Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse.

Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans.

Overexpression of full-length centrobin rescues limb malformation but not male fertility of the hypodactylous (hd) rats.

Rat hypodactyly (hd) mutation is characterized by abnormal spermatogenesis and sperm decapitation, limb malformation (missing digits II and III) and growth retardation. We have previously reported centrobin (centrosome BRCA2-interacting protein) truncation at the C-terminus in the hd mutant. Here, we report data from a transgenic rescue experiment carried out to determine a role of centrobin in pathogenesis of hd. The transgenic construct, consisting of full-length-coding cDNA linked to a ubiquitous strong promoter/enhancer combination, was inserted to chromosome 16 into a LINE repeat. No known gene is present in the vicinity of the insertion site. Transgenic centrobin was expressed in all tissues tested, including testis. Transgenic animals show normal body weight and limb morphology as well as average weight of testis and epididymis. Yet, abnormal spermatogenesis and sperm decapitation persisted in the transgenic animals. Western blotting showed the coexistence of full-length and truncated or partially degraded centrobin in sperm of the rescued transgenic animals. Immunocytochemistry showed a buildup of centrobin and ODF2 (outer dense fiber 2) at the sperm decapitation site in the hd mutant and rescued transgenic rats. Additional findings included bulge-like formations and thread-like focal dissociations along the sperm flagellum and the organization of multiple whorls of truncated sperm flagella in the epididymal lumen. We conclude that centrobin is essential for normal patterning of the limb autopod. Centrobin may be required for stabilizing the attachment of the sperm head to flagellum and for maintaining the structural integrity of the sperm flagellum. We postulate that the presence of truncated centrobin, coexisting with full-length centrobin, together with incorrect timing of transgenic centrobin expression may hamper the rescue of fertility in hd male rats.

GMAP210 and IFT88 are present in the spermatid golgi apparatus and participate in the development of the acrosome-acroplaxome complex, head-tail coupling apparatus and tail.

We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome-acroplaxome complex, the head-tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis-Golgi, whereas IFT88 prevails in the trans-Golgi network. Both proteins colocalize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome-acroplaxome region of the sperm head, whereas GMAP210 is no longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail-less. GMAP210 is visualized in the Ift88 mutant during acrosome-acroplaxome biogenesis. However, GMAP210-stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin-A and nocodazole blocks the progression of GMAP210- and IFT88-stained proacrosomal vesicles to the acrosome-acroplaxome complex but F-actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule-associated golgin GMAP210 and IFT88 participate in acrosome, HTCA, and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development.

Cytoskeletal track selection during cargo transport in spermatids is relevant to male fertility.

Spermatids generate diverse and unusual actin and microtubule populations during spermiogenesis to fulfill mechanical and cargo transport functions assisted by motor and non-motor proteins. Disruption of cargo transport may lead to teratozoospermia and consequent male infertility. How motor and non-motor proteins utilize the cytoskeleton to transport cargos during sperm development is not clear. Filamentous actin (F-actin) and the associated motor protein myosin Va participate in the transport of Golgi-derived proacrosomal vesicles to the acrosome and along the manchette. The acrosome is stabilized by the acroplaxome, a cytoskeletal plate anchored to the nuclear envelope. The acroplaxome plate harbors F-actin and actin-like proteins as well as several other proteins, including keratin 5/Sak57, Ran GTPase, Hook1, dynactin p150Glued, cenexin-derived ODF2, testis-expressed profilin-3 and profilin-4, testis-expressed Fer tyrosine kinase (FerT), members of the ubiquitin-proteasome system and cortactin. Spermatids express transcripts encoding the non-spliced form of cortactin, a F-actin-regulatory protein. Tyrosine phosphorylated cortactin and FerT coexist in the acrosome-acroplaxome complex. Hook1 and p150Glued, known to participate in vesicle cargo transport, are sequentially seen from the acroplaxome to the manchette to the head-tail coupling apparatus (HTCA). The golgin Golgi-microtubule associated protein GMAP210 resides in the cis-Golgi whereas the intraflagellar protein IFT88 localizes in the trans-Golgi network. Like Hook1 and p150Glued, GMAP210 and IFT88 colocalize at the cytosolic side of proacrosomal vesicles and, following vesicle fusion, become part of the outer and inner acrosomal membranes before relocating to the acroplaxome, manchette and HTCA. A hallmark of the manchette and axoneme is microtubule heterogeneity, determined by the abundance of acetylated, tysosinated and glutamylated tubulin isoforms produced by post-translational modifications. We postulate that the construction of the male gamete requires microtubule and F-actin tracks and specific molecular motors and associated non-motor proteins for the directional positioning of vesicular and non-vesicular cargos at specific intracellular sites.

Rnf19a, a ubiquitin protein ligase, and Psmc3, a component of the 26S proteasome, tether to the acrosome membranes and the head-tail coupling apparatus during rat spermatid development.

We report the cDNA cloning of rat testis Rnf19a, a ubiquitin protein ligase, and show 98% and 93% protein sequence identity of testicular mouse and human Rnf19a, respectively. Rnf19a interacts with Psmc3, a protein component of the 19S regulatory cap of the 26S proteasome. During spermatid development, Rnf19a and Psmc3 are initially found in Golgi-derived proacrosomal vesicles. Later on, Rnf19a, Psmc3, and ubiquitin are seen along the cytosolic side of the acrosomal membranes and the acroplaxome, a cytoskeletal plate linking the acrosome to the spermatid nuclear envelope. Rnf19a and Psmc3 accumulate at the acroplaxome marginal ring-manchette perinuclear ring region during spermatid head shaping and in the developing sperm head-tail coupling apparatus and tail. Rnf19a and Psmc3 may interact directly or indirectly with each other, presumably pointing to the participation of the ubiquitin-proteasome system in acrosome biogenesis, spermatid head shaping, and development of the head-tail coupling apparatus and tail.

Expression of full-length and truncated Fyn tyrosine kinase transcripts and encoded proteins during spermatogenesis and localization during acrosome biogenesis and fertilization.

We report that full-length and truncated transcripts of Fyn tyrosine protein kinase are expressed during testicular development. Truncated Fyn (tr-Fyn) transcripts encode a 24 kDa protein with a N-terminal (NT) domain, a complete Src homology (SH) 3 domain and an incomplete SH2 domain. The kinase domain is missing in tr-Fyn. In contrast, full-length Fyn transcripts encode a 59-55 kDa protein. Fractionated spermatids by centrifugal elutriation express tr-Fyn transcripts and protein, but not full-length Fyn transcripts and protein. Neither full-length Fyn nor tr-Fyn transcripts and encoded proteins are detected in elutriated pachytene spermatocytes. Sertoli cells express full-length and truncated Fyn throughout testicular development. In contrast, sperm contain full-length Fyn transcripts and protein but not the truncated form. tr-Fyn protein is visualized at the cytosolic side of Golgi membranes, derived proacrosomal vesicles, along the outer acrosome membrane and the inner acrosome membrane-acroplaxome complex anchoring the acrosome to the spermatid nuclear envelope. Fyn and phosphotyrosine immunoreactivity coexist in the tail of capacitated sperm. During fertilization, the Fyn-containing acroplaxome seen in the egg-bound and egg-fused sperm is no longer detected upon decondensation of the sperm nucleus. tr-Fyn expands the catalog of truncated tyrosine protein kinases expressed during spermiogenesis. We suggest that the NT and SH3 domains of tr-Fyn may recruit adaptor and effector proteins, in particular GTPase activating proteins, required for acrosome-acroplaxome biogenesis, acroplaxome F-actin dynamics and Sertoli cell function. During fertilization, full-length Fyn in the acroplaxome may contribute to a transient local signaling burst during the early events of sperm-egg interaction.

Expression of Fer testis (FerT) tyrosine kinase transcript variants and distribution sites of FerT during the development of the acrosome-acroplaxome-manchette complex in rat spermatids.

We report the association of testicular Fer, a non-receptor tyrosine kinase, with acrosome development and remodeling of the acrosome-associated acroplaxome plate during spermatid head shaping. A single gene expresses two forms of Fer tyrosine kinases in testis: a somatic form (FerS) and a truncated testis-type form (FerT). FerT transcript variants are seen in spermatocytes and spermatids. FerS transcripts are not detected in round spermatids but are moderately transcribed in spermatocytes. FerT protein is associated with the spermatid medial/trans-Golgi region, proacrosomal vesicles, the cytosolic side of the outer acrosome membrane and adjacent to the inner acrosome membrane facing the acroplaxome. FerT coexist in the acroplaxome with phosphorylated cortactin, a regulator of F-actin dynamics. We propose that FerT participates in acrosome development and that phosphorylated cortactin may contribute to structural changes in F-actin in the acroplaxome during spermatid head shaping.

Genomic origin, processing and developmental expression of testicular outer dense fiber 2 (ODF2) transcripts and a novel nucleolar localization of ODF2 protein.

Outer dense fibers are a major constituent of the sperm tail and outer dense fiber 2 (ODF2) protein is one of their major components. ODF2 shares partial homology with cenexin 1 and cenexin 2, regarded as centriolar proteins. We show that ODF2 and cenexin 2 transcripts are the product of differential splicing of a single gene, designated Cenexin/ODF2 and that cenexin 1 is an incomplete clone of ODF2. ODF2 terminates in exon 20b whereas in cenexin 2 this exon is spliced out and translation terminates in exon 24. We demonstrate a transcriptional switch during rat testicular development, from somatic-type to testis-type ODF2 and cenexin transcripts during the onset of meiosis. The switch is completed when spermiogenesis is established. ODF2 immunoreactive sites were visualized in the acroplaxome, along the sperm tail and the centrosome-derived sperm head-to-tail coupling apparatus. An unexpected finding was the presence of ODF2 antigenic sites, but not cenexin antigenic sites, in the dense fibrillar component of the nucleolus of Sertoli cells, spermatogonia and primary spermatocytes. The characterization of the genomic origin, processing and developmental expression of ODF2 transcript isoforms and their protein products can help reconcile differences in the literature on the role of ODF2 and cenexin in the centrosome. Furthermore, the finding of ODF2 in the dense fibrillar component of the nucleolus suggests that this protein, in addition to its presence in sperm outer dense fibers and centrosome, highlights and adds to the nucleolar function during spermatogenesis and early embryogenesis.

Molecular biology of sperm head shaping.

The shaping of the mammalian sperm involves the elongation and condensation of the spermatid nucleus, the development of the acrosome, and the transient appearance of the microtubular manchette. F-actin-containing ectoplasmic hoops of Sertoli cells embrace the upper third of the spermatid head during elongation. During acrosomal biogenesis, proacrosomal vesicles derived from the Golgi apparatus, dock and fuse along the acroplaxome, an F-actin/keratin 5-containing cytoskeletal plate. The acroplaxome consists of a bent plate and a marginal ring encircling the spermatid nucleus. It anchors the developing acrosome to the spermatid nucleus. The manchette, consisting of a perinuclear rings with inserted microtubules, lies subjacent to the marginal ring of the acroplaxome. During spermatid elongation, the two overlapping rings reduce their diameter to fit, in a sleeve-like fashion, the decreasing diameter of the spermatid nucleus. The acroplaxome may provide a planar scaffold to modulate exogenous constriction forces generated by Sertoli cell F-actin hoops during spermatid head elongation. The dynamics of the F-actin cytoskeleton, one of the components of the acroplaxome and Sertoli cell hoops, can be regulated by tyrosine kinases, which target cortactin, an F-actin-associated protein. Tyrosine phosphorylation of cortactin correlates with a reduction in the crosslinking properties of F-actin. Phosphorylated cortactin and tyrosine kinase Fer are present in the acroplaxome, thus supporting a role of this F-actin remodelling pathway during spermatid head shaping. Keratin 5, an additional component of the acroplaxome, may also undergo dynamic reorganization during spermatid head elongation. We postulate that the F-actin/keratin 5 cytoskeleton in the acroplaxome may undergo a dynamic reorganization to modulate exogenous shear forces exerted by Sertoli cell F-actin hoops during spermatid head shaping. The acroplaxome-manchette perinuclear rings may reduce their diameter to balance exogenous constriction forces generated by the embracing Sertoli cell F-actin hoops and guide nuclear elongation.

Role of integrins, tetraspanins, and ADAM proteins during the development of apoptotic bodies by spermatogenic cells.

We have previously reported that Sertoli cell geometric changes induced by a Fas (CD95) agonist or by restricting Sertoli cell spreading can trigger spermatogenic cell detachment from Sertoli cell surfaces and initiate a programmed cell death sequence. Here, we have focused on ADAM proteins, tetraspanins CD9 and CD81, and the integrin beta1 subunit, which is co-expressed in testis with integrin alpha3 and integrin alpha6 subunits, to understand how these molecules may stabilize spermatogenic cell attachment to Sertoli cell surfaces. Like ADAM proteins, integrin beta1, alpha3, and alpha6 subunits, and CD9 and CD81 transcripts are expressed in the fetal testis and throughout testicular maturation, as well as, in Sertoli-spermatogenic cell co-cultures. Prespermatogonia (gonocytes) display CD9 and CD81 immunoreactive sites. Integrin alpha6 subunit transcripts have unusual developmental characteristics: fetal testis expresses the integrin alpha6B isoform exclusively. In contrast, the integrin alpha6B isoform co-exists with the integrin alpha6A isoform in prepubertal testes and Sertoli-spermatogenic cell co-cultures. A blocking anti body targeting the extracellular domain (N-terminal) of the integrin beta1 subunit causes rapid contraction of Sertoli cells leading to the gradual detachment of associated spermatogenic cells. In contrast, predicted active site peptides targeting the disintegrin domain of ADAM 1, ADAM 2, ADAM 3 (cyritestin), ADAM 4, ADAM 5, ADAM 6, and ADAM 15 (metragidin) do not disturb significantly the attachment of spermatogenic cells to Sertoli cell surfaces. Spermatogenic cells dislodged from their attachment sites by the integrin beta1 subunit blocking antibody display annexin V immunoreactivity, a sign of early apoptosis. Time-lapse videomicroscopy demonstrates that the removal by apoptosis of a single member of a spermatogenic cell cohort inter-connected by cytoplasmic bridges does not affect the remaining members of the cohort. During spermatogenic cell apoptosis, integrin beta1, alpha3, and alpha6 subunits, and tetraspanins CD9 and C81 become displaced away from the developing apoptotic bodies. In contrast, the intermediate filament protein Sak57, a keratin 5 ortholog, concentrates in the developing apoptotic bodies. We propose that the redistribution of integrin-tetraspanin complexes during spermatogenic cell apoptosis may be evidence of a signaling cascade initiated by Sertoli cell geometric changes. As a result, Sertoli cell reduction in surface area may be a limiting factor of spermatogenic cell survival and in the developmental regulation of spermatogenic cell progenies in the intact seminiferous epithelium.

The ADAM-integrin-tetraspanin complex in fetal and postnatal testicular cords.

New insights have emerged about the expression, during testicular cord formation, of the ADAM (a disintegrin and metalloprotease) domain family of proteins that combines both cell surface adhesion and proteolytic activity; this family includes integrins alpha3beta1 and alpha6beta1 and tetraspanins, a distinct family of proteins containing four transmembrane domains, a small and a large extracellular loop, and short cytoplasmic tails. ADAM3 (cyritestin), ADAM5, ADAM6, and ADAM15 are expressed in fetal rat testes. In contrast, the expression of the ADAM1/ADAM2 pair (fertilin alpha/fertilin beta, respectively) is not detected in fetal testis. Yet the expression of ADAM1 starts immediately after birth, and is followed within 24 hr by the expression of ADAM2. Therefore, the ADAM1/ADAM2 heterodimer is visualized far in advance of the meiotic and spermiogenic phase of spermatogenesis. A similar expression pattern was observed for integrin subunits alpha3, alpha6, and beta1, as well as for tetraspanins CD9, CD81, and CD98; the latter is a single-pass integrin subunit beta1-binding protein. ADAM2, integrin subunits alpha3, alpha6, and beta1, and tetraspanin CD9 and CD81 immunoreactive sites are observed in prespermatogonia (also known as primordial germ cells or gonocytes). A model is proposed in which the ADAM-integrin-tetraspanin complex, known to constitute a network of membrane microdomains called the tetraspanin web, may be involved in the migration of prespermatogonia from the center to the periphery of the testicular cords and in the reinitiation of mitotic activity during the initial wave of spermatogenesis. A complementary model consists in the rearrangement of the tetraspanin web in prespermatogonia/spermatogonia undergoing spontaneous or Fas-induced apoptosis upon coculturing with Sertoli cells. In this model, the cellular site involved in the formation of preapoptotic bodies is devoid of tetraspanin-integrin clusters, in contrast with nonapoptotic cells, which display a diffuse circumferential distribution. In apoptotic prespermatogonia, immunoreactive clusters are restricted to sites where the attachment of prespermatogonia/spermatogonia to Sertoli cell surfaces is still preserved.

XY chromosomal bivalent: nucleolar attraction.

Nucleolar organization by autosomal bivalents occurs during male meiotic prophase in mammalian species. During late leptotene-early zygotene stages, several autosomal bivalents are engaged in ribosomal RNA synthesis. At pachytene stage, nucleolar masses detach from the sites of primary autosomal origin, relocate close to the XY chromosomal pair, and nucleolar components become segregated. In early pachytene, an extensive synaptonemal complex at the pseudoautosomal region, links X and Y chromosomes in close juxtaposition along most of the length of the Y chromosome, except for a terminal region of the Y that diverges from the pairing region. As meiotic prophase advances, X and Y chromosomes progressively desynapse and, at diplotene, the XY pair is associated end-to-end. Xmr (Xlr-related, meiosis regulated) is a protein component of the nucleolus associated to the XY pair and of the asynapsed portions of the X and Y axial cores. Xmr, like SCP3, is a component of the lateral element of the synaptonemal complex. Both share structural homology in their C-terminal region. This region contains several putative coiled-coil domains known to mediate heterodimeric protein-protein interactions and to provide binding sites to regulatory proteins. Like Xmr, the tumor repressor protein BRCA1 is present along the unsynapsed cores of the XY bivalent. Both Xmr and BRCA1 have been implicated in a mechanism leading to chromatin condensation and transcription inactivation of the XY bivalent. The BRCA1-ATR kinase complex, as recent research suggests, triggers the phosphorylation of histone H2AX, which predominates in the condensed chromatin of the XY chromosomal pair. Xmr is not present in the XY bivalent when the expression of histone H2AX is deficient. The role of Xmr in chromatin condensation of the XY bivalent has not been determined. The partial structural homology of SCP3 and Xmr, their distribution along the unsynapsed axial cores of the X and Y chromosomes, and the presence of Xmr in the XY pair-associated nucleolus raises the possibility that Xmr, and other proteins including protein kinases, may be recruited to the nucleolus to perform functions related to chromosomal synapsis, chromatin condensation and recombination processes, as well as cell cycle progression.

Primordial germ cells: what does it take to be alive?

Specification of primordial germ cells (PGCs) in the proximal epiblast enables about 45 founder PGCs clustered at the base of the allantoic bud to enter the embryo by active cell movement. Specification of the PGC lineage depends on paracrine signals derived from the somatic cell neighbors in the extraembryonic ectoderm. Secretory bone morphogenetic proteins (BMP) 4, BMP8b, and BMP2 and components of the Smad signaling pathway participate in the specification of PGCs. Cells in the extraembryonic ectoderm induce expression of the gene fragilis in the epiblast in the presence of BMP4, targeting competence of PGCs. The fragilis gene encodes a family of transmembrane proteins presumably involved in homotypic cell adhesion. As PGCs migrate throughout the hindgut, they express nanos3 protein. In the absence of nanos3 gene expression, no germ cells are detected in ovary and testis. During migration and upon arrival at the genital ridges, the population of PGCs is regulated by a balanced proliferation/programmed cell death or apoptosis. Paracrine and autocrine mechanisms, involving transforming growth factor-beta1 and fibroblast growth factors exert stimulatory or inhibitory effects on PGCs proliferation, modulated in part by the membrane-bound form of stem cell factor. Apoptosis requires the participation of the pro-apoptotic family member Bax, whose activity is balanced by the anti-apoptotic family member Bcl21/Bcl-x. In addition, a loss of cell-cell contacts in vitro results in the apoptotic elimination of PGCs. It needs to be determined whether apoptosis is triggered by a failure of PGC to establish and maintain appropriate cell-cell contacts with somatic cells or whether undefined survival factors released by adjacent somatic cells cannot reach physiological levels to satisfy needs of the expanding population of PGCs.

The acroplaxome is the docking site of Golgi-derived myosin Va/Rab27a/b- containing proacrosomal vesicles in wild-type and Hrb mutant mouse spermatids.

Acrosome biogenesis involves the transport and fusion of Golgi-derived proacrosomal vesicles along the acroplaxome, an F-actin/keratin 5-containing cytoskeletal plate anchored to the spermatid nucleus. A significant issue is whether the acroplaxome develops in acrosomeless mutant mice. Male mice with a Hrb null mutation are infertile and both spermatids and sperm are round-headed and lack an acrosome. Hrb, a protein that contains several NPF motifs (Asn-Pro-Phe) and interacts with proteins with Eps15 homology domains, is regarded as critical for the docking and/or fusion of Golgi-derived proacrosomal vesicles. Here we report that the lack of an acrosome in Hrb mutant spermatids does not prevent the development of the acroplaxome. Yet the acroplaxome in the mutant contains F-actin but is deficient in keratin 5. We also show that the actin-based motor protein myosin Va and its receptor, Rab27a/b, known to be involved in vesicle transport, are present in the Golgi and Golgi-derived proacrosomal vesicles in wild-type and Hrb mutant mouse spermatids. In the Hrb mutant, myosin-Va-bound proacrosome vesicles tether to the acroplaxome, where they flatten and form a flat sac, designated pseudoacrosome. As spermiogenesis advances, round-shaped spermatid nuclei of the mutant display several nuclear protrusions, designated nucleopodes. Nucleopodes are consistently found at the acroplaxome- pseudoacrosome site. Our findings support the interpretation that the acroplaxome provides a focal point for myosin-Va/ Rab27a/b-driven proacrosomal vesicles to accumulate, coalesce, and form an acrosome in wild-type spermatids and a pseudoacrosome in Hrb mutant spermatids. We suggest that nucleopodes develop at a site where a keratin 5-deficient acroplaxome may not withstand tension forces operating during spermatid nuclear shaping.

The acrosome-acroplaxome-manchette complex and the shaping of the spermatid head.

A combination of exogenous contractile forces generated by a stack of F-actin-containing hoops embracing the apical region of the elongating spermatid nucleus and an endogenous modulating mechanism dependent on the spermatid-containing acrosome-acroplaxome-manchette complex may play a cooperative role in the shaping of the spermatid head. In addition, the manchette is a key element in the transport of vesicles and macromolecules to the centrosome and developing spermatid tails as well as in nucleocytoplasmic transport. The proposed model of spermatid head shaping is based on: 1) currently known structural and molecular components of the F-actin hoops, the main cytoskeletal element of the Sertoli cell ectoplasmic specializations; 2) the molecular features of acrosome biogenesis; 3) the assembly of a subacrosomal cytoskeletal plate called the acroplaxome; and 4) the spatial relationship of the acrosome-acroplaxome complex with the manchette, a transient microtubular/actin-containing structure. During acrosome biogenesis, the acroplaxome becomes the nucleation site to which Golgi-derived proacrosomal vescicles tether and fuse. The acroplaxome has at least two functions: it anchors the developing acrosome to the elongating spermatid head. It may also provide a mechanical scaffolding plate during the shaping of the spermatid nucleus. The plate is stabilized by a marginal ring with junctional complex characteristics, adjusting to exogenous clutching forces generated by the stack of Sertoli cell F-actin-containing hoops applied to the elongating spermatid head. A tubulobulbar complex, formed by cytoplasmic processes protruding from the elongating spermatid head extending into the adjacent Sertoli cell, is located at the concave side of the spermatid head. The tubulobulbar complex might provide stabilizing conditions, together with the actin-afadin-nectin-2/nectin-3 adhesion unit, to enable sustained and balanced clutching exogenous forces applied during the elongation of the spermatid head.

Caspase activity inhibition delays programmed spermatogenic cell death in vitro.

Programmed cell death or apoptosis was analyzed in rat Sertoli-spermatogonial cell cocultures prepared from 2-9 day old rats using time-lapse video microscopy, a cell viability fluorescence microscopy assay, immunocytochemical markers, and cell-permeable caspase inhibitory peptides with reversible and irreversible effects. We show that apoptosis can initially affect a single member of a spermatogonial cell cohort and that single non-viable spermatogonial cells can remain conjoined to viable spermatogonial cells. The integrity of the cytoskeletal F-actin network and the presence on Bcl-2 immunoreactivity are valuable markers of spermatogonial cell viability. Apoptotic bodies released into the culture medium are generally eliminated after culture medium replenishment; however, spermatogonial apoptotic cell remnants can be taken up by Sertoli cells, which are known to represent a phagocytic somatic population within the seminiferous epithelium. Cell permeable caspase-1 and caspase-4 inhibitory peptides with reversible and irreversible action were supplemented to a serum-free hormone-growth factor-supplemented medium. In the absence of the caspase inhibitory peptide, the viability of spermatogonial cells decreases gradually with time in coculture. However, the addition of caspase inhibitory peptides causes a significant accumulation of spermatogenic cells per unit surface area. Although inhibition of caspases, the executors of spermatogonial cell death, results in a substantial increase of spermatogonial cells in the cocultures, it remains to be determined what the differentiation potential of caspase-inhibited spermatogonial cell cohorts is.

Acroplaxome, an F-actin-keratin-containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head.

Nuclear shaping is a critical event during sperm development as demonstrated by the incidence of male infertility associated with abnormal sperm ad shaping. Herein, we demonstrate that mouse and rat spermatids assemble in the subacrosomal space a cytoskeletal scaffold containing F-actin and Sak57, a keratin ortholog. The cytoskeletal plate, designated acroplaxome, anchors the developing acrosome to the nuclear envelope. The acroplaxome consists of a marginal ring containing keratin 5 10-nm-thick filaments and F-actin. The ring is closely associated with the leading edge of the acrosome and to the nuclear envelope during the elongation of the spermatid head. Anchorage of the acroplaxome to the gradually shaping nucleus is not disrupted by hypotonic treatment and brief Triton X-100 extraction. By examining spermiogenesis in the azh mutant mouse, characterized by abnormal spermatid/sperm head shaping, we have determined that a deformity of the spermatid nucleus is restricted to the acroplaxome region. These findings lead to the suggestion that the acroplaxome nucleates an F-actin-keratin-containing assembly with the purpose of stabilizing and anchoring the developing acrosome during spermatid nuclear elongation. The acroplaxome may also provide a mechanical planar scaffold modulating external clutching forces generated by a stack of Sertoli cell F-actin-containing hoops encircling the elongating spermatid nucleus.

Chronological gene expression of ADAMs during testicular development: prespermatogonia (gonocytes) express fertilin beta (ADAM2).

Immediately after birth, primordial germinal cell-derived prespermatogonia (PSG), located in the center of the testicular cords, migrate between adjacent Sertoli cells to establish contact with the cord basal lamina. PSG migration suggests continued assembly and disassembly of cell-cell contacts by a molecular mechanism that may involve integrins and their ligands, the disintegrin domain of spermatogenic cell-specific plasma membrane proteins called ADAMs. We have analyzed the temporal gene expression of selected ADAMs in intact fetal, early postnatal, and pubertal rat testis and Sertoli-spermatogenic cell cocultures by reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunocytochemistry. We report that several ADAM transcripts are expressed in fetal, neonatal, and prepubertal testes. Cyritestin (ADAM3), ADAM5, ADAM6, and ADAM15 are expressed in day 17 fetal testes. In contrast, no expression of fertilin alpha (ADAM1) and fertilin beta (ADAM 2) was detected in fetal testes. Fertilin beta gene expression starts after postnatal day 2, subsequent to the expression of fertilin alpha, which occurs on postnatal day 1. After postnatal day 2, all the indicated ADAMs, including the fertilin alpha and fertilin beta, continue to be expressed. Transcripts of spermatogenic cell-specific fertilin alpha, fertilin beta, ADAM3, and ADAM5 were detected during the coculture of PSG with Sertoli cells for up to 72 hr after plating. The presence of fertilin beta mRNA and protein in cocultured PSG was visualized by in situ hybridization and immunocytochemistry, respectively. These observations indicate that PSG in coculture with Sertoli cells provide a suitable approach for analyzing cell-cell adhesive responses involving spermatogenic cell-specific ADAMs.

Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes.

It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (gamma-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/ approximately kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.

Ran, a GTP-binding protein involved in nucleocytoplasmic transport and microtubule nucleation, relocates from the manchette to the centrosome region during rat spermiogenesis.

Ran, a Ras-related GTPase, is required for transporting proteins in and out of the nucleus during interphase and for regulating the assembly of microtubules. cDNA cloning shows that rat testis, like mouse testis, expresses both somatic and testis-specific forms of Ran-GTPase. The presence of a homologous testis-specific form of Ran-GTPase in rodents implies that the Ran-GTPase pathway plays a significant role during sperm development. This suggestions is supported by distinct Ran-GTPase immunolocalization sites identified in developing spermatids. Confocal microscopy demonstrates that Ran-GTPase localizes in the nucleus of round spermatids and along the microtubules of the manchette in elongating spermatids. When the manchette disassembles, Ran-GTPase immunoreactivity is visualized in the centrosome region of maturing spermatids. The circumstantial observation that fractionated manchettes, containing copurified centrin-immunoreactive centrosomes, can organize a three-dimensional lattice in the presence of taxol and GTP, points to the role of Ran-GTPase and associated factors in microtubule nucleation as well as the potential nucleating function of spermatid centrosomes undergoing a reduction process. Electron microscopy demonstrates the presence in manchette preparations of spermatid centrosomes, recognized as such by their association with remnants of the implantation fossa, a dense plate observed only at the basal surface of developing spermatid and sperm nuclei. In addition, we have found importin beta1 immunoreactivity in the nucleus of elongating spermatids, a finding that, together with the presence of Ran-GTPase in the nucleus of round spermatids and the manchette, suggest a potential role of Ran-GTPase machinery in nucleocytoplasmic transport. Our expression and localization analysis, correlated with functional observations in other cell systems, suggest that Ran-GTPase may be involved in both nucleocytoplasmic transport and microtubules assembly, two critical events during the development of functional sperm. In addition, the manchette-to-centrosome Ran-GTPase relocation, together with the similar redistribution of various proteins associated to the manchette, suggest the existence of an intramanchette molecular transport mechanism, which may share molecular analogies with intraflagellar transport.