Sperm Bundles Research Articles

The Cap and the Spermatostyle Protecting the Sperm Bundle Have a Similar Origin—Ultrastructural Study of the Spermatogenesis from the Ground Beetle Carabus (Chaetocarabus) lefebvrei Dejean, 1826 (Adephaga Carabidae)

The males of Carabus lefebvrei produce sperm bundles of quite variable size as a consequence of a sexual selection via sperm competition. The ultrastructural features of spermatogenesis of the species and the sequence of events leading to sperm bundle formation along the deferent duct lumen revealed the origin and evolution of the apical structure protecting the sperm bundles. Elongated layers of the electron-dense material of variable size and thickness are visible over the microvilli of the distal deferent duct epithelium. They likely correspond to sections through different levels of discoidal secretions. Then, the sperm present in the duct lumen attach to one side of such elongated secreted structures. Later on, these structures bent to take a cap shape and increase their size and thickness by apposition of additional secretions produced by the proximal deferent duct epithelium. These results reconcile the different opinions about the formation of the structures called the cap and the rod (spermatostyle). In our work, we in fact obtained evidence that both these structures have the same origin, even though at the end of their formation they exhibit a different appearance. The reported results indicate that, differently from the spermatostyle, in the initial stages of cap formation, the sperm adhere only to one side of the cap rather than around the whole structure, as it occurs instead along the spermatostyle rod. Under this aspect, the cap structure is similar to the protection present in the sperm of some whirligig beetle.

Evidence of antiparallel sperm bundles and limited sperm production in adult males of Phaleria testacea Say, 1824 (Tenebrionidae, Diaperinae)

Evidence of antiparallel sperm bundles and limited sperm production in adult males of Phaleria testacea Say, 1824 (Tenebrionidae, Diaperinae)

The spermatheca ultrastructure of the ground beetle Clinidium canaliculatum (Costa) (Carabidae, Rhysodinae)

The ground beetle Clinidium canaliculatum is a member of Rhysodinae, a taxon with still discussed systematic position. The spermatheca of this species is a small cylindrical structure connected to the common oviduct by a thin duct. The ultrastructure of the organ has revealed that the apical receptacle is provided with an epithelium lined by a thick cuticle from the deeper region of which several finger-like cuticular structures extend into the cytoplasm. On these structures adhere microtubule bundles that cross the whole cytoplasm to anchor on short densities along the basal plasma membrane. These specializations are strongly reminiscent of the hemidesmosomes, possibly playing a mechanical role enabling the cells to resist to the muscle contractions pushing the sperm towards the spermathecal duct. The cells are rich in mitochondria and glycogen granules and they are possibly involved in fluid uptake from the spermathecal lumen. The spermathecal duct has a simple epithelium lined by a soft cuticle. The sperm present in the apical receptacle and in the duct lumen maintain the structure described in the male genital apparatuses. They are generally free and embedded in a homogeneous electron-dense material. Occasionally, a sperm bundle, still with an apical cap, was visible in the spermathecal receptacle.

Monarch butterfly conservation through male germplasm cryopreservation

Monarch butterfly (Danaus plexippus L.) populations have declined in North America. The International Union for Conservation of Nature (IUCN) recently classified the species as endangered, sparking public concern and conservation efforts. Our approach to conservation is through cryopreservation of germinal cells and tissue. The goal of this study was to develop a cryopreservation protocol for monarch spermatozoa to ensure successful long-term storage. Cryopreserved sperm cells would provide a reserve of monarch germplasm, which could be utilized in the event of population loss. In this study, sperm cell bundles collected from male monarch butterflies were cryopreserved in a cryoprotective medium and stored in liquid nitrogen. To determine the post-cryopreservation sperm cell viability, a subsample of preserved sperm bundles were thawed rapidly, and their viability was qualified using a sperm live/dead stain. We are presenting a protocol to preserve and store genetic material and viable sperm bundles of the monarch butterfly. To date, this is the first report of successful cryopreservation of monarch germplasm which sets the foundation for cryostorage and could be extensible to other vulnerable lepidopterans.

Functional Analysis of Forkhead Transcription Factor Fd59a in the Spermatogenesis of Drosophila melanogaster.

Spermatogenesis is critical for insect reproduction and is regulated by many different genes. In this study, we found that Forkhead transcription factor Fd59a functions as a key factor in the spermatogenesis of Drosophila melanogaster. Fd59a contains a conversed Forkhead domain, and it is clustered to the FoxD subfamily with other FoxD members from some insect and vertebrate species. Mutations in Fd59a caused swelling in the apical region of the testis. More importantly, fewer mature sperm were present in the seminal vesicle of Fd59a mutant flies compared to the control flies, and the fertility of Fd59a2/2 mutant males was significantly lower than that of the control flies. Immunofluorescence staining showed that the homeostasis of the testis stem cell niche in Fd59a2/2 mutant and Fd59a RNAi flies was disrupted and the apoptosis of sperm bundles was increased. Furthermore, results from RNA sequencing and qRT-PCR suggested that Fd59a can regulate the expression of genes related to reproductive process and cell death. Taken together, our results indicated that Fd59a plays a key role in the spermatogenesis of Drosophila.

The mechanoreceptor Piezo is required for spermatogenesis in Bombyx mori

BackgroundThe animal sperm shows high diversity in morphology, components, and motility. In the lepidopteran model insect, the silkworm Bombyx mori, two types of sperm, including nucleate fertile eupyrene sperm and anucleate unfertile apyrene sperm, are generated. Apyrene sperm assists fertilization by facilitating the migration of eupyrene spermatozoa from the bursa copulatrix to the spermatheca. During spermatogenesis, eupyrene sperm bundles extrude the cytoplasm by peristaltic squeezing, while the nuclei of the apyrene sperm bundles are discarded with the same process, forming matured sperm.ResultsIn this study, we describe that a mechanoreceptor BmPiezo, the sole Piezo ortholog in B. mori, plays key roles in larval feeding behavior and, more importantly, is essential for eupyrene spermatogenesis and male fertility. CRISPR/Cas9-mediated loss of BmPiezo function decreases larval appetite and subsequent body size and weight. Immunofluorescence analyses reveal that BmPiezo is intensely localized in the inflatable point of eupyrene sperm bundle induced by peristaltic squeezing. BmPiezo is also enriched in the middle region of apyrene sperm bundle before peristaltic squeezing. Cytological analyses of dimorphic sperm reveal developmental arrest of eupyrene sperm bundles in BmPiezo mutants, while the apyrene spermatogenesis is not affected. RNA-seq analysis and q-RT-PCR analyses demonstrate that eupyrene spermatogenic arrest is associated with the dysregulation of the actin cytoskeleton. Moreover, we show that the deformed eupyrene sperm bundles fail to migrate from the testes, resulting in male infertility due to the absence of eupyrene sperm in the bursa copulatrix and spermatheca.ConclusionsIn conclusion, our studies thus uncover a new role for Piezo in regulating spermatogenesis and male fertility in insects.

GASZ is indispensable for gametogenesis in the silkworm, Bombyx mori.

The prominent role of the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway in animals is to silence transposable elements and maintain genome stability, ensuring proper gametogenesis in gonads. GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) is an evolutionarily conserved protein located on the outer mitochondrial membrane of germ cells and plays vital roles in the piRNA pathway and spermatogenesis in mammals. In the model insect Drosophila melanogaster, GASZ is essential for piRNA biogenesis and oogenesis, whereas its biological functions in non-drosophilid insects are still unknown. Here, we describe a comprehensive investigation of GASZ functions in the silkworm, Bombyx mori, a lepidopteran model insect, by using a binary transgenic CRISPR/Cas9 system. The BmGASZ mutation did not affect growth and development, but led to sterility in both males and females. Eupyrene sperm bundles of mutant males exhibited developmental defects, while the apyrene sperm bundles were normal, which were further confirmed through double copulation experiments with sex-lethal mutants, which males possess functional eupyrene sperm and abnormal apyrene sperm. In female mutant moths, ovarioles were severely degenerated and the eggs in ovarioles were deformed compared with that of wild type (WT). Further RNA-seq and RT-qPCR analysis revealed that amounts of piRNAs and transposon expression were dysregulated in gonads of mutants. In summary, this study has demonstrated vital roles of BmGASZ in gametogenesis through regulating the piRNA pathway in B. mori.

Verification of AKT and CDK5 Gene and RNA Interference Combined with Irradiation to Mediate Fertility Changes in Plutella xylostella (Linnaeus).

Plutella xylostella (Linnaeus) mainly damages cruciferous crops and causes huge economic losses. Presently, chemical pesticides dominate its control, but prolonged use has led to the development of high resistance. In contrast, the sterile insect technique provides a preventive and control method to avoid the development of resistance. We discovered two genes related to the reproduction of Plutella xylostella and investigated the efficacy of combining irradiation with RNA interference for pest management. The results demonstrate that after injecting PxAKT and PxCDK5, there was a significant decrease of 28.06% and 25.64% in egg production, and a decrease of 19.09% and 15.35% in the hatching rate compared to the control. The ratio of eupyrene sperm bundles to apyrene sperm bundles also decreased. PxAKT and PxCDK5 were identified as pivotal genes influencing male reproductive processes. We established a dose-response relationship for irradiation (0-200 Gy and 200-400 Gy) and derived the irradiation dose equivalent to RNA interference targeting PxAKT and PxCDK5. Combining RNA interference with low-dose irradiation achieved a sub-sterile effect on Plutella xylostella, surpassing either irradiation or RNA interference alone. This study enhances our understanding of the genes associated with the reproduction of Plutella xylostella and proposes a novel approach for pest management by combining irradiation and RNA interference.

Dual roles of α1,4-galactosyltransferase1 in spermatogenesis of Drosophila melanogaster.

Spermatogenesis is critical for insect reproduction and the process is regulated by multiple genes. Glycosyltransferases have been shown to participate in the development of Drosophila melanogaster; however, their role in spermatogenesis is still unclear. In this study, we found that α1,4-galactosyltransferase1 (α4GT1) was expressed at a significantly higher level in the testis than in the ovary of Drosophila. Importantly, the hatching rate was significantly decreased when α4GT1 RNA interference (RNAi) males were crossed with w1118 females, with only a few mature sperm being present in the seminal vesicle of α4GT1 RNAi flies. Immunofluorescence staining further revealed that the individualization complex (IC) in the testes from α4GT1 RNAi flies was scattered and did not move synchronically, compared with the clustered IC observed in the control flies. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay showed that apoptosis signals in the sperm bundles of α4GT1 RNAi flies were significantly increased. Moreover, the expression of several individualization-related genes, such as Shrub, Obp44a and Hanabi, was significantly decreased, whereas the expression of several apoptosis-related genes, including Dronc and Drice, was significantly increased in the testes of α4GT1 RNAi flies. Together, these results suggest that α4GT1 may play dual roles in Drosophila spermatogenesis by regulating the sperm individualization process and maintaining the survival of sperm bundles.

Morphology of the Male Reproductive System and Spermatozoa of Mahanarva fimbriolata (Stål, 1854) (Hemiptera: Cercopidae) with evidence of a spermatostyle in the sperm conjugation.

Changes in harvesting methods in the early 1990s led to Mahanarva fimbriolata becoming the most prevalent pest in sugarcane plantations. Encouraged by the scarcity of research on the reproductive system and spermatology of Auchenorrhyncha, we provide a detailed description of the male reproductive system and spermatozoa of this cercopid. This will not only aid in taxonomic purposes but also help in understanding the reproductive biology of the species. The male reproductive system, spermatozoa, and sperm bundles of M. fimbriolata were investigated using light microscopy. Our results reveal a reproductive system consisting of a pair of testes, each with 36 follicles, two vasa deferentia, two well-developed seminal vesicles, a single pair of accessory glands, and an ejaculatory duct. Here, we also describe and explore the particularities of the formation of secondary sperm bundles, where cells interestingly bind only to the apical region of the spermatostyle. We also propose some possible benefits of this unusual organization for the reproductive success of the species.

Underestimated diversity: Integrative taxonomy of Mesenchytraeus (Enchytraeidae, Clitellata) from Changbai Mountain, China

AbstractThe holarctic genus Mesenchytraeus is one of the species‐rich genera in the family Enchytraeidae. Changbai Mountain supports high diversity of Mesenchytraeus species, making it an ideal area to explore species boundaries. We explored species boundaries of Mesenchytraeus using an integrative approach. Morphological taxonomy recognized 10 species in this region, five of them new to science. In contrast, molecular species delimitation analyses showed that there are at least 16 species, 11 of them new to science. Clear genetic gaps were observed among species with high interspecific distances (10%–21.2%) and low intraspecific distances (0.2%–6.7%) based on uncorrected p‐distance of the COI gene. Morphological species complex M. spermatoglomeratus consists of three species, M. spermatoglomeratus Zhang, Lu & Xie, 2018 sensu stricto, M. rijina sp. n., and M. manchu sp. n. The M. duodiverticulus complex consists of M. duodiverticulus sp. n. and M. similiduodiverticulus sp. n. The M. monodiverticulus complex consists of M. monodiverticulus Shen, Chen & Xie, 2012 sensu stricto, M. ngulen sp. n., M. zhenggulen sp. n., and M. fokulen sp. n. Further new species, distinguishable with morphological as well as molecular methods, are M. similigigachaetus sp. n., M. parvidiverticulus sp. n., M. digitalisdiverticulus sp. n., and M. infradiverticulus sp. n. Finally, we inferred the morphogenetic processes of spermathecae and sperm bundles, and filtrated some morphological characters which are useful to identify species. As the first attempt in this genus, our study provides an opportunity to discuss the currently used taxonomic criteria and acquire new ideas for the taxonomy of enchytraeids.

The sperm structure of the Scraptiidae (Coleoptera; Tenebrionoidea)

The sperm ultrastructure of two members of the Scraptiidae Anaspis pulicaria and A. lurida was studied. The results confirm the general organization of the sperm in the superfamily Tenebrionoidea. The sperm bundles at the end of the spermiogenesis show the same peculiar antiparallel distribution at the two opposite poles of the germ cyst, observed in other Tenebrionoidea. The sperm have a bi-layered acrosome, a long cylindrical nucleus with two infoldings at its basal region, two elliptical equal mitochondrial derivatives and two triangular accessory bodies. The flagellar axoneme has the common 9 + 9 + 2 microtubular pattern that at the tail end results disorganized. All these sperm characteristics are quite similar to those found in Pythidae, a closely related family, according to molecular data.

Reproductive Biology of Muga Silk Moth <i>Antheraea assamensis</i> Helfer

Results of morphological and morphometric study of male and female reproductive systems of Antheraea assamensis Helfer (Saturniidae: Lepidoptera), a silk moth cultivated exclusively in the North Eastern Region of India are reported herein along with some observations on its reproductive performance in four seasons. The male reproductive system consists of a pair of testes, paired vasa deferentia, seminal vesicle, ductus ejaculatorius duplex, ductus ejaculatorius simplex, an aedeagus and a pair of male accessory glands. The female reproductive system consists of a pair of ovaries, each with four ovarioles, paired lateral oviducts, a common oviduct which has a dorsal protrusion, the vestibulum, a bursa copulatrix, a spermatheca and a pair of female accessory glands with their reservoirs. Fecundity has been found to be positively correlated with female pupal weight in each season. Hatching % of eggs has been found lowest and significantly different (p<0.001) in summer as compared to autumn, winter and spring seasons. Morphology of eupyrene and apyrene sperm bundles and sperm has also been reported.

Ultrastructure of the female reproductive organs of the diving beetle Deronectes moestus incospectus (Leprieur, 1876) (Dytiscidae, Hydroporinae)

We describe the ultrastructure of the female reproductive organs of Deronectes moestus (Dytiscidae Hydroporinae). The long spermathecal duct has a simple epithelium lined internally by a thin cuticle and externally by a thick layer of muscle cells. The wide duct lumen contains electron-dense material, among which remnants of extracellular material are visible. This material consists of tubular structures assembled around sperm bundles previously described in the male deferent ducts. The so-called gland, disposed along the spermathecal duct, is a structure with epithelial cells lined by an irregular cuticle bearing a rich system of microvilli. Many mitochondria are visible in the apical cytoplasm of the epithelial cells, and a few spheroidal bodies are close to the basal nuclei. Since the epithelial ultrastructure of the gland suggests it is involved in fluid uptake from the lumen rather than secretory activity, the term gland, coined by other authors to describe this organ, is inappropriate. The spermatheca is a large structure with a complex epithelium showing secretory and duct-forming cells. The lumen of this organ contains sperm with the distinctive ultrastructural features of those described in the male deferent ducts, namely having a mitochondrial matrix with a small crystallized area and electron-dense dots. Because to its overall organization, the spermatheca of D. moestus can be considered a more integrated organ than those in previously studied hydroporine species.

Insights into spermatogenesis in the migratory locust, Locusta migratoria (Linnaeus, 1758) (Orthoptera: Acrididae), following histological and ultrastructural features of the testis

The migratory locust, Locusta migratoria (Linnaeus, 1758), is one of the most destructive agricultural pests globally, and this species is particularly localized in several regions of Egypt. However, so far, very little attention has been paid to the characteristics of the testes. Furthermore, spermatogenesis requires careful analysis to characterize and track developmental episodes. We thus investigated, for the first time, the histological and ultrastructural properties of the testis in L. migratoria employing a light microscope, a scanning electron microscope (SEM), and a transmission electron microscope (TEM). Our results revealed that the testis comprises several follicles, emerging with distinct outer surface wrinkle patterns for each follicle throughout the length of the follicular wall. Furthermore, histological examination of the follicles showed that each has three developmental zones. Each zone has cysts with characteristic spermatogenic elements, beginning with the spermatogonia at the distal end of each follicle and ending with the spermatozoa at the proximal end. Moreover, spermatozoa are arranged in spermatozoa bundles called spermatodesms. Overall, this research provides novel insights into the structure of the testes of L. migratoria, which will significantly contribute to formulating effective pesticides against locusts.

Diet impacts the reproductive system’s maturation in the male moth Agrotis ipsilon (Noctuidae, Lepidoptera)

In male moth Agrotis ipsilon, sexual maturation occurs between the third and the fifth day of adult life and is characterized by the development of the reproductive organs such as testes and accessory sex glands. Since sexual maturation requires considerable energy investment, we hypothesized that diet would be an essential regulatory factor in this developmental process. Indeed, the links between the male diet and reproductive physiology have not been described as in females. To test the previous hypothesis, we offered male moths diets corresponding to different flower nectars found in nature, and measured morphological and functional changes in the testes and accessory sex glands. In comparison to a diet composed of sucrose only, males fed with a diet composed of diverse sugars, including glucose, supplemented with sodium led to an earlier increase in the length and the protein content of accessory sex glands, as well as a reduction of the testicular volume accompanied by an acceleration of the sperm bundle transfer from the testes to the duplex. These results show that these specific diets accelerate the maturation of the reproductive system in male moth Agrotis ipsilon.

Mob4 is essential for spermatogenesis in Drosophila melanogaster.

Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of interconnected spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. Here, we show that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role in female fertility. We show that Mob4 is required for proper axonemal structure and its loss leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of developing spermatids following mob4RNAi revealed expansion of the outer axonemal microtubules such that the 9 doublets no longer remained linked to each other and defective mitochondrial organization. Mob4 is a STRIPAK component, and male fertility is similarly impaired upon depletion of the STRIPAK components, Strip and Cka. Expression of the human Mob4 gene rescues all phenotypes of Drosophila mob4 downregulation, indicating that the gene is evolutionarily and functionally conserved. Together, this suggests that Mob4 contributes to the regulation of the microtubule- and actin-cytoskeleton during spermatogenesis through the conserved STRIPAK complex. Our study advances the understanding of male infertility by uncovering the requirement for Mob4 in sperm individualization.

Sperm structure of the diving beetle Deronectes moestus incospectus (Leprieur, 1876) (Hydroporinae, Dytiscidae) and considerations on extracellular material surrounding sperm bundles

The sperm cells of the diving beetle Deronectes moestus incospectus are characterized by sperm conjugation leading to the formation of sperm bundles of 64 units each. These bundles are formed at the end of spermatocyte cell divisions occurring in the testes and can be detected in the anterior region of the deferent ducts (first type of sperm conjugation). Fusions of some sperm bundles can occur at the end of the deferent ducts. The sperm bundles show sperm-head stacks (sperm rouleaux) and are surrounded by a cup of extracellular material secreted by the epithelial cells of the deferent ducts. This material extends posteriorly around the sperm bundle to cover the nuclei and the initial region of the sperm flagella. The cup extracellular material consists of fine tubules, and is no longer visible in sperm bundles at the posterior end of the deferent ducts. The sperm cells of D. moestus incospectus have an axoneme with a 9 + 9 + 2 pattern and unusual mitochondrial derivatives having a matrix showing dense dots and a small crystallized domain. Two thin elongated accessory bodies are located between the mitochondrial derivatives and the axoneme. The extracellular material can have different morphologies in the various families of Adephaga, but all are produced by the epithelium of the deferent ducts. Thus it is reasonable to assume that it has the same function in the different groups.

Cellular mechanisms of the lufenuron growth regulator in adults of Anthonomus grandis (Coleoptera: Curculionidae)

Abstract Growth-regulating insecticides are used for several cotton pests when immature, and although not registered for Anthonomus grandis, their use causes a satisfactory population reduction in adults. However, it is not known which mechanisms are involved in this reduction. Thus, we studied which tissue and cellular changes may be involved in this process. Adults were fed with flower buds treated with lufenuron (4 ml commercial product /l) for 24 h. Then, the histology of the gonads, oxidative stress and apoptosis were evaluated after periods of 24 h and 48 h. Our study revealed that lufenuron activates the oxidative stress pathway in A. grandis, causing significant changes at the cellular level. These changes may have been caused by stimulation of the production of oxygen ions, free radicals and hydrogen peroxides, resulting in an increase in lipid peroxidation 48 h after treatment. These effects were confirmed by the presence of histopathologies in the gonads of this pest such as disorganization of the follicular cell epithelium, reduction of the yolk, disappearance of the germinal vesicle, reduction of sperm bundles and cysts. Such alterations injured the gonads and impaired tissue homeostasis. It is concluded that together these factors lead to a desirable population reduction when it comes to managing the pest in the field, because the aim is to keep it below the control level. In addition to this desired effect, it is worth noting that lufenuron is safer when compared to other commonly used products, another feature that makes its use quite interesting.

Morphological organization and ultrastructural evaluation of the oocyte–sperm bundle of the Southwestern Atlantic coral Mussismilia harttii

Morphological organization and ultrastructural evaluation of the oocyte–sperm bundle of the Southwestern Atlantic coral Mussismilia harttii