Chimeric Mice with Deletion of Cfl2 that Encodes Muscle-Type Cofilin (MCF or Cofilin-2) Results in Defects of Striated Muscles, Both Skeletal and Cardiac Muscles.

Cofilin, a member of the ADF/cofilin family, is an actin-binding protein which is widely distributed among eukaryotic organisms and involved in actin filament dynamics in a variety of cell types. In mammalian striated muscles, muscle-type cofilin (MCF or cofilin-2) is predominantly expressed. Previous investigations have shown that MCF plays an essential role in the regulation of assembly of contractile apparatus in skeletal muscle, but its role in cardiac muscle has remained unclear. In the present study, in order to further clarify the role of MCF in organization of myofibrillar structure in vivo, we generated chimeric mice with a combination of MCF-deficient cells that were generated by Cfl2-knockout (Cfl2-/-) and wild type cells containing MCF, and examined the effect of MCF deficiency on striated muscles, especially on the fine structures of contractile apparatus in cardiac muscle. We found that mice chimeric for MCF deficient cells exhibited structural defects in their skeletal muscles as previously reported. Histological analysis showed that MCF deficiency leads to degradation of myofibers and promotion of muscle regeneration. Electron microscopic observation of cardiac muscle of the chimeric mice showed coexistence of the cells with normal sarcomeres and those with disorganized myofibrils in a chimeric pattern. In these cofilin-deficient cells, myofilaments were scattered in the cytoplasm and myofibrillar structures were severely disrupted. These results provide strong evidence for that MCF plays a critical role in the formation and the maintenance of myofibril structure not only in skeletal muscle but also in cardiac muscle.

Adhesion between plasma membrane and mitochondria with linking filaments in relation to migration of cytoplasmic droplet during epididymal maturation in guinea pig spermatozoa.

High-resolution microscopy has been used to investigate the mechanism of the migration of cytoplasmic droplets during epididymal maturation of guinea pig spermatozoa. On testicular spermatozoa, droplets are located at the neck and, after passage through the middle cauda epididymidis, migrate only as far as the center of the midpiece. Initially, the space between the plasma membrane and outer mitochondrial membranes outside the droplet is 30.8+/-11.0 nm, whereas on mature spermatozoa, it significantly (P<0.01) narrows to a more consistent 15.9+/-1.3 nm. This is accompanied by the appearance of thin filaments cross-linking the two membranes above and below the droplet. Changes also occur in the arrangement of intramembranous particles (IMPs) in the plasma membrane overlying the midpiece. At the spermatid stage, linear arrays of IMPs are absent but appear on immature spermatozoa, where they are short with an irregular orientation, in the epididymis. On mature spermatozoa, numerous parallel linear arrays are present at the region where the plasma membrane adheres to the mitochondria. The membrane adhesion process can thus be observed two-dimensionally. The initial migration of the droplet from the neck is probably attributable to diffusion, with the formation of cross-linking filaments between the two membranes in the proximal midpiece preventing any backward flow and squeezing the droplet distally until it is arrested at the central midpiece by the filaments formed in the distal midpiece. The filaments might also stabilize the flagellum against hypo-osmotic stress encountered during ejaculation and within the female tract.

A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin.

It is important to establish a reliable and progressive model of the acrosome reaction. Here, we present a progression model of the acrosome reaction centering around the acrosomal membrane-anchored protein equatorin (MN9), comparing the staining pattern traced by MN9 antibody immunofluorescence with that traced by Arachis hypogaea agglutinin (PNA)-FITC. Prior to the acrosome reaction, equatorin was present in both the anterior acrosome and the equatorial segment. Since sperm on zona pellucida showed various staining patterns, MN9-immunostaining patterns were classified into four stages: initial, early, advanced, and final. As the acrosome reaction progressed from the initial to the early stage, equatorin spread from the peripheral region of the anterior acrosome toward the center of the equatorial segment, gradually over the entire region of the equatorial segment during the advanced stage, and finally uniformly at the equatorial segment at the final stage. In contrast, the PNA-FITC signals spread more quickly from the peripheral region of the acrosome toward the entire equatorial segment, while decreasing in staining intensity, and finally became weak at the final stage. MN9-immunogold electron microscopy showed equatorin on the hybrid vesicles surrounded by amorphous substances at advanced stage of acrosome reaction. Equatorin decreased in molecular mass from 40-60 to 35 kDa, and the signal intensity of 35 kDa equatorin increased as the acrosome reaction progressed. Thus, the established equatorin-based progression model will be useful for analyzing not only the behavior of equatorin but also of other molecules of interest involved in the acrosome reaction.

Oocyte activation ability correlates with head flatness and presence of perinuclear theca substance in human and mouse sperm.

BACKGROUND: Recent studies indicate that round-headed sperm cannot activate oocytes and lack the postacrosomal sheath (PAS) or perinuclear theca (PT), although normal flat-headed sperm can activate oocytes and do have PAS (PT). In this study, we investigated how oocyte activation ability correlates with sperm head morphology (round and flat) and the presence of PT, by studying MN13, a representative molecule of the PT. METHODS: We analyzed sperm with flat and round heads from infertile patients with globozoospermia (n = 1) and teratozoospermia (n = 1), and also from GOPC(-/-) mice, an animal model of human globozoospermia. Differential interference contrast image analysis, immunocytochemistry with MN13 antibody, transmission electron microscopy and an oocyte activation assay (assessing pronucleus formation) with ICSI were used. RESULTS: Flat-headed (control) sperm from both a healthy fertile volunteer man and wild-type mice had MN13 and PAS (PT). Flat-headed sperm (<5% of the population) from GOPC(-/-) mice also had both MN13 and PAS (PT), and they showed high oocyte activation ability. In contrast, round-headed sperm from a globozoospermia patient (100%) and GOPC(-/-) mice (>95% of the population) had neither MN13, nor PAS (PT), nor oocyte activation ability. Oocyte activation was higher in flat- versus round-headed sperm from GOPC(-/-) mice (P < 0.05). CONCLUSIONS: Oocyte activation ability may be related to sperm head flatness and presence of MN13 and PAS (PT) in human and mouse sperm. This information is a first step towards the possibility of selecting good-quality sperm with high oocyte activation ability for ICSI.

Suppression of cardiac troponin T induces reduction of contractility and structural disorganization in chicken cardiomyocytes.

We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnT-siRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5-2 nmol CTnT-siRNA towards 1-3 x 10(6) cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnT-suppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.

The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins.

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

Disruption of ectoplasmic specializations between Sertoli cells and maturing spermatids by anti-nectin-2 and anti-nectin-3 antibodies.

AIM: To understand the biological functions of the ectoplasmic specializations between Sertoli cells and maturing spermatids in seminiferous epithelia. METHODS: In order to disrupt the function of the ectoplasmic specializations, nectin-2, which is expressed at the specialization, was neutralized with anti-nectin-2 antibody micro-injected into the lumen of the mouse seminiferous tubule. Anti-nectin-3 antibody was also micro-injected into the lumen in order to neutralize nectin-3, which is expressed at the specialization. RESULTS: The actin filaments at the specialization disappeared, and exfoliation of maturing spermatids was observed by electron microscopy. CONCLUSION: Nectin-2 was neutralized by anti-nectin-2 antibody and nectin-3 was neutralized by anti-nectin-3 antibody, respectively. Inactivated nectin-2 and nectin-3 disrupted the nectin-afadin-actin system, and finally the actin filaments disappeared. As a result, the specialization lost the holding function and detachment of spermatids was observed. One of the functions of the specialization seems to be to hold maturing spermatids until spermiation.

Factors maintaining normal sperm tail structure during epididymal maturation studied in Gopc-/- mice.

Gopc (Golgi-associated PDZ- and coiled-coil motif-containing protein)(-/-) mice are infertile, showing globozoospermia, coiled tails, and a stratified mitochondrial sheath. Transmission electron microscope (TEM) images of the spermatozoa were studied quantitatively to analyze disorganization processes during epididymal passage. Factors maintaining straight tail and normal mitochondrial sheath were also studied by TEM and immunofluorescent microscopy. Sperm tails retained a normal appearance in the proximal caput epididymidis. Tail disorganization started between the proximal and the middle caput epididymidis, and the latter is the major site for it. The tail moved up through the defective posterior ring and coiled around the nucleus to various degrees. Tail coiling occurred in the caput epididymidis suggesting it was triggered by cytoplasmic droplet migration. SPATA19/spergen-1, a candidate mitochondrial adhesion protein, remained on the stratified mitochondria, while GPX4/PHGPx, a major element of the mitochondrial capsule, was unevenly distributed on them. From these findings, we speculate GPX4 is necessary to maintain normal sheath structure, and SPATA19 prevents dispersal of mitochondria, resulting in a stratified mitochondrial sheath formation in Gopc(-/-) spermatozoa. The epididymal epithelium was normal in structure and LRP8/apoER2 expression suggesting that tail abnormality is due to intrinsic sperm factors. Three cell structures are discussed as requisite factors for maintaining a straight tail during epididymal maturation: 1) a complete posterior ring to prevent invasion of the tail into the head compartment, 2) stable attachment of the connecting piece to the implantation fossa, and 3) a normal mitochondrial sheath supported by SPATA19 and supplied with sufficient and normally distributed GPX4.

The involvement of immunoglobulin superfamily proteins in spermatogenesis and sperm-egg interaction.

The immunoglobulin superfamily (IgSF) proteins are expressed on the plasma membrane between Sertoli cells and germ cells in the testis. IgSF proteins are specifically present at the apical Sertoli-germ cell junction, that is, ectoplasmic specialization and are involved in germ cell differentiation. Some IgSF proteins are present on the surface of germ cells and undergo further biochemical modifications during sperm maturation. These IgSF proteins undergo final modifications during capacitation and/or the acrosome reaction. The function and expression of IgSF proteins in the testis and spermatozoa, as they relate to spermatogenesis and sperm-egg interaction, are discussed. (Reprod Med Biol 2006; 5: 87-93).

Impairment of spermatogenesis leading to infertility.

Highly differentiated spermatozoa are generated through multiple cellular and molecular processes maintained by Sertoli cells. The cellular events associated with germ cells include proliferation, protein folding and transportation, as well as sequential changes in chromatin and cell organelles. These processes are strictly controlled by the expression of specific genes, including transcription and DNA replication/repair. This complex spermatogenesis is impaired by a mutation such as gene knockout, which leads to a variety of morphological and functional abnormalities found in mature spermatozoa. An overview of spermatogenesis impairment induced by gene knockout is provided in the present review.

Stage-specific expression of mouse germ cell-less-1 (mGCL-1), and multiple deformations during mgcl-1 deficient spermatogenesis leading to reduced fertility.

A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1), is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-1 in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum et al., 2003), postacrosomal sheath, and posterior ring apparently formed. In addition, MN7 and MN13, essential substances for fertilization that are located in sperm heads, were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility.

Failure to assemble the peri-nuclear structures in GOPC deficient spermatids as found in round-headed spermatozoa.

Deletion of the GOPC gene encoding mouse GOPC (Golgi-associated PDZ- and coiled-coil motif-containing protein) causes infertile round-headed spermatozoa, which have acrosome-less round heads and deformed tails (Yao et al, 2002). This study investigated how GOPC deficient spermatids fail to assemble the peri-nuclear structures in round-headed spermatozoa during spermiogenesis in GOPC knockout mouse testes. In step 1-8 spermatids, Golgi-derived proacrosomal vesicles that are transported to the perinuclear region formed acrosome-like vesicles of various sizes, called pseudoacrosomes. The marginal ring of the acroplaxome, which is generally formed between the descending edge of a developing acrosome and nuclear envelope in a wild spermatid, was poorly formed between the pseudoacrosome and nuclear envelope. In step 9-11 elongating spermatids, a majority of pseudoacrosomes were detached from the nucleus and disappeared from the perinuclear region by spermiation. Concomitantly, several failures occurred on the nucleus, manchette, postacrosomal sheath (perinuclear theca), and posterior ring. Ectoplasmic specializations were poorly formed, and did not always associate with developing spermatids. Consequently, spermatid nuclear elongation to form round-headed spermatozoa developed was impaired. In addition to these sequential failures, the posterior ring deficiency was attributed to the tail deformation destined to occur during epididymal maturation as reported in an accompanying paper (Suzuki-Toyota et al, 2004 in this issue), its eventual phenotype being reminiscent of the round-headed spermatozoa of human infertile globozoospermia.

The coiled tail of the round-headed spermatozoa appears during epididymal passage in GOPC-deficient mice.

Male mice deleting the gene encoding GOPC (Golgi-associated PDZ- and coiled-coil motif-containing protein) are infertile, showing globozoospermia with a coiled tail (Yao et al., 2002). We confirmed how and where tail anomalies were produced in spermatids and epididymal spermatozoa by light and electron microscopy. During spermiogenesis, tail formation occurred normally, but a defect was found at the posterior ring. Thereafter, remarkable sperm tail deformations were induced during epididymal passage. In the proximal caput epidiymidis, the tails remained normal and straight, but most of them coiled around the nucleus in the cauda epididymidis. Coiling is presumed to occur with the migration of the cytoplasmic droplet by the absence of the posterior ring. The connecting piece of the coiled tail was often dislocated or separated from the implantation fossa. Many mitochondria were separated from the outer dense fibers (ODFs) and formed a stratified mitochondrial sheath. Due to this, the distal part of the midpiece became bared of the mitochondrial sheath. The bared ODFs were often bent and disorganized. Tail deformities are attributed to weak or incomplete adhesion between the following structures: 1) plasma membrane and nuclear envelope at the posterior ring, 2) connecting piece and implantation fossa, and 3) mitochondria and ODFs. These defects result in a coiled tail, tail dislocation from the implantation fossa, and the stratified mitochondrial sheath accompanying bared ODFs in the midpiece, respectively. Thus the posterior ring is significant in preventing coiled tail formation. The GOPC-deficient spermatozoa provide a valuable model not only for head but also for tail anomalies.

Adverse effects of bisphenol A to spermiogenesis in mice and rats.

Either a 20 or 200 microg/kg body weight/injection of bisphenol A (BPA) was subcutaneously administered to adult mice and rats for 6 days, and the effects on the testes were investigated by electron and light microscopy. Abnormalities were observed in the spermatids: acrosomal vesicles, acrosomal caps, acrosomes and nuclei of the spermatids were severely deformed. The ectoplasmic specialization between the Sertoli cell and spermatids were also affected: incomplete specialization, redundant ectopic specialization and aplasia were observed. Rats and mice responded similarly to BPA. There were no dose dependencies between the 20- and 200 microg/kg body weight/injection groups. The ectoplasmic specialization between adjoining Sertoli cells, or blood-testis barrier, was not affected. Since similar adverse effects were observed when adult mice were treated with beta-estradiol 3-benzoate, the effects of BPA reported here seem to reflect the estrogenic effects on the testes. Animals kept for an additional two months after cessation of the administration were shown to be fertile and the testes showed normal histology, indicating that the adverse effects were transitory.

Pathological effects on mice by gambierol, possibly one of the ciguatera toxins.

Gambierol was isolated from Gambierdiscus toxicus, which causes ciguatera fish poisoning. The acute toxicological effects induced in mice by synthesized gambierol were studied. The lethal doses were about 80 microg/kg by i.p. and i.v., and 150 microg/kg by p.o. The main injury by this toxin was observed in the lung, and secondary in the heart, resulting in systemic congestion. Another toxic effect was seen in the stomach, inducing hypersecretion and ulceration. With survival from the severe stage during the initial 3 h, recovery was favorable, especially after 4 days. Additional effects were not evident during 1-week post-administration observation.