ﾗｯﾄ後期精子細胞の tubulobulbar complex におけるﾎﾟﾘﾕﾋﾞｷﾁﾝ化ｼｸﾞﾅﾙの免疫電顕的局在

後期精子細胞の頭部には､核から隣接するｾﾙﾄﾘ細胞の中に伸びる tubulobulbar complex(小管小胞複合体)と名付けられた複雑な構造が知られている｡この構造ははじめ精子細胞頭部をｾﾙﾄﾘ細胞に繋留する装置と考えられたが､後に酸ﾎｽﾌｧﾀｰｾﾞの存在から､核で不要になった物質を分解する装置と提案された｡今回､われわれはこの装置にﾎﾟﾘﾕﾋﾞｷﾁﾝ化ﾀﾝﾊﾟｸ質のｼｸﾞﾅﾙが存在することを免疫電顕法によりはじめて見出した｡また､装置を取り巻くｾﾙﾄﾘ細胞にﾌﾟﾛﾃｱｿｰﾑが局在することも明らかになった｡これらの事実は､後期精子細胞核の不要ﾀﾝﾊﾟｸ質がﾎﾟﾘﾕﾋﾞｷﾁﾝ化されて､ｾﾙﾄﾘ細胞に送られ､そこでﾌﾟﾛﾃｱｿｰﾑで分解されることを示唆する｡To approach to the involvement of the tubulobulbar complex in ubiquitin-proteasome degradation of unnecessary nuclear proteins, the localization of polyubiquitin and the proteasome was studied by immunofluorescence and immunoelectron microscopy. Polyubiquitin signals were localized to this device and the proteasome α subunit was stained in the cytoplasm of Sertoli cells surrounding the device. The results suggest that the tubulobulbar complex participates in ubiquin-proteasome degradation of unnecessary proteins during the rapid nuclear compaction of late spermatids

Immunological detection of alkaline-diaminobenzidine-negative peroxisomes of the nematode Caenorhabditis elegans: Purification and unique pH optima of peroxisomal catalase

We purified catalase-2 of the nematode Caenorhabditis elegans and identified peroxisomes in this organism. The peroxisomes of C. elegans were not detectable by cytochemical staining using 3,3'-diaminobenzidine, a commonly used method depending on the peroxidase activity of peroxisomal catalase at pH 9 in which genuine peroxidases are inactive. The cDNA sequences of C. elegans predict two catalases very similar to each other throughout the molecule, except for the short C-terminal sequence; catalase-2 (500 residues long) carries a peroxisomal targeting signal 1-like sequence (Ser-His-Ile), whereas catalase-1 does not. The catalase purified to near homogeneity from the homogenate of C. elegans cells consisted of a subunit of 57 kDa and was specifically recognized by anti-(catalase-2) serum but not by anti-(catalase-1) serum. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected catalase-2 inside vesicles judged to be peroxisomes using morphological criteria. The purified enzyme (220 kDa) was tetrameric, similar to many catalases from various sources, but exhibited unique pH optima for catalase (pH 6) and peroxidase (pH 4) activities; the latter value is unusually low and explains why the peroxidase activity was undetectable using the standard alkaline diaminobenzidine- staining method. These results indicate that catalase-2 is peroxisomal and verify that it can be used as a marker enzyme for C. elegans peroxisomes

Type-II 3-oxoacyl-CoA thiolase of the nematode Caenorhabditis elegans is located in peroxisomes, highly expressed during larval stages and induced by clofibrate

We examined the expression and localization of type-II 3-oxoacyl-CoA thiolase in the nematode Caenorhabditis elegans. Type-II thiolase acts on 3- oxoacyl-CoA esters with a methyl group at the α carbon, whereas conventional thiolases do not. Mammalian type-II thiolase, which is also termed sterol carrier protein x (SCPx) or SCP2/3-oxoacyl-CoA thiolase, is located in the peroxisomes and involved in phytanic acid degradation and most probably in bile acid synthesis. The nematode enzyme lacks the SCP2 domain, which carries the peroxisomal-targeting signal, but produces bile acids in a cell-free system. Northern and Western blot analyses demonstrated that C. elegans expressed type-II thiolase throughout its life cycle, especially during the larval stages, and that the expression was significantly enhanced by the addition of clofibrate at 5 mM or more to the culture medium. Whole-mount in situ hybridization and immunostaining of L4 larvae revealed that the enzyme was mainly expressed in intestinal cells, which are multifunctional like many of the cell types in C. elegans. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected the enzyme in the matrix of peroxisomes. These results indicate the fundamental homology between mammalian SCPx and the nematode enzyme regardless of whether the SCP2 part is fused, suggesting their common physiological roles

Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells

Localization of the dynamin-related GTPase Drp1 to mitochondria relies on the mitochondrial fission factor Mff

OAZ-t/OAZ3 Is Essential for Rigid Connection of Sperm Tails to Heads in Mouse

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. Although considerable amounts of polyamines are synthesized and stored in the testes, their roles remain unknown. Ornithine decarboxylase antizymes (OAZs) control the intracellular concentration of polyamines in a feedback manner. OAZ1 and OAZ2 are expressed ubiquitously, whereas OAZ-t/OAZ3 is expressed specifically in germline cells during spermiogenesis. OAZ-t reportedly binds to ornithine decarboxylase (ODC) and inactivates ODC activity. In a prior study, polyamines were capable of inducing a frameshift at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t. To investigate the physiological role of OAZ-t, we generated OAZ-t–disrupted mutant mice. Homozygous OAZ-t mutant males were infertile, although the polyamine concentrations of epididymides and testes were normal in these mice, and females were fertile. Sperm were successfully recovered from the epididymides of the mutant mice, but the heads and tails of the sperm cells were easily separated in culture medium during incubation. Results indicated that OAZ-t is essential for the formation of a rigid junction between the head and tail during spermatogenesis. The detached tails and heads were alive, and most of the headless tails showed straight forward movement. Although the tailless sperm failed to acrosome-react, the heads were capable of fertilizing eggs via intracytoplasmic sperm injection. OAZ-t likely plays a key role in haploid germ cell differentiation via the local concentration of polyamines