Lymphatic vessels around the uterus: D2-40 (podoplandin) immunohistochemistry using elderly cadavers.

Using D2-40 immunohistochemistry, we examined the morphology of lymphatic vessels (LVs) in, along and around the uterus of 10 donated female cadavers (mean age, 85 years). All these women had 1 or 2 children with vaginal delivery, but the other obstetrics information was unknown. When compared with the bladder, vagina and the subperitoneal tissue, the percentage area of LVs in a 3 × 2 mm square including the hot spot was extremely high along the uterine artery and superficial uterine vein, in spite of the silent physiology of the elderly uterus. Notably, the LVs along the uterine artery and superficial uterine vein were highly dilated and embedded in the tight connective tissue around the artery and vein. In contrast, the LVs were separated from the artery and vein in the so-called vesico- -uterine ligament. Thus, surgical separation of the LVs from the artery and vein, i.e., skeletonisation, appears very difficult along the uterine artery and superficial uterine vein. This may become a major factor limiting the future application of robot-assisted surgery for uterine cancers.

Initiation and stimulation of spermatogenesis in vitro by mammalian follicle-stimulating hormone in the Japanese newt, Cynops pyrrhogaster.

In order to elucidate the molecular mechanisms by which spermatogenesis is regulated, especially the roles of hormones and somatic cells in the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments rich in secondary spermatogonia were cultured in control medium for three weeks, most of the testicular cysts still remained as secondary spermatogonia. On the other hand, in the medium supplemented with follicle-stimulating hormone (FSH) alone, DNA syntheses in secondary spermatogonia and Sertoli cells were stimulated and secondary spermatogonia differentiated into primary spermatocytes (zygotene-pachytene) in more than half of the cysts by the second week. When newt testes fragments rich in primary spermatocytes were cultured in a control medium for three weeks only round spermatids were observed at the most advanced stage. On the other hand, in the medium supplemented with FSH alone, elongated spermatids appeared by the second week. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5 alpha-dihydrotestosterone) to the control medium stimulated differentiation for either step. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH for either stage of the testes (secondary spermatogonia and primary spermatocytes). Preliminary results indicate that FSH does not bind to germ cells but to somatic cells (most probably Sertoli cells). These and our unpublished data suggest that FSH triggers proliferation and differentiation of spermatogonia into elongated spermatids by acting on Sertoli cells which in turn act on germ cells.

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix.

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.

IGF-I, IGF-II and insulin promote differentiation of spermatogonia to primary spermatocytes in organ culture of newt testes.

Recombinant human insulin-like growth factors (rhIGF-I and rhIGF-II) and human insulin promoted the differentiation of spermatogonia into primary spermatocytes in newt testes fragments cultured in a chemically defined medium. The biological potency for promoting differentiation was dose-dependent for all the ligands with the highest potency displayed by IGF-I, followed by IGF-II, and the least by insulin. The difference in potency was larger between IGF-II and insulin than that between IGF-I and IGF-II. This order of biological potency was in good accordance with the order of affinity in binding specificity of [125I]IGF-I to the testicular membrane fractions: IGF-II and insulin competed the binding of [125I]IGF-I only at concentrations 20-fold and 100-fold higher, respectively, than IGF-I. Specific binding was observed in both somatic cells (mostly Sertoli cells) and germ cells (spermatogonia and primary spermatocytes), though the binding to somatic cells was about 2.7 times higher than that to germ cells. These results indicate that (1) specific binding sites for IGF-I are present in the newt testes, (2) IGF-II and insulin also bind to these receptors but to a lesser degree, and (3) IGF-II and insulin as well as IGF-I promote spermatogonial differentiation into primary spermatocytes by binding to the IGF-I receptor.

Suppression of P450 aromatase gene expression in sex-reversed males produced by rearing genetically female larvae at a high water temperature during a period of sex differentiation in the Japanese flounder (Paralichthys olivaceus).

The phenotypic sex of many teleost fishes including flounders can be experimentally altered by treating embryos or larvae with varied temperatures or sex-steroid hormones. To analyse the sex determination mechanism, especially the role of cytochrome P450 aromatase (P450arom), an enzyme that catalyses the conversion of androgens to estrogens, in temperature-dependent gonadal sex differentiation in the Japanese flounder, we generated two populations of larvae, both having XX (genetic females) but each growing up to display all phenotypic females or males, by rearing the larvae at normal (18 degrees C) or high (27 degrees C) water temperatures from days 30 to 100 after hatching respectively. The larvae (XX) were produced artificially by mating normal females (XX) with gynogenetic diploid males (XX) which had been sex-reversed to phenotypic males by 17alpha-methyltestosterone. To study the role of P450arom in sex determination in the flounder, we first isolated a P450arom cDNA containing the complete open reading frame from the ovary. RT-PCR showed that P450arom mRNA was highly expressed in the ovary and spleen but weakly in the testis and brain. Semi-quantitative analyses of P450arom mRNA in gonads during sex differentiation showed that there was no difference in the levels of P450arom mRNA between the female and male groups when the gonad was sexually indifferent (day 50 after hatching). However, after the initiation of sex differentiation (day 60), the mRNA levels increased rapidly in the female group, whereas they decreased slightly in the male group. Similarly, estradiol-17beta levels rose remarkably in the female group, yet remained constant in the male group. These results suggest that induction of sex reversal of genetically female larvae to phenotypic males by rearing them at a high water temperature caused a suppression of P450arom gene expression. Furthermore, we suggest that the maintenance of P450arom mRNA at very low levels is a prerequisite for testicular differentiation, while the increased levels are indispensable for ovarian differentiation.

Improved production of phenomycin by a genetically engineered Escherichia coli.

We established an improved production of an antitumor polypeptide anti biotic, phenomycin (PHM), by using a genetically engineered Escherichia coli. Phenomycin consists of 89 natural amino acids without intramolecular disulfide bridge. PHM gene was synthesized as a fusion gene in which PHM at the C-terminus and the residues 1 approximately 20 of Hirudin variant 1 (HV1) at the N-terminus connected by a factor Xa recognition sequence (Ile-Glu-Gly-Arg). E coli JM 109 transformed with a plasmid containing the synthesized gene expressed a fusion protein and the trypsinization of the fusion protein purified by ultrafiltration and ion-exchange chromatography gave efficiently recombinant PHM at a final yield of 50 mg/liter of culture. This PHM yield was six times higher than that obtained by a natural PHM producing strain of Streptoverticillium baldacci. Recombinant PHM was not distinguishable from natural PHM in all aspects observed.

Cloning of full length cDNA, high-level expression and biological characterization of feline IL-12.

A full length cDNA of feline interleukin(IL)-12 p35 and p40 subunits was cloned. By transferring the plasmids containing both the subunit genes to mammalian cells, we expressed biologically active feline IL-12. The expressed feline IL-12 has interferon-gamma-inducing activity against both human and feline peripheral blood mononuclear cells (PBMC) and stimulates cytotoxic T lymphocyte activity against herpes simplex virus-infected human PBMCs. There were two kinds of molecules (p75, p80) in the purified recombinant feline IL-12, and both molecules exhibited biological activity. The difference between p75 and p80 was the degree of the glycosylation of the p35 chain. Moreover, when we modified the cDNA of p35 by changing some codons and deleted the 5' and 3' non-coding regions, the expression level of IL-12 increased about 100 fold.

Loss of programmed cell death 4 induces apoptosis by promoting the translation of procaspase-3 mRNA.

The programmed cell death 4 (Pdcd4), a translation inhibitor, plays an essential role in tumor suppression, but its role in apoptosis remains unclear. Here we show that Pdcd4 is a critical suppressor of apoptosis by inhibiting the translation of procaspase-3 mRNA. Pdcd4 protein decreased more rapidly through microRNA-mediated translational repression following apoptotic stimuli than did the activation of procaspase-3, cleavage of poly(ADP)ribose polymerase (PARP) by active caspase-3, and nuclear fragmentation. Strikingly, the loss of Pdcd4 by the specific RNA interference increased procaspase-3 expression, leading to its activation and PARP cleavage even without apoptotic stimuli, and sensitized the cells to apoptosis. Thus, our findings provide insight into a novel mechanism for Pdcd4 as a regulator of apoptosis.

Meiosis of primary spermatocytes and early spermiogenesis in the resultant spermatids in newt, Cynops pyrrhogaster in vitro.

Dissociated spermatogenic cells were cultivated within the collagen matrix at low cell density. The largest cell type in the culture was identified as the primary spermatocytes by their size and the morphological characteristics revealed by ultra-thin sections. Chromosome analysis showed that about 90% of the cells examined were either in first or second meiosis. Within the collagen matrix, the fates of 282 single primary spermatocytes at meiotic stage in diakinesis or metaphase were followed. In a few days, most of them gave rise to four spermatids, passing through first and second meiotic divisions. About 80% of the spermatids formed motile flagella. They grew about 20-60 micrometers a day. The final state of the differentiation attained in our culture conditions was the spermatids with localized spherical nuclei and motile flagella, about 500 micrometers in length after 1-month's culture. Ultra-thin sections of the spermatids show that the rings, neck-pieces, and acrosomes developed in the cells.

Inhibition of second meiotic division and a switching over to flagellar formation in secondary spermatocytes of newt by cycloheximide.

10.0 micro M cycloheximide (CH) was found to completely inhibit the second meiotic division of newt spermatocytes. Under continuous incubation with CH from the beginning of interphase II, secondary spermatocytes fail to initiate chromosomal condensation and thus remain in interphase II. After 12-15 h of incubation, a single motile flagellum, about 5 micrometers in length, was observed on each of the secondary spermatocytes. These flagella grew to a length of 60-80 micrometers, but thereafter ceased to grow, whereas ordinarily spermatids grew flagella up to 500 micrometers in length in the absence of CH [1]. When CH was applied within 2 h following telophase I, the percentage of meiosis II inhibition was almost 100% and when applied even later, it became less, which showed that the early half period during interphase II was sensitive to CH. Regardless of the length of incubation time with CH, flagella were found to grow within a period of 12-15 h following telophase I. Upon removal of CH, even after 60 h of incubation, the flagella of the secondary spermatocytes shortened and disappeared completely. These spermatocytes underwent the second meiotic divisions. Also, flagella grew on the resulting spermatids. The possibility that a particular centriole which participated in the first meiotic division changes into a basal body for flagellar formation under the influence of CH and vice versa upon removal of it, is discussed in the following.

The occurrence of a gene-encoded variant of nuclear basic protein (SP4) in sperm of Xenopus laevis.

In our recent analyses [1] of five tandemly arranged genes encoding a major sperm-specific basic nuclear protein (SP4) of Xenopus laevis, we found a gene containing a single base substitution which will give rise to the replacement of the 69th residue among the 78 amino acids of SP4. In this study, the polypeptide from sperm nuclei which were separated by reversed-phase HPLC as a distinct entity from SP4 were collected for their peptide mapping with V8 protease and partial amino acid sequence analyses. It resulted that a polypeptide exhibiting an amino acid replacement at exactly the same position as predicted from a single base substitution of SP4 occurs in approximately one-fourth of the amount of SP4. This finding suggests that the relative amount of SP4 and its variant directly depends on the relative number of genes of SP4 and its variant.

cDNA cloning and expression of Xenopus sperm-specific basic nuclear protein 5 (SP5) gene.

As part of our continuing program to understand the molecular mechanisms controlling the synthesis of sperm-specific nuclear proteins (SPs1-6) during spermatogenesis in Xenopus, we report here on the isolation of a cDNA clone for SP5, the partial sequencing of the amino acids in the SPs, and the expression of the mRNA for SP5. A cDNA clone (pXSP633) was isolated from a cDNA library, previously prepared from poly (A)+ mRNA obtained from Xenopus round spermatids. Determination of the amino acid sequence of the N-terminal regions of all the SPs(1-6) suggested that pXSP633 encodes SP5, whereas SPs3, 4, and 6 are derived from a second mRNA species, and SPs1 and 2 from a third mRNA species. Thus it seems likely that the six SPs are derived from three different mRNA species. Northern blot analyses of RNA, extracted from primary spermatocytes and round spermatids, was performed with oligonucleotide probes specific for SPs4 and 5 mRNAs. The results showed that whereas both SPs4 and 5 mRNAs are expressed in primary spermatocytes, the amount of SP5 mRNA is only about one-fifth of that of SP4 mRNA. However, both mRNA species undergo a similar size change in the length of their poly (A) tracts during spermatogenesis: the size of the mRNA in cultured round spermatids on day 0 was longer than that in primary spermatocytes, but the size of the mRNA in round spermatids on day 6 was shorter than that in round spermatids on day 0.

Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus).

The sex of Japanese flounder (Paralichthys olivaceus) is easily altered by water temperature or sex steroid hormone treatment during the period of sex determination. We have previously shown that rearing the genetically female larvae at high water temperature caused the suppression of P450 aromatase (P450arom) gene expression in the gonad and phenotypic sex-reversal of the individuals to males (Kitano et al. 1999. J Mol Endocrinol 23:167-176). In the present study, we show that treatment of genetically female larvae with fadrozole (aromatase inhibitor) or 17alpha-methyltestosterone induces sex-reversal as well as suppression of P450arom gene expression. The effect of fadrozole was counteracted by co-administration of estradiol-17beta. Effective periods for fadrozole treatment to induce sex-reversal were similar to those for high water temperature treatment. RT-PCR did not detect P450arom mRNA in gonad of the sex-reversed, phenotypic males. These results indicate that sex-reversal of the genetically female larvae by aromatase inhibitor (or 17alpha-methyltestosterone) may be due to the suppression of P450arom gene expression and the resultant decrease in the amount of estrogen.

Cloning of cDNA for newt WT1 and the differential expression during spermatogenesis of the Japanese newt, Cynops pyrrhogaster.

To investigate the function of Wilms' tumor 1 (WT1) during spermatogenesis, cDNA for newt WT1 homolog was cloned and the expression of WT1 in newt testes was examined. The cDNA is 2089 bp in length and encodes 426 amino acid (aa) residues. The deduced aa sequence shares 76 and 79% homology with human and Xenopus WT1, respectively. Northern blot analysis shows that WT1 mRNA, 3.2 and 4.5 kb in length, are expressed in the testis and kidney. Both WT1 mRNA species are detected in various stages of spermatogenesis, but the 3.2 kb mRNA is highly expressed in spermatogonia and mature sperm stages, while the amount of 4.5 kb mRNA is almost constant throughout spermatogenesis. In situ hybridization reveals that WT1 mRNA is localized in Sertoli cells. Moreover, immunohistochemical analysis shows that WT1 protein is highly expressed in the nuclei of Sertoli cells in early spermatogonia and mature sperm stages, but not in pericystic cells or germ cells. These results suggest that WT1 is involved in the regulation of gene expression in Sertoli cells, depending on the spermatogenic stage.

Elevation of plasma prolactin concentrations by low temperature is the cause of spermatogonial cell death in the newt, Cynops pyrrhogaster.

Temperature plays an important role in reproduction of urodeles. Spermatogenesis in newts is arrested when the environmental temperature lowers. We found that transfer of newts, Cynops pyrrhogaster, to low temperature (8 and 12 degrees C) caused cell death of spermatogonia just before meiosis and elevation of prolactin concentration in the newt plasma. Injection of a dopamine antagonist (pimozide), which is known to increase the plasma prolactin concentration, to the newt caused significant increase of spermatogonial degeneration, whereas treatment with an agonist (bromocryptin), which is known to decrease the prolactin concentration, suppressed the cell death. Finally, injection of anti-prolactin serum into the newts which had been transferred to low temperature almost completely inhibited the spermatogonial degeneration for as long as 3 days. These results demonstrate that low temperature caused elevation of prolactin concentration in the newt blood, which induced cell death of spermatogonia just before meiosis.

Differentiation of lens-like structures from newt iris epithelial cells in vitro.

Dissociated cells of pigmented iris epithelium from adult newts grew intensively in monolayer cultures after a lag of two to three weeks. During the lag period, depigmentation occurred in many cells. When cultures became confluent five to six weeks after seeding, many tiny lens-like structures (30-70 per plate) differentiated from dense foci of amelanotic epithelial cells. These lens-like structures appeared in all cultures originated from cells of ventral as well as dorsal iris. The identification of these structures as lens was established by both immunological and ultrastructural techniques.

Mammalian follicle-stimulating hormone and insulin-like growth factor I (IGF-I) up-regulate IGF-I gene expression in organ culture of newt testis.

We previously showed that porcine follicle-stimulating hormone (pFSH) and human recombinant insulin-like growth factor (rhIGF-I) promote the differentiation of secondary spermatogonia into primary spermatocytes in organ cultures of newt testes, respectively. To elucidate the molecular action of FSH and IGF-I, we cloned cDNAs for newt IGF-I and IGF-I receptor (IGF-IR), and examined their mRNA expression in organ culture during newt spermatogenesis. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that IGF-I mRNA was highly expressed in somatic cells (mostly Sertoli cells) at the secondary spermatogonial stage but barely in germ cells, and that IGF-IR mRNA was expressed in both germ and somatic cells at all stages examined. The addition of pFSH to newt testis markedly increased IGF-I mRNA expression. Also, rhIGF-I increased IGF-I mRNA expression, whereas IGF-IR mRNA expression declined slightly. These results suggest that the ability of FSH to promote the differentiation of secondary spermatogonia is at least partly mediated by somatic cell-derived IGF-I, and that IGF-I mRNA expression in somatic cells is auto-upregulated.

Caspase activity in newt spermatogonial apoptosis induced by prolactin and cycloheximide.

We previously showed in vivo and in vitro, that among the spermatogenic stages of the newt, prolactin (PRL) induces apoptosis specifically in the penultimate stage of secondary spermatogonia. In the current report, we demonstrate in vitro that cycloheximide (CHX), an inhibitor of protein synthesis, induces morphological apoptotic changes similar to those caused by PRL, such as chromatin condensation and apoptotic body formation. Next, we found that Z-VAD-fmk, an inhibitor of various caspases, suppressed the apoptosis induced by PRL and CHX, but ICE inhibitor Ac-YVAD-CHO or caspase-3 inhibitor Ac-DEVD-CHO did not. As high caspase activity was present in extracts of testes treated with CHX, we suggest that an unidentified caspase induces the morphological changes of apoptosis in newt spermatogonia.

Expression of a novel matrix metalloproteinase gene during Cynops early embryogenesis.

Matrix metalloproteinases (MMPs) are thought to play important roles in the gastrulation of Cynops pyrrhogaster embryos. MMP cDNAs were cloned from Cynops pyrrhogaster and we report here a novel MMP called CyMMP, which has strong similarity to MMP-21 (XMMP) in Xenopus. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that CyMMP mRNA was already present in cleavage stage embryos. The amount of the mRNA then gradually decreased, but increased again starting in late gastrula. There were regional differences in the level of CyMMP mRNA expression at late gastrula: the involved archenteron roof was the predominant site of expression of the gene, while there was weak expression in the neuroectoderm and epidermal ectoderm. We also found that the gene was activated in artificially mesodermalized ectoderm. The present findings indicate that CyMMP mRNA expression is activated in differentiating mesoderm during gastrulation, suggesting that CyMMP plays a role in gastrulation-related cell movement.

Primary structures of sperm-specific basic nuclear proteins and gene expression in Japanese newt, Cynops pyrrhogaster.

Electrophoretic analyses of acid extracts from mature sperm of newt, Cynops pyrrhogaster, on acid/urea/Triton X-100 polyacrylamide gel showed the exclusive occurrence of sperm-specific nuclear basic proteins (SBPs), which moved faster than somatic histones on the gel. These SBPs were eluted separately by reversed phase-high-performance liquid chromatography as two large peaks and a few small peaks. Of these, only the small peaks disappeared with treatment of the acid extracts with alkaline phosphatase before they were injected into the column, so that there were only two distinct components: NP1 and NP2. Determination of amino acid sequences by the Edman method as well as by sequencing of cDNA for both components indicated that each protein consisted of 43 (NP1) or 48 (NP2) amino acid residues, rich in arginine residues (53.5% in NP1; 47.9% in NP2), forming the clusters. They had molecular masses of 5,386 Da (NP1) and 5,748 Da (NP2), respectively. Northern blot analysis using cDNAs as probes indicated that mRNAs for both NP1 and NP2 occurred not in primary spermatocytes but in round spermatids. In situ hybridization analyses using antisense RNA for NP1 as a probe clearly showed the first appearance of NP1 mRNA at the late stage of round spermatid.