Hideki Ohtake

Purification and the primary structure of sperm-activating peptides from the jelly coat of sea urchin eggs

Abstract Two different peptides, which stimulate the respiration of spermatozoa, were purified from the jelly coat of sea urchin, Hemicentrotus pulcherrimus eggs. Analysis of the sequence by Edman degradation indicated that those sequences are Gly-Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val and Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly.

Circadian rhythms of vasopressin release in primary cultures of rat suprachiasmatic nucleus

We have developed a suprachiasmatic nucleus (SCN) cell culture system to study the cellular and molecular bases of the mammalian circadian pacemaker. The SCN regions were dissected from 4- to 6-day-old rat pups and dissociated cells were cultured in a defined medium. In all the cultures, the release of vasopressin showed clear circadian oscillation, which appeared within a few days in culture and lasted for more than a month. The peak of vasopressin release was observed at subjective day. These findings suggest that this culture system provides a valuable model for elucidating the mechanism of the circadian pacemakers.

Characterization of a cAMP-dependent protein kinase catalytic subunit from rainbow trout spermatozoa.

The cyclic AMP-dependent phosphorylation of proteins is essential for the initiation of sperm motility in salmonid fishes. This study isolated cDNA for the catalytic subunit of a cAMP-dependent protein kinase (PKA-C) from rainbow trout testis. The deduced amino acid sequence shows 75-80% identity to sequences previously reported in other organisms. However, the N-terminal regions of PKA-C from the testis as well as ovary in the trout appear slightly shorter than those from other tissues, suggesting that small PKA-C might be specific to germ cells. An immunofluorescence study using polyclonal antibody against trout testis PKA-C shows that it localizes along sperm flagellum. Furthermore, immunoelectron microscopy revealed that PKA-C is anchored to the outer arm dynein of flagellar axonemes. These results suggest that PKA-C is involved in regulating the flagellar motility of sperm via phosphorylation of a subunit of the outer arm dynein.

Type I and type III procollagen gene expressions in the early phase of ligament healing in rabbits: an in situ hybridization study.

The purpose of this study is to observe type I and type III procollagen gene expressions in the healing ligament using in situ hybridization histochemistry. The rabbit medial collateral ligaments were incised and harvested at 3, 7, 14, and 28 days postoperatively. The healing ligament showed increased expression of both procollagen genes through this period compared with the unoperated ligament. The peak expression level was observed at 7 or 14 days for type I and at 7 days for type III, respectively. The strongest expression of both genes was detected in the scar tissue created between the ends of the old ligament. Although type III procollagen gene expression was observed almost only in the newly created scar tissue, type I procollagen gene was expressed not only in the scar tissue, but also at the ends of the previously normal ligament. These results suggest that type I collagen synthesis begins shortly after ligament injury and occurs at the ends of the injured ligament as well as in the scar tissue, and that type III collagen is largely synthesized in the scar tissue around one week after injury but continues being synthesized for at least four weeks after injury.

Heterogenous expression of ornithine decarboxylase gene in the proximal tubule of the mouse kidney following testosterone treatment

The expression of the ornithine decarboxylase (ODC) gene in the mouse kidney following testosterone treatment was examined using in situ hybridization histochemistry. Testosterone (n=5) or vehicle (n=5) was subcutaneously injected (1 mg/animal) into male BALB/c mice (8 weeks in age) 14 h before sacrifice. Animals were sacrificed under ether anesthesia, their kidneys were removed and immediately frozen in liquid nitrogen. Frozen sections (10-μm-thick) were cut on a cryostat. Sections were hybridized with 35S-labeled sense or antisense RNA probe. The hybridization continued for 24 h at 50° C and emulsion autoradiography was subsequently performed. A marked increase in ODC mRNA was exclusively detected in the proximal tubule of the renal cortex in the testosterone-treated animals. The hybridization signal was greater in the outer portion of the proximal tubule than in the inner portion. No significant hybridization signal was detected either in the distal tubule, renal corpuscle or peritubular tissues. These results indicate that testosterone induces the expression of the ODC gene in the proximal tubule of the renal cortex, leading to the increase in ODC activity in the same region.

LOCALIZATION OF D-AMINO ACID OXIDASE MRNA IN THE MOUSE KIDNEY AND THE EFFECT OF TESTOSTERONE TREATMENT

Abstractd-Amino acid oxidase (DAO), which catalyzes oxidative deamination ofd-amino acids, is known to be highly expressed in the kidney. This study was designed to examine the localization of DAO mRNA in the mouse kidney using in situ hybridization histochemistry (ISH). For comparison, ISH for mRNA of ornithine decarboxylase (ODC), which is also highly expressed in the mouse kidney, was simultaneously performed. Adult, male mice which received 1 mg of testosterone propionate or vehicle injection, were sacrificed 14 h after injection and their kidneys were removed and processed for ISH. Hybridization signals for both mRNAs were exclusively located over the epithelial cells of the proximal tubule in the vehicle-treated animals. Signals for the DAO mRNA were observed at nearly the same hybridization intensity throughout the proximal tubule, whereas hybridization signals for the ODC mRNA were observed exclusively in the pars convoluta. Following testosterone treatment, ODC mRNA in the pars convoluta was expressed with a stronger intensity than that in the vehicle-injected animals. ODC mRNA was also expressed in the pars recta with a weaker intensity than in the pars convoluta. On the other hand, DAO mRNA expression was little affected by testosterone treatment. These results indicate that, although both genes are possibly expressed in the same cells, the expression of these genes is regulated by different mechanisms.

Structure and action of sperm activating peptides from the egg jelly of a sea urchin, Anthocidaris crassispina.

Three Sperm Activating Peptides (SAPs) have been isolated to homogeneity from the jelly coat of the eggs of a Japanese sea urchin, Anthocidaris crassispina, and two of them have been sequenced. At pH 6.8 they can stimulate the sperm respiration 20-30 fold, to the level in normal seawater (pH 8.2), and the half-maximal activation was achieved by SAPs as low as around 100 pM. The stimulative activity was both pH- and Na+-dependent. The chymotryptic fragments (res. 3-10) were 10(4)-10(5) times less active, and the thermolytic fragment (res. 4-10) was 10(6) times less active than the parent SAP. CD spectra of SAPs indicate that they have unordered structure in aqueous solution.

Purification and characterization of prolyl endopeptidase from the Pacific herring, Clupea pallasi, and its role in the activation of sperm motility.

Protease activities with specificity toward synthetic substrates, Suc‐Gly‐Pro‐Leu‐Gly‐Pro‐MCA for prolyl endopeptidase or collagenase‐like peptidase, and Suc‐Ala‐Ala‐Pro‐Phe‐MCA for chymotrypsin were identified in the detergent‐soluble fraction of herring spermatozoa. The enzyme activities increased in the presence of herring sperm‐activating protein (HSAP). Among them a prolyl endopeptidase [EC. 3. 4. 21. 26] was purified to near homogeneity from herring testis. The molecular mass of the enzyme was 79 kDa and the properties of the enzyme were quite similar to prolyl endopeptidase from other tissues or cells. Both the enzyme activation and the sperm motility activation by HSAP were inhibited by benzyloxycarbonyl‐L‐thioproline‐thioprolinal, a specific inhibitor for prolyl endopeptidase. Furthermore, the motility activation by HSAP was inhibited by substrates of the prolyl endopeptidase. Western blotting with mouse anti‐prolyl endopeptidase serum revealed the presence of 79 kDa prolyl endopeptidase in the tail fraction of herring sperm. These results suggest that prolyl endopeptidase exists on the surface of the sperm tail and interacts with the HSAP.

Effect of Perinatal Hypothyroidism on Expression of Cytochrome C Oxidase Subunit I Gene, which is Cloned by Differential Plaque Screening from the Cerebellum of Newborn Rat

Early development of the central nervous system is influenced by several hormones including thyroid hormone. This study was designed to clone the gene whose expression is changed in association with perinatal hypothyroidism in the rat cerebellum. Rats were sacrificed at 15 day‐old postnatal age (P15) and their cerebella were removed. Poly (A)+ RNA was extracted to construct a cDNA library using λgt 10 cloning vector. Differential plaque screening was then performed using 32P‐labeled antisense cDNA synthesized from poly (A)+ RNA of the methimazole‐treated (hypothyroid) P15 rat cerebellum (hypothyroid probe), and of the euthyroid P15 rat cerebellum (euthyroid probe). The clones, which hybridized strongly to the euthyroid probe and weakly or not at all to the hypothyroid probe, were isolated. Sequence analysis of these clones revealed that all isolated clones encode cytochrome c oxidase subunit I (COX I), which is located in the mitochondria1 DNA. The decrease in COX I gene expression was not seen in the animals, which received methimazole treatment and daily replacement of thyroid hormone. In situ hybridization detection showed not only overall decrease in COX I gene expression but also change in distribution of hybridization signal in the cerebellar cortex of hypothyroid rat. Such change was not observed in the T4‐replaced animals. Based on the evidence that thyroid hormone greatly influences brain development, the results of the present study indicate that the terminal enzyme of mitochondria1 respiratory chain, COX I is one of the important target molecules regulated by thyroid hormone in the newborn rat cerebellum.

Localization of prolactin and its receptor messenger RNA in the human decidua.

Prolactin (PRL) is known as an anterior pituitary hormone. On the other hand, PRL is also produced in the human decidualized endometrium. The physiological role and site of action of endometrial PRL have not yet been clarified. This study was designed to investigate the localization of PRL receptor (PRL-R) gene-expressing cells in the human decidualized endometrium using in situ hybridization histochemistry. Sense and antisense35S-labeled RNA probes for human PRL-R mRNA were hybridized with cryostat sections of human decidua, which were obtained from patients undergoing therapeutic abortion at 8–10 weeks of gestation. Hybridization signals for PRL-R mRNA were seen over the decidual cells. No labeled cells were seen in the chorion, amnion, or trophoblast. Comparing the localization of PRL-R gene-expressing cells to that of PRL gene-expressing cells using adjacent sections, their distributions were quite similar. These results indicate that not only PRL but also PRL-R transcripts are located in the decidual cells.