Micro-Computed Tomography Reveals a Remarkable Twin Intromittent Organ in Spiders – A Novelty for Arachnids With Direct Sperm Transfer

The modification of male pedipalps into secondary sexual intromittent organs is one of the hallmark characteristics of spiders, yet understanding the development and evolution of male genitalia across the order remains a challenging prospect. The embolus – the sclerite bearing the efferent spermatic duct or spermophor, and used to deliver sperm directly to the female genitalia during copulation – has always been considered the single unambiguously homologous palpal sclerite shared by all spider species, fundamental to the bauplan of the order and to the evolution and functional morphology of spider reproductive systems. Indeed, after two centuries of comparative research on spider reproduction, the presence of a single spermophor and embolus on each of a male spider’s two pedipalps remains a central tenet of evolutionary arachnology. Our findings challenge this premise, and reveal a remarkable twin intromittent organ sperm transfer system in a lineage of Australian palpimanoid spiders, characterized by a bifurcate spermophor and the presence of two efferent ducts leading to a pair of embolic sclerites on each pedipalp. This is the first time such a remarkable conformation has been observed in any group of arachnids with direct sperm transfer, complicating our understanding of palpal sclerite homologies, and challenging ideas about the evolution of spider genitalia.

The Rete Testis: Development and Role in Testis Function

The rete testis connects seminiferous tubules in which germ cells develop to the efferent ducts and the epididymis, where gametes mature and gain mobility. Several recent studies have thoroughly explored the morphogenesis of this structure in mice during embryonic and postnatal periods. A part of the rete testis has been shown to derive from the precursors of gonad somatic cells before sex determination. The other part forms from embryonal Sertoli cells of testis cords adjacent to the mesonephros. The transformation of Sertoli cells into rete testis cells is apparently not limited to the embryonic stage of development and continues during postnatal testis development. Recently, it was found that the rete testis participates in the formation and maintenance of specialized Sertoli cells in terminal segments of seminiferous tubules, transitional zones. Current views suggest that the transitional zones of the seminiferous tubules may represent a niche for spermatogonial stem cells, the site of the prolonged proliferation of Sertoli cells in the pubertal and postpubertal periods of testis development, and also could be a generator of spermatogenic waves. To sum up, the rete testis transports gametes from the testis to the epididymis, maintains pressure within seminiferous tubules, regulates the composition of the testicular fluid, and impacts the spermatogenic process itself.

Development of Testis Cords and the Formation of Efferent Ducts in Xenopus laevis: Differences and Similarities with Other Vertebrates

The knowledge of testis development in amphibians relative to amniotes remains limited. Here, we used Xenopus laevis to investigate the process of testis cord development. Morphological observations revealed the presence of segmental gonomeres consisting of medullary knots in male gonads at stages 52–53, with no distinct gonomeres in female gonads. Further observations showed that cell proliferation occurs at specific sites along the anterior-posterior axis of the future testis at stage 50, which contributes to the formation of medullary knots. At stage 53, adjacent gonomeres become close to each other, resulting in fusion; then (pre-)Sertoli cells aggregate and form primitive testis cords, which ultimately become testis cords when germ cells are present inside. The process of testis cord formation in X. laevis appears to be more complex than in amniotes. Strikingly, steroidogenic cells appear earlier than (pre-)Sertoli cells in differentiating testes of X. laevis, which differs from earlier differentiation of (pre-)Sertoli cells in amniotes. Importantly, we found that the mesonephros is connected to the testis gonomere at a specific site at early larval stages and that these connections become efferent ducts after metamorphosis, which challenges the previous concept that the mesonephric side and the gonadal side initially develop in isolation and then connect to each other in amphibians and amniotes.

Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack outer dynein arms, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

The Distal Appendage Protein CEP164 Is Essential for Efferent Duct Multiciliogenesis and Male Fertility

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. Multiciliated cells (MCCs) are a specialized cell type with hundreds of motile multicilia, lining the brain ventricles, airways, and reproductive tracts to propel fluids/substances across the epithelial surface. In the male reproductive tract, MCCs in efferent ducts (EDs) move in a whip-like motion to stir the luminal contents and prevent sperm agglutination. Previously, we demonstrated that the essential distal appendage protein CEP164 recruits Chibby1 (Cby1), a small coiled-coil-containing protein, to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in MCCs (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility, however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. Consistent with these findings, multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that deletion of CEP164 in the MCCs of EDs causes basal body docking defects and loss of multicilia, leading to sperm agglutination, obstruction of EDs, and male infertility. Our study therefore unravels an essential role of the distal appendage protein CEP164 in male fertility.Author SummaryMulticilia are tinny hair-like microtubule-based structures that beat in a whip-like pattern to generate a fluid flow on the apical cell surface. Multiciliated cells are essential for the proper function of major organs such as brain, airway, and reproductive tracts. In the male reproductive system, multiciliated cells are present in the efferent ducts, which are small tubules that connect the testis to the epididymis. However, the importance of multiciliated cells in male fertility remains poorly understood. Here, we investigated the role of the critical ciliary protein CEP164 in male fertility using a mouse model lacking CEP164 in multiciliated cells. Male mice are infertile with reduced sperm counts. We demonstrate that, in the absence of CEP164, multiciliated cells are present in the efferent ducts but fail to extend multicilia due to basal body docking defects. Consistent with this, the recruitment of key ciliary proteins is perturbed. As a result, these mice show sperm agglutination, obstruction of sperm transport, and degeneration of germ cells in the testis, leading to infertility. Our study therefore reveals essential roles of CEP164 in the formation of multicilia in the efferent ducts and male fertility.

Neutrophil Extracellular Traps Promote the Development and Growth of Human Salivary Stones

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. The mechanism behind the formation of sialoliths has been elusive. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland, and often results in sialadenitis with the need for surgical intervention. Here, we show by the use of immunohistochemistry, immunofluorescence, computed tomography (CT) scans and reconstructions, special dye techniques, bacterial genotyping, and enzyme activity analyses that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths in humans. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in the dense aggregation of the latter, and the subsequent mineralization creates alternating layers of dense mineral, which are predominantly calcium salt deposits and DNA. The further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis, in which an immune system-mediated response essentially participates in the physicochemical process of concrement formation and growth.

An atlas of human proximal epididymis reveals cell-specific functions and distinct roles for CFTR

Spermatozoa released from the testis are unable to fertilize an egg without a coordinated process of maturation in the lumen of the epididymis. Relatively little is known about the molecular events that integrate this critical progression along the male genital ducts in man. Here, we use single cell RNA-sequencing to construct an atlas of the human proximal epididymis. We find that the CFTR, which is pivotal in normal epididymis fluid transport, is most abundant in surface epithelial cells in the efferent ducts and in rare clear cells in the caput epididymis, suggesting region-specific functional properties. We reveal transcriptional signatures for multiple cell clusters, which identify the individual roles of principal, apical, narrow, basal, clear, halo, and stromal cells in the epididymis. A marked cell type–specific distribution of function is seen along the duct with local specialization of individual cell types integrating processes of sperm maturation.

Development and Growth of Human Salivary Stones by Neutrophil Extracellular Traps

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland and often results in sialadenitis with the need for surgical intervention. The mechanism behind the formation of sialoliths has been elusive. Here we show that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in their dense aggregation, and the subsequent mineralization creates alternating layers of dense mineral, predominantly calcium salt deposits and DNA. Further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis in which an immune system mediated response essentially participates in the physico-chemical process of concrement formation and growth.