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.

OBJECTIVE: To investigate the expression of Zfx gene in spermatogenic cells.
METHODS: The testes of d6, d8, d17 and adult mice were collected, single cell suspension was prepared by combinatorial enzyme digestion, spermatogenic cells were isolated by BSA density gradient method, and Zfx expression was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western Blot (WB).
RESULTS: Single cell suspension prepared by combination enzyme digestion method and density gradient method laid with BSA can obtain various types of spermatogenic cells with purity>85%; The expression level of the Zfx gene is low in primitive type A spermatogonia, type A spermatogonia, and type B spermatogonia, whereas it is high in preleptotene spermatocytes, pachytene spermatocytes, and round spermatid cells. It is not expressed in elongating spermatids and mature sperm.
CONCLUSIONS: Zfx gene exhibits periodic expression in various levels of spermatogenic cells and may be an important transcription factor involved in regulating meiosis in spermatogenic cells.

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin-related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interfaces create an important intercellular bridge whose adhesive function is in turn supported by Fjx1, a nonreceptor Ser/Thr protein kinase. This concept is derived from earlier studies of Drosophila, which has been confirmed in this and earlier reports as well. Herein, we use the approach of knockdown of Fat1 by RNAi using primary cultures of Sertoli cells that mimicked the blood-testis barrier (BTB) in vivo, and a series of coherent experiments including functional assays to monitor the Sertoli cell tight junction (TJ) permeability barrier and a functional in vitro TJ integrity assay to assess the role of Fat1 in the testis. It was shown that planar cell polarity (PCP) protein Fat1 affected Sertoli cell function through its modulation of actin and microtubule cytoskeletal function, altering their polymerization activity through the Fat1/Fjx1 complex. Furthermore, Fat1 is intimately associated with β-catenin and α-N-catenin, as well as with Prickle 1 of the Vangl1/Prickle 1 complex, another PCP core protein to support intercellular interactions to confer PCP. In summary, these findings support the notion that the Fat:Dchs and the Vangl2:Fzd PCP intercellular bridges are tightly associated with basal ES/TJ structural proteins to stabilize PCP function at the Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interface to sustain spermatogenesis.

BACKGROUND: The melon fly, Zeugodacus cucurbitae Coquillett, is one of the major pests attacking Cucurbitaceae crops. Identifying critical genes or proteins regulating fertility is essential for sustainable pest control and a research hotspot in insect physiology. MicroRNAs (miRNAs) are short RNAs that do not directly participate in protein translation, but instead function in post-transcriptional regulation of gene expression involved in male fertility.
RESULTS: We found that miR-927-5p is highly expressed in the testes and investigated its function in spermatogenesis in Z. cucurbitae. Fluorescence in situ hybridization (FISH) showed miR-927-5p in the transformation and maturation region of the testis, and overexpression of miR-927-5p reduced the number of sperms by 53%. In continuation, we predicted 12 target genes of miR-927-5p using bioinformatics combined with transcriptome sequencing data, and found that miR-927-5p targets the new gene Stalky in insects, which was validated by quantitative real-time PCR, RNA pull-down and dual luciferase reporter assays. FISH also confirmed the co-localization of miR-927-5p and the transcript Stalky_1 in the testis. Moreover, silencing of Stalky_1 by RNA interference reduced the number of sperms by 32% and reduced sperm viability by 39% in physiologically mature male adults. Meanwhile, the silencing of Stalky_1 also resulted in low hatchability.
CONCLUSIONS: Our work not only presents a new, so far unreported mechanism regulating spermatogenesis by miR-927-5p targeting a new unknown target, Stalky, which is providing new knowledge on the regulatory network of insect spermatogenesis, but also lays a foundation for the development of SIT against important tephritid fly pests. © 2024 Society of Chemical Industry.

Ribosomal protein 25 (RPS25) has been related to male fertility diseases in humans. However, the role of RPS25 in spermatogenesis has yet to be well understood. RpS25 is evolutionarily highly conserved from flies to humans through sequence alignment and phylogenetic tree construction. In this study, we found that RpS25 plays a critical role in Drosophila spermatogenesis and its knockdown leads to male sterility. Examination of each stage of spermatogenesis from RpS25-knockdown flies showed that RpS25 was not required for initial germline cell divisions, but was required for spermatid elongation and individualization. In RpS25-knockdown testes, the average length of cyst elongation was shortened, the spermatid nuclei bundling was disrupted, and the assembly of individualization complex from actin cones failed, resulting in the failure of mature sperm production. Our data revealed an essential role of RpS25 during Drosophila spermatogenesis through regulating spermatid elongation and individualization.

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer with no targeted therapy. Spermatid perinuclear RNA binding protein (STRBP), a poorly characterized RNA-binding protein (RBP), has an essential role in normal spermatogenesis and sperm function, but whether and how its dysregulation contributing to cancer progression has not yet been explored. Here, we report that STRBP functions as a novel oncogene to drive TNBC progression. STRBP expression was upregulated in TNBC tissues and correlated with poor disease prognosis. Functionally, STRBP promoted TNBC cell proliferation, migration, and invasion in vitro, and enhanced xenograft tumor growth and lung colonization in mice. Mechanistically, STRBP interacted with Dicer, a core component of the microRNA biogenesis machinery, and promoted its proteasomal degradation through enhancing its interaction with E3 ubiquitin ligase UBR5. MicroRNA-sequencing analysis identified miR-200a-3p as a downstream effector of STRBP, which was regulated by Dicer and affected epithelial-mesenchymal transition. Importantly, the impaired malignant phenotypes of TNBC cells caused by STRBP depletion were largely rescued by knockdown of Dicer, and these effects were compromised by transfection of miR-200a-3p mimics. Collectively, these findings revealed a previously unrecognized oncogenic role of STRBP in TNBC progression and identified STRBP as a promising target against TNBC.

The centrosome is critical for maintaining the sperm head-tail connection and the formation of flagellar microtubules. In this study, we found that in mouse testes, CCDC159 (coiled-coil domain-containing protein 159) is specifically localized to the head-tail coupling apparatus (HTCA) of spermatids, a structure that ensures sperm head-tail tight conjunction. CCDC159 contains a C-terminal coiled-coil domain that functions as the centrosomal localization signal. Gene knockout (KO) of Ccdc159 in mice resulted in acephalic spermatozoa, abnormal flagella, and male infertility. To explore the mechanism behind CCDC159 regulating spermatogenesis, we identified CCDC159-binding proteins using a yeast two-hybrid screen and speculated that CCDC159 participates in HTCA assembly by regulating protein phosphatase PP1 activity. Further RNA-sequencing analyses of Ccdc159 KO testes revealed numerous genes involved in male gamete generation that were downregulated. Together, our results show that CCDC159 in spermatids is a novel centrosomal protein anchoring the sperm head to the tail. Considering the limitation of KO mouse model in clarifying the biological function of CCDC159 in spermatogenesis, a gene-rescue experiment will be performed in the future.

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

Successful completion of spermatogenesis is crucial for the perpetuation of the species. In Drosophila, spermatid individualization, a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm. Ant2, encoding a mitochondrial adenine nucleotide translocase, is highly expressed in male testes and plays a role in energy metabolism in the mitochondria. However, its molecular function remains unclear. Here, we identified an important role of Ant2 in spermatid individualization. In Ant2 knockdown testes, spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution, and mature sperms were absent in the seminal vesicle, thus leading to male sterility. The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function. Excessive apoptotic cells were also observed in Ant2-knockdown testes. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA level, 868 differentially expressed genes were identified, of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown. iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins, of which 117 proteins were upregulated and 233 were downregulated. The expression of glutathione transferase (GstD5, GstE5, GstE8, and GstD3), proteins involved in reproduction were significantly regulated at both the mRNA and protein levels. These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis.

Kisspeptin, a neuropeptide encoded by the Kiss1 gene, combines with its receptor Kiss1R to regulate the onset of puberty and male fertility by the hypothalamic-pituitary-gonadal axis. However, little is known regarding the expression signatures and molecular functions of Kiss1 in the testis. H&E staining revealed that well-arranged spermatogonia, spermatocytes, round and elongated spermatids, and spermatozoa, were observed in 4-, 6-, and 8-month-old testes compared to 1- and 3-month-old testes of Hezuo pigs; however, these were not observed in Landrance until 6 months. The diameter, perimeter, and cross-sectional area of seminiferous tubules and the perimeter and area of the tubular lumen increased gradually with age in both pigs. Still, Hezuo pigs grew faster than Landrance. The cloning results suggested that the Hezuo pigs\' Kiss1 CDS region is 417 bp in length, encodes 138 amino acids, and is highly conserved in the kisspeptin-10 region. qRT-PCR and Western blot indicated that the expression trends of Kiss1 mRNA and protein were essentially identical, with higher expression levels at post-pubertal stages. Immunohistochemistry demonstrated that the Kiss1 protein was mainly located in Leydig cells and post-pubertal spermatogenic cells, ranging from round spermatids to spermatozoa. These studies suggest that Kiss1 is an essential regulator in the onset of puberty and spermatogenesis of boars.