenesis and individualization during spermatogenesis in Drosophila melanogaster.

The Drosophila testis is an excellent system for studying the process from germ stem cells to motile sperm, including the proliferation of male germ cells, meiosis of primary spermatocytes, mitochondrial morphogenesis, and spermatid individualization. We previously demonstrated that ocnus (ocn) plays an essential role in male germ cell development. Among those genes and proteins whose expression levels were changed as a result of ocn knockdown, cytochrome c1-like (cyt-c1L) was downregulated significantly. Here, we show that cyt-c1L is highly expressed in the testis of D. melanogaster. Knockdown or mutation of cyt-c1L in early germ cells of flies resulted in male sterility. Immunofluorescence staining showed that cyt-c1L knockdown testes had no defects in early spermatogenesis; however, in late stages, in contrast to many individualization complexes (ICs) composed of F-actin cones that appeared at different positions in control testes, no actin cones or ICs were observed in cyt-c1L knockdown testes. Furthermore, no mature sperm were found in the seminal vesicle of cyt-c1L knockdown testes whereas the control seminal vesicle was full of mature sperm with needle-like nuclei. cyt-c1L knockdown also caused abnormal mitochondrial morphogenesis during spermatid elongation. Excessive apoptotic signals accumulated in the base of cyt-c1L knockdown fly testes. These results suggest that cyt-c1L may play an important role in spermatogenesis by affecting the mitochondrial morphogenesis and individualization of sperm in D. melanogaster.

Comparative phosphoproteomics reveal new candidates in the regulation of spermatogenesis of Drosophila melanogaster.

The most common phenotype induced by the endosymbiont Wolbachia in insects is cytoplasmic incompatibility, where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females. This suggests that some modifications are induced in host sperms during spermatogenesis by Wolbachia. To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster, we applied isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic strategy combined with titanium dioxide (TiO2 ) enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male reproductive systems in D. melanogaster. We identified 182 phosphopeptides, defining 140 phosphoproteins, that have at least a 1.2 fold change in abundance with a P-value of <0.05. Most of the differentially abundant phosphoproteins (DAPPs) were associated with microtubule cytoskeleton organization and spermatid differentiation. The DAPPs included proteins already known to be associated with spermatogenesis, as well as many not previously studied during this process. Six genes coding for DAPPs were knocked down, respectively, in Wolbachia-free fly testes. Among them, Slmap knockdown caused the most severe damage in spermatogenesis, with no mature sperm observed in seminal vesicles. Immunofluorescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted. These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction. By identifying phospho-modulated proteins we also provide a significant candidate set for future studies on their roles in spermatogenesis.

shrub is required for spermatogenesis of Drosophila melanogaster.

Shrub (CG8055) encodes the vps32/snf7 protein, a filament-forming subunit of the ESCRT (endosomal sorting complexes required for transport)-III complex involved in inward membrane budding. It was reported that shrub was required for abscission in female germline stem cells. In this study, we showed that the expression level of shrub in the testis was significantly higher than that in the ovary of 1-day-old Drosophila melanogaster, suggesting a role in male reproduction. Then we used nosGal4 driver to knockdown shrub specifically in the fly testis and found that this resulted in a significantly lower paternal effect egg hatch rate relative to the control group. Immunofluorescence staining showed that shrub knockdown in fly testes caused an accumulation of early-stage germ cells and lack of spectrin caps. In the late stages (spermiogenesis), the control testis contained multiple compacted spermatid bundles and individualization complexes (ICs) consisting of actin cones, whereas there were scattered spermatid nuclei and only a few ICs with disorganized actin cones in the shrub knockdown testis. Finally, the control seminal vesicle was full of mature sperms with needle-like heads, but in shrub knockdown testis 75% of seminal vesicles had no mature sperms. We also found that knockdown of shrub in fly testes led to upregulated expression of several cytoskeleton-associated genes, and an accumulation of ubiquitylated proteins. These results suggest that knockdown of shrub in fly testes might damage spermatogenesis by affecting transportability.

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.

Small RNA analysis provides new insights into cytoplasmic incompatibility in Drosophila melanogaster induced by Wolbachia.

Wolbachia is a genus of endosymbiotic bacteria that induce a wide range of effects on their insect hosts. Cytoplasmic incompatibility (CI) is the most common phenotype mediated by Wolbachia and results in embryonic lethality when Wolbachia-infected males mate with uninfected females. Studies have revealed that bacteria can regulate many cellular processes in their hosts using small non-coding RNAs, so we investigated the involvement of small RNAs (sRNAs) in CI. Comparison of sRNA libraries between Wolbachia-infected and uninfected Drosophila melanogaster testes revealed 18 novel microRNAs (miRNAs), of which 12 were expressed specifically in Wolbachia-infected flies and one specifically in Wolbachia-uninfected flies. Furthermore, ten miRNAs showed differential expression, with four upregulated and six downregulated in Wolbachia-infected flies. Of the upregulated miRNAs, nov-miR-12 exhibited the highest upregulation in the testes of D. melanogaster. We then identified pipsqueak (psq) as the target gene of nov-miR-12 with the greatest complementarity in its 3' untranslated region (UTR). Wolbachia infection was correlated with reduced psq expression in D. melanogaster, and luciferase assays demonstrated that nov-miR-12 could downregulate psq through binding to its 3'UTR region. Knockdown of psq in Wolbachia-free fly testes significantly reduced egg hatching rate and mimicked the cellular abnormalities of Wolbachia-induced CI in embryos, including asynchronous nuclear division, chromatin bridging, and chromatin fragmentation. These results suggest that Wolbachia may induce CI in insect hosts by miRNA-mediated changes in host gene expression. Moreover, these findings reveal a potential molecular strategy for elucidating the complex interactions between endosymbionts and their insect hosts, such as Wolbachia-driven CI.

[Treatment of Chronic Endometritis by Penning Granule: a Clinical Randomized Controlled Trial].

Objective To observe the mechanism of Penning Granule ( PG) for treating chronic endometritis (CE). Methods Totally 38 CE patients in line with inclusive criteria were assigned to the PG group (23 cases) and the control group (15 cases) according to random digit table. PG (consisting of sargentgloryvine stem, Herba Patriniae, Hedyotis Diffusa, red peony root, Angelica Sinensis, prepared mastiche, prepared myrrh, common burreed tuber, zedoary root, Spina Gleditsiae, Danshen root, Radix Bupleuri, asarum , Astragalus, Pangolin) was administered to patients in the PG group, 105 g each time, twice per day for 4 successive weeks. Those in the control group took Levofloxacin (0.5 g, once per day) and Metronidazole Tablet (0. 5 g, twice daily for 1 successive week). They were followed-up for half a year after ending treatment. The morphological changes of uterine endometrium were observed by hyst- eroscopy before and after treatment, and endometrium biopsy performed at fixed position. The expres- sions of mucin-1 (MUC-1 ) and hypoxia induced facter-1 α (HIF-1α) in inflammatory endometrium were analyzed. The clinical effects were compared between PG and antibiotics from improvement of clinical symptoms, hysteroscopic manifestations, pathological analyses, and molecular levels. Results The markedly effective rate of clinical symptoms in the PG group was 91. 3% (21/23) , higher than that of the control group [60. 0% (9/15) ; P <-0. 05]. The plasma cell CD38 infiltration in endometrial stroma of the PG group were significantly decreased, showing better effect than antibiotics in the control group (P < 0.05). The positive expression of MUC-1 was increased and the expression of HIF-1 α was decreased in the PG group, showing better effect than antibiotics in the control group (P <0. 05). Conclusion PG showed obvious effects for treating chronic endometritis , and it was superior to that of antibiotics alone.

Activity of encapsulated Lactobacillus bulgaricus in alginate-whey protein microspheres

In this work, alginate-whey protein was used as wall materials for encapsulating Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The characteristics of encapsulated and free L. bulgaricus showed that the free L. bulgaricus lost viability after 1 min exposure to simulated gastric fluid (SGF) at pH 2.0 and 2.5. However, the viability of encapsulated L. bulgaricus did not decrease in SGF at pH 2.5 for 2 h incubation. The viable numbers of encapsulated L. bulgaricus decreased less than 1.0 log unit for 2 h incubation in SGF at pH 2.0. For bile stability, only 1.2 log units and 2.0 log units viability of the encapsulated L. bulgaricus was lost in 1 and 2% bile for 1 h exposure, respectively, compared with no survival of free L. bulgaricus under the same conditions. Encapsulated L. bulgaricus was completely released from the microspheres in simulated intestinal fluid (SIF, pH 6.8) in 3 h. The viability of the encapsulated L. bulgaricus retained more 8.0 log CFU/g after stored at 4°C for four weeks. However, for free L. bulgaricus, only around 3.0 log CFU/mL was found at the same storage conditions. Results showed that the encapsulation could improve the stability of L. bulgaricus.