A novel function for the presenilin family member <Emphasis Type="Italic">spe-4</Emphasis>: inhibition of spermatid activation in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>.

Background  

Sperm cells must regulate the timing and location of activation to maximize the likelihood of fertilization. Sperm from most species, including the nematode Caenorhabditis elegans, activate upon encountering an external signal. Activation for C. elegans sperm occurs as spermatids undergo spermiogenesis, a profound cellular reorganization that produces a pseudopod. Spermiogenesis is initiated by an activation signal that is transduced through a series of gene products. It is now clear that an inhibitory pathway also operates in spermatids, preventing their premature progression to spermatozoa and resulting in fine-scale control over the timing of activation. Here, we describe the involvement of a newly assigned member of the inhibitory pathway: spe-4, a homolog of the human presenilin gene PS1. The spe-4(hc196) allele investigated here was isolated as a suppressor of sterility of mutations in the spermiogenesis signal transduction gene spe-27.     

spe-12 encodes a sperm cell surface protein that promotes spermiogenesis in Caenorhabditis elegans

During spermiogenesis, Caenorhabditis elegans spermatids activate and mature into crawling spermatozoa without synthesizing new proteins. Mutations in the spe-12 gene block spermatid activation, rendering normally self-fertile hermaphrodites sterile. Mutant males, however, are fertile. Surprisingly, when mutant hermaphrodites mate with a male, their self-spermatids activate and form functional spermatozoa, presumably due to contact with male seminal fluid. Here we show that, in addition to its essential role in normal activation of hermaphrodite-derived spermatids, SPE-12 also plays a supplementary but nonessential role in mating-induced activation. We have identified the spe-12 gene, which encodes a novel protein containing a single transmembrane domain. spe-12 mRNA is expressed in the sperm-producing germ line and the protein localizes to the spermatid cell surface. We propose that SPE-12 functions downstream of both hermaphrodite- and male-derived activation signals in a spermatid signaling pathway that initiates spermiogenesis.     

The genetic and molecular analysis of spe-19, a gene required for sperm activation in Caenorhabditis elegans

During the process of spermiogenesis (sperm activation) in Caenorhabditis elegans, the dramatic morphological events that ultimately transform round sessile spermatids into polar motile spermatozoa occur without the synthesis of any new gene products. Previous studies have identified four genes (spe-8, spe-12, spe-27 and spe-29) that specifically block spermiogenesis and lead to hermaphrodite-specific fertility defects. Here, we report the cloning and characterization of a new component of the sperm activation pathway, spe-19, that is required for fertility in hermaphrodites. spe-19 is predicted to encode a novel single-pass transmembrane protein. The spe-19 mutant phenotype, genetic interactions and the molecular nature of the gene product suggest SPE-19 to be a candidate for the receptor/co-receptor necessary for the transduction of the activation signal across the sperm plasma membrane.     

Spermiogenesis initiation in Caenorhabditis elegans involves a casein kinase 1 encoded by the spe-6 gene

Immature spermatids from Caenorhabditis elegans are stimulated by an external activation signal to reorganize their membranes and cytoskeleton to form crawling spermatozoa. This rapid maturation, termed spermiogenesis, occurs without any new gene expression. To better understand this signal transduction pathway, we isolated suppressors of a mutation in the spe-27 gene, which is part of the pathway. The suppressors bypass the requirement for spe-27, as well as three other genes that act in this pathway, spe-8, spe-12, and spe-29. Eighteen of the suppressor mutations are new alleles of spe-6, a previously identified gene required for an early stage of spermatogenesis. The original spe-6 mutations are loss-of-function alleles that prevent major sperm protein (MSP) assembly in the fibrous bodies of spermatocytes and arrest development in meiosis. We have isolated the spe-6 gene and find that it encodes a predicted protein-serine/threonine kinase in the casein kinase 1 family. The suppressor mutations appear to be reduction-of-function alleles. We propose a model whereby SPE-6, in addition to its early role in spermatocyte development, inhibits spermiogenesis until the activation signal is received. The activation signal is transduced through SPE-8, SPE-12, SPE-27, and SPE-29 to relieve SPE-6 repression, thus triggering the formation of crawling spermatozoa.     

A New Player in the Spermiogenesis Pathway of Caenorhabditis elegans

Precise timing of sperm activation ensures the greatest likelihood of fertilization. Precision in Caenorhabditis elegans sperm activation is ensured by external signaling, which induces the spherical spermatid to reorganize and extend a pseudopod for motility. Spermatid activation, also called spermiogenesis, is prevented from occurring prematurely by the activity of SPE-6 and perhaps other proteins, termed “the brake model.” Here, we identify the spe-47 gene from the hc198 mutation that causes premature spermiogenesis. The mutation was isolated in a suppressor screen of spe-27(it132ts), which normally renders worms sterile, due to defective transduction of the activation signal. In a spe-27(+) background, spe-47(hc198) causes a temperature-sensitive reduction of fertility, and in addition to premature spermiogenesis, many mutant sperm fail to activate altogether. The hc198 mutation is semidominant, inducing a more severe loss of fertility than do null alleles generated by CRISPR-associated protein 9 (Cas9) technology. The hc198 mutation affects an major sperm protein (MSP) domain, altering a conserved amino acid residue in a β-strand that mediates MSP–MSP dimerization. Both N- and C-terminal SPE-47 reporters associate with the forming fibrous body (FB)-membranous organelle, a specialized sperm organelle that packages MSP and other components during spermatogenesis. Once the FB is fully formed, the SPE-47 reporters dissociate and disappear. SPE-47 reporter localization is not altered by either the hc198 mutation or a C-terminal truncation deleting the MSP domain. The disappearance of SPE-47 reporters prior to the formation of spermatids requires a reevaluation of the brake model for prevention of premature spermatid activation.     

spe-29 encodes a small predicted membrane protein required for the initiation of sperm activation in Caenorhabditis elegans

Caenorhabditis elegans spermatids complete a dramatic morphogenesis to crawling spermatozoa in the absence of an actin- or tubulin-based cytoskeleton and without synthesizing new gene products. Mutations in three genes (spe-8, spe-12, and spe-27) prevent the initiation of this morphogenesis, termed activation. Males with mutations in any of these genes are fertile. By contrast, mutant hermaphrodites are self-sterile when unmated due to a failure in spermatid activation. Intriguingly, mutant hermaphrodites form functional spermatozoa and become self-fertile upon mating, suggesting that spermatids can be activated by male seminal fluid. Here we describe a mutation in a fourth gene, spe-29, which mimics the phenotype of spe-8, spe-12, and spe-27 mutants. spe-29 sperm are defective in the initiation of hermaphrodite sperm activation, yet they maintain the ability to complete the morphogenetic rearrangements that follow. Mutant alleles of spe-12, spe-27, and spe-29 exhibit genetic interactions that suggest that the wild-type products of these genes function in a common signaling pathway to initiate sperm activation. We have identified the spe-29 gene, which is expressed specifically in the sperm-producing germ line and is predicted to encode a small, novel transmembrane protein.     

Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans

In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.     

Genetic and Molecular Analysis of Spe-27, a Gene Required for Spermiogenesis in Caenorhabditis Elegans Hermaphrodites

Hermaphrodites with mutations in the spe-27 gene are self-sterile, laying only unfertilized eggs; mutant males are fertile. Hermaphrodites make spermatids that fail to activate to crawling spermatozoa so passing oocytes sweep them out of the spermatheca. These spermatids do activate and produce self-progeny if young mutant hermaphrodites are mated by fertile (or sterile) males. Spermatids isolated from either mutant males or hermaphrodites initiate activation in vitro when treated with proteases, but then arrest with spiky membrane projections that resemble those of a normal intermediate in pseudoped formation. These phenotypes are identical to spe-8 and spe-12 mutants. They can be explained if males and hermaphrodites have distinct pathways for spermatid activation, and these three genes are necessary only for the hermaphrodite pathway. Consistent with this model, when spe-27 mutant male spermatids without seminal fluid are artificially inseminated into hermaphrodites, they fail to activate. The spe-27 gene has been isolated, sequenced and its regulatory regions identified. The sequence predicts a 131 amino acid polypeptide that has no striking structural motifs and no resemblance to known proteins. Two of the mutations in spe-27 alter mRNA splicing; a third mutation is a temperature-sensitive missense mutation.     

Ultrastructural Analysis of Spermiogenesis in Segregation Distorter Males of DROSOPHILA MELANOGASTER: The Homozygotes

We have studied spermiogenesis at the ultrastructural level in males of genotype SD(NH)-2/SD-72, which are nearly sterile owing to the dysfunction of virtually all of their sperm. Ultrastructural aspects of spermiogenesis in these homozygous SD males are qualitatively similar to those found among dysfunctional sperm produced by heterozygous SD males. In particular, chromatin condensation and/or compaction has been found to be abnormal. However, major quantitative differences have been noted. Most of the dysfunctional sperm in SD(NH)-2/SD-72 males are individualized and coiled. Then, the sperm evidently undergo degeneration, as few mature sperm can be found in the seminal vesicle. The relevance of these findings to the mechanism leading to near sterility in homozygous SD males is discussed.     

Ultrastructure of spermiogenesis and spermatozoa in Prorhynchus sp. (Platyhelminthes,Lecithoepitheliata, Prorhynchidae)

Summary

Mature sperm of Prorhynchus sp. have an elongated nucleus, multiple mitochondria and dense bodies, and two free axonemes which are located in grooves of the main shaft for much of their length. The axonemes are subterminally inserted and have the typical 9+ ‘1’ arrangement unique to Platyhelminthes and synapomorphic for taxa of Trepaxonemata. The testis follicles examined had small numbers of developing spermatids and very few mature sperm were present. During spermiogenesis, spermatids remain joined in clusters by distinctive bridges. In each spermatid two centrioles (with an intercentriolar body between them) give rise to free axonemes which grow out in opposite directions from each other. Indistinct ciliary rootlets are present. The axonemes are carried distally from the main spermatid mass on an elongating process and turn back towards the main spermatid mass. Nucleus, mitochondria and dense bodies move into the shaft, and the spermatid elongates before detaching from others in the cluster. This is the first detailed study of sperm and spermiogenesis in Lecithoepitheliata. Mature sperm are distinctly different from those of prolecithophorans, to which they are reputedly related, the latter having aflagellate sperm without dense bodies.     

Mammalian Fused is essential for sperm head shaping and periaxonemal structure formation during spermatogenesis

During mammalian spermatogenesis, the diploid spermatogonia mature into haploid spermatozoa through a highly controlled process of mitosis, meiosis and post-meiotic morphological remodeling (spermiogenesis). Despite important progress made in this area, the molecular mechanisms underpinning this transformation are poorly understood. Our analysis of the expression and function of the putative serine–threonine kinase Fused (Fu) provides critical insight into key steps in spermatogenesis. In this report, we demonstrate that conditional inactivation of Fu in male germ cells results in infertility due to diminished sperm count, abnormal head shaping, decapitation and motility defects of the sperm. Interestingly, mutant flagellar axonemes are intact but exhibit altered periaxonemal structures that affect motility. These data suggest that Fu plays a central role in shaping the sperm head and controlling the organization of the periaxonemal structures in the flagellum. We show that Fu localizes to multiple tubulin-containing or microtubule-organizing structures, including the manchette and the acrosome–acroplaxome complex that are involved in spermatid head shaping. In addition, Fu interacts with the outer dense fiber protein Odf1, a major component of the periaxonemal structures in the sperm flagellum, and Kif27, which is detected in the manchette. We propose that disrupted Fu function in these structures underlies the head and flagellar defects in Fu-deficient sperm. Since a majority of human male infertility syndromes stem from reduced sperm motility and structural defects, uncovering Fu?s role in spermiogenesis provides new insight into the causes of sterility and the biology of reproduction.     

Arginine-rich nucleoprotein transition occurs in the two size classes of spermatozoa of Drosophila subobscura males

The normal male of Drosophila subobscura displays polymegaly, which is the presence of two sizes of spermatozoa in the same testis. It is still unknown whether both kinds of sperm are able to fertilize the egg. An indicator of normal functioning of Drosophila spermatozoa is the replacement of the somatic histones by sperm-specific arginine-rich nucleoproteins during spermiogenesis. The appearance of these arginine-rich nucleoproteins in the two kinds of sperm was investigated using the fluorescent dye sulfoflavine, which stains basic proteins at pH 8. In the spherical nuclei of early spermatids of Drosophila subobscura the somatic histones fluoresced strongly, but fluorescence could not be detected in later stages when the spermatid nuclei were elongating. After elongation, however, the nuclei of both kinds of sperm, long and short, fluoresced brightly again, due to the presence of sperm-specific arginine-rich nucleoproteins. Half of the cysts of both types contained spermatid nuclei with aberrant fluorescent pattern including 5–9% of both cyst types which do not undergo histone transition at all. These results indicate that both sperm types may be functional.     

Dynamic aspects of spermiogenic chromatin condensation patterning by phase separation during the histone-to-protamine transition in charalean algae and relation to bryophytes

During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules → fibers → contorted lamellae → condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid–liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha.     

A microtubule organizing centre (MTOC) is responsible for the production of the sperm flagellum in Matsucoccus feytaudi (Hemiptera: Coccoidea)

A microtubule organizing centre (MTOC) has been described in the spermatid of the hemipteran Matsucoccus feytaudi (Coccoidea). This structure, revealed as a fluorescent ring by treatment with γ-tubulin antibody, gives rise to a bundle of microtubules which surrounds the elongated cylindrical nucleus. This microtubule bundle has been considered an atypical sperm flagellum provided with sperm motility. A comparison of the M. feytaudi MTOC with the material associated with the centriole of Drosophila melanogaster spermatids confirms the great similarity between the two structures, both involved in the nucleation of microtubules. Like the D. melanogaster material associated with the centriole, the M. feytaudi MTOC is a transient structure which disappears or degenerates at the end of spermiogenesis and is no longer visible in the mature sperm.     

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small ''bag'' called the spermatheca. Later on, hermaphrodites continually produce oocytes¹. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for >50% of the total cells in a typical adult worm². Therefore, isolating sperm from males is easier than from that of hermaphrodites.Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome³. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population³.Males can be easily distinguished from hermaphrodites by observing the tail morphology⁴. Hermaphrodite''s tail is pointed, whereas male tail is rounded with mating structures.Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids².Males are directly dissected on a small drop of ''Sperm Medium''. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes⁵, and Nelson⁶ previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process⁷. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.Download video file.^{(33M, flv)}     

The spe-42 gene is required for sperm-egg interactions during C. elegans fertilization and encodes a sperm-specific transmembrane protein

Fertilization, the union of sperm and egg to form a new organism, is a critical process that bridges generations. Although the cytological and physiological aspects of fertilization are relatively well understood, little is known about the molecular interactions that occur between gametes. C. elegans has emerged as a powerful system for the identification of genes that are necessary for fertilization. C. elegans spe-42 mutants are sterile, producing cytologically normal spermatozoa that fail to fertilize oocytes. Indeed, male mating behavior, sperm transfer to hermaphrodites, sperm migration to the spermatheca, which is the site of fertilization and sperm competition are normal in spe-42 mutants. spe-42 mutant sperm make direct contact with oocytes in the spermatheca, suggesting that SPE-42 plays a role during sperm-egg interactions just prior to fertilization. No other obvious defects were observed in spe-42 mutant worms. Cloning and sequence analysis revealed that SPE-42 is a novel predicted 7-pass integral membrane protein with homologs in many metazoan species, suggesting that its mechanism of action could be conserved.     

The sperm of Matsucoccus feytaudi (Insecta,Coccoidea): Can the microtubular bundle be considered as a true flagellum?

In the present work the spermiogenesis and sperm structure of Matsucoccus feytaudi, a primary pest of the maritime pine in southern eastern Europe, is studied. In addition to the already known characteristics of coccid sperm, such as the absence of the acrosome and mitochondria, and the presence of a bundle of microtubules responsible for sperm motility, a peculiar structure from which the microtubule bundle takes origin is described. Such a structure – a short cylinder provided with a central hub surrounded by several microtubules with a dense wall – is regarded as a Microtubule Organizing Centre (MTOC). During spermiogenesis, quartets of fused spermatids are formed; from each spermatid, a bundle of microtubules, generated by the MTOC, projects from the cell surface. Each cell has two centrioles, suggesting the lack of a meiotic process and the occurrence of parthenogenesis. At the end of the spermiogenesis, when the cysts containing bundles of sperm are formed, part of the nuclear material together with the MTOC structure is eliminated. Based on the origin of the microtubular bundle from the MTOC, the nature of the bundle as a flagellum is discussed.     

Developmental Stages of Genome Elimination Resulting in Transmission Ratio Distortion of the T-007 Male Recombination (MR) Chromosome of DROSOPHILA MELANOGASTER

T-007 is a Male Recombination (MR) second chromosome that induces transmission ratio distortion (at its own expense) when heterozygous with many laboratory marker chromosomes. The developmental timing of elimination of T-007 chromosomes has been investigated. About 21% of the T-007 chromosomes expected to be recovered among the progeny of heterozygous T-007 males are lost at some point between fertilization and eclosion (representing 29% of the total distortion observed in young males). Another 52% of the expected number of T-007 chromosomes are lost as a result of spermatid abortion during spermiogenesis (representing 71% of the total distortion). Abnormalities in both the number of spermatids per bundle and the structure of spermatid tails are seen at the earliest stages of spermiogenesis in T-007 males.