Mamiko Maekawa

Mice Deficient in the Axonemal Protein Tektin-t Exhibit Male Infertility and Immotile-Cilium Syndrome Due to Impaired Inner Arm Dynein Function

ABSTRACT The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

HANP1/H1T2, a Novel Histone H1-Like Protein Involved in Nuclear Formation and Sperm Fertility

ABSTRACT We cloned a testis-specific cDNA from mice that encodes a histone H1-like, haploid germ cell-specific nuclear protein designated HANP1/H1T2. The HANP1/H1T2 protein was specifically localized to the nuclei of murine spermatids during differentiation steps 5 to 13 but not to the nuclei of mature sperm. HANP1/H1T2 contains an arginine-serine-rich domain and an ATP/GTP binding site, and it binds to DNA, ATP, and protamine. To investigate the physiological role of HANP1/H1T2, we generated Hanp1/H1T2-disrupted mutant mice. Homozygous Hanp1/H1T2 mutant males were infertile, but females were fertile. Although a substantial number of sperm were recovered from the epididymides, their shape and function were abnormal. During sperm morphogenesis, the formation of nuclei was disturbed and protamine-1 and -2 were only weakly detectable in the nuclei. The chromatin packaging was aberrant, as demonstrated by electron microscopy and biochemical analysis. The mutant sperm exhibited deficient motility and were not competent to fertilize eggs under in vitro fertilization conditions; however, they were capable of fertilizing eggs via intracytoplasmic sperm injection that resulted in the birth of healthy progeny. Thus, we found that HANP1/H1T2 is essential for nuclear formation in functional spermatozoa and is specifically involved in the replacement of histones with protamines during spermiogenesis. At the time of submission of the manuscript, we found an independent publication by Martianov et al. (I. Martianov, S. Brancorsini, R. Catena, A. Gansmuller, N. Kotaja, M. Parvinen, P. Sassone-Corsi, and I. Davidson, Proc. Natl. Acad. Sci. USA 102:2808-2813, 2005) that reported similar results.

Oligo-astheno-teratozoospermia in mice lacking RA175/TSLC1/SynCAM/IGSF4A, a cell adhesion molecule in the immunoglobulin superfamily.

ABSTRACT RA175/TSLC1/SynCAM/IGSF4A (RA175), a member of the immunoglobulin superfamily with Ca2+-independent homophilic trans-cell adhesion activity, participates in synaptic and epithelial cell junctions. To clarify the biological function of RA175, we disrupted the mouse Igsf4a (Ra175/Tslc1/SynCam/Igsf4a Ra175) gene. Male mice lacking both alleles of Ra175 (Ra175− / −) were infertile and showed oligo-astheno-teratozoospermia; almost no mature motile spermatozoa were found in the epididymis. Heterozygous males and females and homozygous null females were fertile and had no overt developmental defects. RA175 was mainly expressed on the cell junction of spermatocytes, elongating and elongated spermatids (steps 9 to 15) in wild-type testes; the RA175 expression was restricted to the distal site (tail side) but not to the proximal site (head side) in elongated spermatids. In Ra175 − / − testes, elongated and mature spermatids (steps 13 to 16) were almost undetectable; round spermatids were morphologically normal, but elongating spermatids (steps 9 to 12) failed to mature further and to translocate to the adluminal surface. The remaining elongating spermatids at improper positions were finally phagocytosed by Sertoli cells. Furthermore, undifferentiated and abnormal spermatids exfoliated into the tubular lumen from adluminal surfaces. Thus, RA175-based cell junction is necessary for retaining elongating spermatids in the invagination of Sertoli cells for their maturation and translocation to the adluminal surface for timely release.

Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor α in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor α and cyclin D1.

Fyn Tyrosine Kinase in Sertoli Cells Is Involved in Mouse Spermatogenesis

Abstract Fyn is a member of the Src family of non-receptor-type tyrosine kinases and plays an important role in signal transductions regulating cell proliferation and differentiation. Fyn immunoreactivity was localized in the Sertoli cells of mouse testes. Although fyn-deficient adult male mice were fertile, a significant reduction in testis weight and degenerated germ cells were observed at 3 and 4 wk of age. Electron microscopic examination revealed that fyn −/− testis has ultrastructural abnormalities in the specialized junctional structures of the Sertoli cells, the ectoplasmic specializations. Unusual vesicular structures were found in the actin filament layers of the ectoplasmic specializations of mutant mice. Immunohistochemical studies demonstrated that both Fyn and actin filaments were concentrated in the areas of ectoplasmic specializations. At these sites, a high level of phosphotyrosine was also immunostained in wild-type testes, whereas phosphotyrosine immunoreactivity was reduced in fyn −/− testes. Immunoblot analyses revealed that Fyn was mainly distributed within the Triton X-100-insoluble cytoskeletal fraction prepared from wild-type testes, suggesting that Fyn might be associated with cytoskeletal proteins such as actin filaments. These findings suggest that Fyn kinase functions at the ectoplasmic specializations of the Sertoli cells in the testes, regulating the dynamics of cytoskeletal proteins. Fyn-mediated signal transduction in the Sertoli cells may affect the survival and differentiation of germ cells at a specific stage during spermatogenesis.

Histological characterization of defective spermatogenesis in mice lacking the basigin gene.

Basigin is a transmembrane protein belonging to the immunoglobulin superfamily. In the light of the fact that knockout mice lacking the basigin gene (Bsg) are azoospermic, the phenotype in the male reproductive system was extensively examined in this study. Spermatogenesis in Bsg (−/−) mice was found to be disrupted, and arrested at the metaphase of the first meiotic division. A few germ cells differentiated into young spermatids, but they were exfoliated. The lumens of the male reproductive system were filled with round degenerated cells. Using the TUNEL method and electron microscopy, some of the degenerated cells in the testis and epididymal head were shown to be apoptotic. Crystalloids of fine tubules and unusual ectoplasmic specializations were also observed in the Sertoli cells of Bsg (−/−) mice. These specializations displayed unusual ‘circular’ structures. Furthermore, unusual ectoplasmic specializations covering the spermatocytes rather than the mature spermatids were found. These structures were formed as a result of the lack of mature spermatids in the Bsg (−/−) testis. Results from analyses of azoospermia in the Bsg (−/−) mice suggest that basigin, through the interactions between germ cells and Sertoli cells, is an essential factor in the growth and/or survival of spermatids.

Factors Maintaining Normal Sperm Tail Structure During Epididymal Maturation Studied in Gopc−/− Mice

Abstract 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.

Fyn Is Required for Haloperidol-induced Catalepsy in Mice

Fyn-mediated tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D2 receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D2 receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D2 receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.

Development of the blood–testis barrier in the mouse is delayed by neonatally administered diethylstilbestrol but not by β-estradiol 3-benzoate

Summary.  A group of newborn mice were treated with 1 µg dose−1 individual−1 of diethylstilbestrol (DES) on alternate days, from days 1 to 11 postnatally. Another group of mice were treated similarly with 125 ng dose−1 individual−1 of β‐estradiol 3‐benzoate (E2B). The testes were sequentially examined up to 84 days of age using light and electron microscopy. Spermatogenic cells in the DES‐treated mice differentiated normally from birth until 17 days of age, when they differentiated into pachytene spermatocytes and remained at this meiotic prophase for the next 10 days approximately. The cells then began to differentiate further, ultimately forming spermatozoa by 49 days of age. Confocal and electron microscopy showed that the blood–testis barrier did not form until 28 days of age in the DES‐treated mice, and a delay in the functional maturation of this structure, as the blood–testis barrier, was confirmed by intercellular tracer experiments. The arrest of spermatogenesis at the meiotic prophase may have been attributable to the DES‐induced defective formation of the blood–testis barrier. No delay of the blood–testis barrier formation was detected in the E2B‐treated mice. Thus, DES and E2B, both of which are known as potent oestrogenic compounds, had different effects on the Sertoli cells.

Equatorin: Identification and Characterization of the Epitope of the MN9 Antibody in the Mouse

Equatorin (MN9 antigenic molecule) is a widely distributed acrosomal protein in mammalian sperm. During the acrosome reaction, some amount of equatorin translocates to the plasma membrane, covering the equatorial region. From the results of studies of both in vitro and in vivo fertilization inhibition using the MN9 antibody, equatorin has been suggested to be involved in fusion with the oolemma. In the present study, we cloned equatorin and, using mass spectrometry and carbohydrate staining, found it to be a highly glycosylated protein. Equatorin is a sperm-specific type 1 transmembrane protein, and glycosidase treatment and recombinant protein assays verified that it is an N,O-sialoglycoprotein. In addition, the gamete interaction-related domain recognized by the MN9 antibody is posttranslationally modified. The modified domain was identified near threonine 138, which was most likely to be O-glycosylated when analyzed by amino acid substitution, dephosphorylation, and O-glycosylation inhibitor assays. Immunogold electron microscopy localized the equatorin N-terminus, where the MN9 epitope is present, on the acrosomal membrane facing the acrosomal lumen. These biochemical properties and the localization of equatorin are important for further analysis of the translocation mechanism leading to gamete interaction.