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.

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.

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.

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.

Rat hd mutation reveals an essential role of centrobin in spermatid head shaping and assembly of the head-tail coupling apparatus.

The hypodactylous (hd) locus impairs limb development and spermatogenesis, leading to male infertility in rats. We show that the hd mutation is caused by an insertion of an endogenous retrovirus into intron 10 of the Cntrob gene. The retroviral insertion in hd mutant rats disrupts the normal splicing of Cntrob transcripts and results in the expression of a truncated protein.

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.

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.

Testis-expressed profilins 3 and 4 show distinct functional characteristics and localize in the acroplaxome-manchette complex in spermatids.

Background: Multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. The testis-specific isoforms profilin-3 (PFN3) and profilin-4 (PFN4) may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the "somatic" profilins, profilin-1 (PFN1) and profilin-2 (PFN2).

Results: Ligand interactions and spatial distributions of PFN3 and PFN4 were compared by biochemical, molecular and immunological methods; PFN1 and PFN2 were employed as controls.

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.

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.

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.

Cell-cycle regulation and mammalian gametogenesis: a lesson from the unexpected.

The progression of mammalian gametogenesis requires a precise balance between cell-cycle activities and elimination of defective gametogenic cells to ensure the perpetuation of species. Both spermatogonia and oogonia are stem cell populations committed to meiosis with the aim of generating haploid gametes for fertilization. At puberty, mitotically dividing spermatogonial cell cohorts maintain the ability of cell renewal and occupy niches in the seminiferous tubule.

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.

Tyrosine protein kinases and spermatogenesis: truncation matters.

Protein phosphorylation on serine/threonine or tyrosine residues represents a significant regulatory mechanism in signal transduction during spermatogenesis, oogenesis, and fertilization. There are several families of tyrosine protein kinases operating during spermatogenesis: the Src family of tyrosine protein kinases; the Fujinami poultry sarcoma/feline sarcoma (Fps/Fes) and Fes-related protein (Fer) subfamily of non-receptor proteins; and c-kit, the transmembrane tyrosine kinase receptor that belongs to the family of the PDGF receptor. A remarkable characteristic is the coexistence of full-length and truncated tyrosine kinases in testis.

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.

Sperm tail abnormalities in mutant mice with neo(r) gene insertion into an intron of the keratin 9 gene.

Keratin 9 (K9) is one of the components of the perinuclear ring of the manchette found in developing spermatids but is predominantly expressed in the epidermis of the footpad (palm and sole in human epidermis). As an initial step to determine the function of K9 protein in sperm development, we have generated a mutant mouse by homologous recombination of the targeting vector containing the disrupted K9 gene in which the neo(r) gene was inserted into the intron 6. This insertion resulted in the expression of two K9 mRNAs: a wild-type K9 mRNA, in which intron 6 with the neo(r) gene was completely spliced out, and a mutated form in which only a portion of the intron 6 between neo(r) gene and exon 7 was spliced out.

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.

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.

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.

Polycystins: what polycystic kidney disease tells us about sperm.

Experimental evidence indicates that the membrane-associated proteins polycystin-1 and polycystin-2 operate as a receptor-calcium channel complex that regulates signaling pathways essential for modulation of renal tubulogenesis. Polycystic kidney disease is characterized by defective renal tubular structure and results from mutations in either PKD1 or PKD2 genes. Recent data suggest that polycystin-1 and polycystin-2 might localize to primary cilium in principal cells of renal collecting tubules and are thought to act as mechanosensors of fluid flow and contents.

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.

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.

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.

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.

Epididymal G protein-coupled receptor (GPCR): two hats and a two-piece suit tailored at the GPS motif.

G protein-coupled receptors (GPCRs) are involved in cell recognition and signaling and their function has been experimentally determined by ligand activation and site-directed mutagenesis. Structurally, GPCRs consist of an extracellular N-terminus and an intracellular C-terminus separated by seven helical transmembrane domains (TM7). The extracellular region is highly glycosylated.