Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as \'extracystic spermiogenesis\'. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage.

Mammalian spermatogenesis, probably the most complex of all cellular developmental processes, is an ideal model both for studying the specific mechanism of gametogenesis and for understanding the basic rules governing all developmental processes, as it entails both cell type-specific and housekeeping molecular processes. Spermatogenesis can be viewed as a mission with many tasks to accomplish, and its success is genetically programmed and ensured by the collaboration of a large number of genes. Here, I present an overview of mammalian spermatogenesis and the mechanisms underlying each step in the process, covering the cellular and molecular activities that occur at each developmental stage and emphasizing their gene regulation in light of recent studies.

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.

In the adult mouse testis, germ cells of various developmental cell states co-exist. FACS isolation of cells stained with the DNA dye Hoechst 33342 has been used for many years to sub-divide these cells based on their DNA content. This approach provides an efficient way to obtain broad categories of male germ cells: pre-meiotic spermatogonia, meiotic spermatocytes and post-meiotic spermatids. The addition of a red filter for Hoechst staining enables further sub-division of spermatocytes depending on sub-stages of meiotic prophase. However, separation of different stage spermatids using Hoechst staining alone is not possible. We recently reported a methodology, combining Hoechst staining with a second DNA dye (SYTO16) that enables the further separation of these cells into three sub-populations: round, early elongating, and late elongating spermatids (Gill et al., Cytometry A 101:529-536, 2022). This method makes it possible to obtain rapidly and simply pure fractions of male germ cells from multiple developental stages from the same animal.

The mammalian X chromosome exhibits enrichment in genes associated with germ cell development. Previously, we generated a rat model of Becker muscular dystrophy (BMD) characterized by an in-frame mutation in the dystrophin gene, situated on the X chromosome and responsible for encoding a protein crucial for muscle integrity. Male BMD rats are infertile owing to the absence of normal spermatids in the epididymis. Within the seminiferous tubules of BMD rats, elongated spermatids displayed abnormal morphology. To elucidate the cause of infertility, we identified a putative gene containing an open reading frame situated in the intronic region between exons 6 and 7 of the dystrophin gene, specifically deleted in male BMD rats. This identified gene, along with its encoded protein, exhibited specific detection within the testes, exclusively localized in round to elongated spermatids during spermiogenesis. Consequently, we designated the encoded protein as dystrophin-locus-derived testis-specific protein (DTSP). Given the absence of DTSP in the testes of BMD rats, we hypothesized that the loss of DTSP contributes to the infertility observed in male BMD rats.

Biallelic loss-of-function mutation of NUP210L, encoding a testis-specific nucleoporin, has been reported in an infertile man whose spermatozoa show uncondensed heads and histone retention. Mice with a homozygous transgene intronic insertion in Nup210l were infertile but spermatozoa had condensed heads. Expression from this insertion allele is undefined, however, and residual NUP210L production could underlie the milder phenotype. To resolve this issue, we have created Nup210lem1Mjmm , a null allele of Nup210l, in the mouse. Nup210lem1Mjmm homozygotes show uniform mild anomalies of sperm head morphology and decreased motility, but nuclear compaction and histone removal appear unaffected. Thus, our mouse model does not support that NUP210L loss alone blocks spermatid nuclear compaction. Re-analyzing the patient\'s exome data, we identified a rare, potentially pathogenic, heterozygous variant in nucleoporin gene NUP153 (p.Pro485Leu), and showed that, in mouse and human, NUP210L and NUP153 colocalize at the caudal nuclear pole in elongating spermatids and spermatozoa. Unexpectedly, in round spermatids, NUP210L and NUP153 localisation differs between mouse (nucleoplasm) and human (nuclear periphery). Our data suggest two explanations for the increased phenotypic severity associated with NUP210L loss in human compared to mouse: a genetic variant in human NUP153 (p.Pro485Leu), and inter-species divergence in nuclear pore function in round spermatids.

Spastic paraplegia 7 (SPG7) is an m-AAA protease subunit involved in mitochondrial morphology and physiology. However, its function in animal reproduction is yet to be evaluated. In this study, its molecular features, subcellular localization, and expression dynamics were investigated to analyze its potential function in the reproduction of male Phascolosoma esculenta, an economically important marine species in China. The full-length cDNA of P. esculenta spg7 (Pe-spg7) measures 3053 bp and encodes an 853-amino acid protein (Pe-SPG7). Pe-SPG7 includes two transmembrane domains, an AAA domain and a proteolytic domain. Amino acid sequence alignment revealed that SPG7 was conserved during evolution. The mRNA and protein expression of spg7 indicated its involvement in reproduction. Its expression was the highest in coelomic fluid, where spermatids develop, and it was significantly higher in the breeding stage than in the nonbreeding stage. SPG7 was mainly found in the mitochondria of spermatids in the coelomic fluid, indicating that it functions in this organelle in spermatids. Immunofluorescence experiments showed that SPG7 was expressed and colocalized in the mitochondria during spermiogenesis, suggesting its involvement in P. esculenta spermiogenesis. Therefore, SPG7 may participate in spermiogenesis by functioning in the mitochondria and regulate the reproduction of male P. esculenta. This study provided insights into the function of SPG7 in animal reproduction and P. esculenta gametogenesis.

BACKGROUND: Chrysopa pallens is one of the most beneficial and effective natural predators, and is famous for its extensive distribution, wide prey spectrum, and excellent reproductive performance. This study examined the anatomy and fine structure of the C. pallens reproductive system and spermatogenesis.
RESULTS: The male reproductive system of C. pallens comprises a pair of testes, a vas deferens, seminal vesicles, accessory glands, and short ejaculatory ducts. The testes were already mature on the day of emergence, but the accessory glands did not mature until 5 days post-emergence. In early spermatids, the flagellum had an axoneme on one side of the two mitochondrial derivatives. The nucleus was surrounded by parallel crystalline and paracrystalline materials. The spermatid envelope extends towards the paracrystalline material in a tail-shaped wing. In mature spermatids, the axoneme is located between the two accessory bodies and mitochondrial derivative sets. The parallel-crystalline and paracrystalline materials disappeared. In the testes, the wall of seminal cysts consists of a layer of epithelium, a muscular-connective sheath, and several vesicles of different sizes. The mature seminal cysts contained 128 spermatozoa. The accessory gland is composed of six parts: ventral papilla-like protuberance, anterior glandular lobe, lateral glandular lobe, seminal cyst, posterior kidney-shaped lobe, and posterior papilla-like protuberance. Muscle fibers and secretory granules are extensive.
CONCLUSIONS: This study provides information on the reproductive system of C. pallens and offers a resource for taxonomy and reproductive biology.

Gene knockout experiments have shown that many genes are dispensable for a given biological function. In this review, we make an assessment of male and female germ cell-specific genes dispensable for the function of reproduction in mice, the inactivation of which does not affect fertility. In particular, we describe the deletion of a 1 Mb block containing nineteen paralogous genes of the oogenesin/Pramel family specifically expressed in female and/or male germ cells, which has no consequences in both sexes. We discuss this notion of dispensability and the experiments that need to be carried out to definitively conclude that a gene is dispensable for a function.

Valosin-containing protein (VCP) is a versatile and ubiquitously expressed AAA+ ATPase that regulates multiple stages of Drosophila spermatogenesis. While VCP has documented roles in mitotic spermatogonia and meiotic spermatocytes, it is also highly expressed in post-meiotic spermatids, suggesting potential late-stage developmental functions as well. However, tools to assess late-stage activities of pleiotropic spermatogenesis genes such as VCP are lacking. Available germline-specific Gal4 drivers activate in stem cells or spermatogonia; consequently, knocking down VCP using one of these drivers disrupts or blocks early germ-cell development, precluding analysis of VCP in later stages. A Gal4 driver that activates later in development, such as at the meiotic spermatocyte stage, may permit functional analyses of VCP and other factors in post-meiotic stages. Here, we describe a germline-specific Gal4 driver, Rbp4-Gal4, which drives transgene expression beginning in the early spermatocyte stage. We find that Rbp4-Gal4-driven knockdown of VCP causes defects in spermatid chromatin condensation and individualization without affecting earlier developmental stages. Interestingly, the defect in chromatin condensation appears linked to errors in the histone-to-protamine transition, a key event in spermatid development. Overall, our study reveals roles for VCP in spermatid development and establishes a powerful tool to dissect the functions of pleiotropic spermatogenesis genes.