Transfer of spermatogenesis-related cDNAs into eel testis germ-somatic cell coculture pellets by electroporation: methods for analysis of gene function.

Genes encoding spermatogenesis-related substance (eSRSs) show unique expression patterns during spermatogenesis. To analyze their function, we developed a new assay system using gene transfer techniques combined with coculture of the eel germ-somatic cells. First, we investigated the efficacy of in vitro electroporation transfer of gene into germ-somatic cell pellets using green fluorescent protein (GFP) gene. Second, in order to define the function of the eSRSs, we electrophoretically transferred eel spermatogonial stem cell renewal factor (eSRS34) and eel spermatogenesis-preventing substance (eSRS21) genes into germ-somatic cell pellets. Presence of the transferred cDNA was examined by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, proliferating cells were detected histologically, after labeling with BrdU. Transfer of the eSRS34 gene induced spermatogonial stem cell renewal in the pellets. Moreover, 11-ketotestosterone (11-KT) treatment stimulated the proliferation of spermatogonia, which resulted in the appearance of late type B spermatogonia in the pellets. The proliferation of spermatogonia by 11-KT stimulation was suppressed by transfer of the eSRS21 gene. These results indicate that the transferred eSRS34 and 21genes were functional in the pellets. Thus, an efficient in vitro gene transfer technique for coculture system of germ and somatic cell of Japanese eel was established.

Antiparasitic effect of calcium and magnesium ion-free buffer treatments against a common monogenean Neobenedenia girellae.

This study investigated a new effective method for controlling the capsalid monogenean Neobenedenia girellae. We examined in vitro and in vivo the effect on the percentage survival of N. girellae in buffers containing different metallic ions. Decreased survival was observed in buffer solutions lacking two ions. In particular, the percentage survival of N. girellae was significantly decreased after 10 min exposure to buffer containing neither Ca(2+) nor Mg(2+). Transmission electron microscopic observations showed that treatment with this buffer disrupted intercellular junctions. This significant effect on percentage survival of N. girellae using Ca(2+)/Mg(2+)-free buffer was confirmed in an in vivo assay. Ca(2+)/Mg(2+)-free buffer had no effect on the condition of the host, spotted halibut Verasper variegates (Pleuronectidae). These results suggest that treatment with Ca(2+)/Mg(2+)-free buffer is a new effective control method, which could replace existing control methods.

The effect of para-nonylphenol on Japanese eel (Anguilla japonica) spermatogenesis in vitro.

Endocrine disrupters have been recognized to interfere with endocrine systems that regulate reproduction, for example, by mimicking or inhibiting the action of endogenous sex steroid hormones including estradiol-17beta (E2). In the present study, we examined the effect of an endocrine disrupter, para-nonylphenol (p-NP) on spermatogenesis, and compared it with the action of E2, using an eel testicular organ culture system. p-NP alone stimulated early spermatogonial renewal in the same manner as E2. Neither induced further progress in spermatogenesis. In the presence of 11-ketotestosterone (11-KT), the major androgen in teleosts, p-NP did not prevent the 11-KT-induced progress in spermatogenesis. However, this treatment enlarged the Sertoli cells. Electron microscopic observation revealed that enlarged Sertoli cells contained well-developed organelles. Moreover, the proportion of germ cells appeared to have decreased as a result of Sertoli cell hypertrophy. These results clearly show that p-NP has an effect on Sertoli cells in the presence of an androgen (11-KT), potentially disturbing 11-KT-induced spermatogenesis.

Involvement of sex steroid hormones in the early stages of spermatogenesis in Japanese huchen (Hucho perryi ).

In higher vertebrates, considerable progress has been made in understanding the endocrine regulation of puberty; however, in teleosts, the regulatory mechanisms of spermatogenesis during the first annual cycle remain unclear. The present study was conducted to understand the regulatory mechanisms of spermatogenesis throughout the different stages of the first spermatogenic cycle and to check the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in Japanese huchen (Hucho perryi ). The results indicate that the serum level of 11-ketotestosterone (11-KT) was positively associated with germ cell type; the level first began to rise with the appearance of late-type B spermatogonia and continued to increase gradually throughout the active spermatogenic stages and spermiogenesis, reaching a peak value 2 wk before spawning, and then declined. During the spermatogenic stages, the serum concentration of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) was undetectable. Only a small peak was detected with the appearance of spermatocytes and spermatids, and at the time of spawning, the level increased dramatically, reaching its maximum value with the onset of milt production. Despite the high variation in serum levels of 17beta-estradiol (E2) both between months and among the individuals, E2 was found during the whole reproductive cycle. From these results, we concluded that 1) 11-KT is necessary for the initiation of spermatogenesis and sperm production, and it probably plays a role in spermiation, 2) 17alpha,20beta-DP is essential for the final maturation stage, could play a significant role in the mitosis phase and meiosis process, and probably participates in the regulation of spawning behavior, and 3) estrogen is an indispensable male hormone that plays a physiological role in some aspects of testicular functions, especially during the mitotic phase. The three steroids were also able to induce DNA synthesis, spermatogonial renewal, and/or spermatogonial proliferation in vitro.

A randomized, double-masked, placebo-controlled trial of recombinant granulocyte colony-stimulating factor administration to preterm infants with the clinical diagnosis of early-onset sepsis.

OBJECTIVE: We performed a randomized, double-masked, parallel-groups, placebo-controlled trial of recombinant granulocyte colony-stimulating factor (rG-CSF) administration to 44 preterm neonates who had blood cultures obtained and antibiotics begun because of the clinical diagnosis of early-onset sepsis. Two primary outcome variables were tested 1) mortality and 2) development of nosocomial infections over the 2-week period after dosing. DESIGN AND METHODS: The treatment group (n = 22) received 10 microgram/kg/day of intravenous rG-CSF once daily for 3 days and the placebo group (n = 22) received the same volume of a visually indistinguishable vehicle. Mortality and culture-proven nosocomial infections were recorded. Immediately before the first, second, and third doses, and again 10 days after the first dose, serum concentrations were determined for tumor necrosis factor-alpha, interleukin 6, granulocyte-macrophage colony stimulating factor, and G-CSF, and blood leukocyte counts, absolute neutrophil counts, immature/total neutrophil ratios, platelet counts, and hemoglobin concentrations were measured. RESULTS: The treatment and placebo groups were of similar gestational age (29 +/- 3 vs 31 +/- 3 weeks) and birth weight (1376 +/- 491 vs 1404 +/- 508 g), and had similar Apgar scores and 24-hour Score for Neonatal Acute Physiology scores. The mortality rate was not different between treatment and placebo groups. However, the occurrence of a subsequent nosocomial infection was lower in the rG-CSF recipients (relative risk:.19; 95% confidence interval:.05-.78). rG-CSF treatment did not alter the serum concentrations of the cytokines measured (except for G-CSF). Serum G-CSF levels and blood neutrophil counts were higher in the treatment than in the placebo group 24 hours and 48 hours after dosing. CONCLUSIONS: Administration of 3 daily doses of rG-CSF (10 microgram/kg/day) to premature neonates with the clinical diagnosis of early-onset sepsis did not improve mortality but was associated with acquiring fewer nosocomial infections over the subsequent 2 weeks.

Levels of specific antigen (gp43), specific antibodies, and antigen-antibody complexes in saliva and serum of paracoccidioidomycosis patients.

The present study analyses human immunoglobulin G (IgG) antibodies directed against the Paracoccidioides brasiliensis exoantigen, gp43, as well as the presence of gp43-IgG immune complexes (ICs) in 31 samples of saliva and serum from 19 patients with paracoccidioidomycosis (PCM) and 12 normal donors. Additional analysis of secretory IgA (sIgA) was performed on the same saliva samples. Consistent with previous findings, a significant increased specific IgG level was observed in PCM patients' saliva and serum (P < 0.05). The analysis of serum gp43 and gp43-IgG IC demonstrated a higher level in patients with PCM (P < 0.05); however, this difference was not statistically significant with regard to gp43 and gp43-IgG in saliva when compared to the healthy donors. A high level of sIgA in saliva of PCM patients compared to that of normal donors was also observed (P < 0.05). Patients exhibiting low levels of serum IgG but with high titres of IC were observed, thus strengthening the idea of the necessity to use more than one marker for diagnosis and treatment monitoring of PCM. This is the first report of sIgA in PCM patients' saliva and may be indicative of a protective role in neutralizing antigens on mucosal surfaces.

[Assessing the efficacy of the recombinant human granulocyte colony-stimulating factor "rhG-CSF" in the treatment of early neonatal sepsis in premature neonates] / Avaliação da eficácia do fator estimulador recombinante humano de colônias de granulócitos "rhG-CSF" no tratamento da sepse de início precoce, em recém-nascidos prematuros.

OBJECTIVE: To evaluate the efficacy of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the treatment of early-onset neonatal sepsis among premature infants.MATERIALS AND METHODS: A double-blind, randomized, placebo-controlled trial was performed among forty-four preterm neonates who had "clinical diagnosis" of early-onset sepsis. The treatment group (n=22) received 10 micro g/kg/d of rhG-CSF, IV once daily for three consecutive days, and the placebo group (n=22) received the same volume of a visually-indistinguishable vehicle. Prior to the first dose, and prior to the second and third doses, and again 10 days after the first dose, we measured tumor necrosis factor-a, interleukin-6, granulocyte-macrophagocyte colony-stimulating factor, G-CSF, leukocyte count, absolute neutrophil count, immature/total neutrophil ratio, platelet count, and hemoglobin concentration. A bone marrow aspiration was performed seven days after the first dose, and both the neutrophil storage pool (NSP) percent and the NSP/NPP (neutrophil proliferative pool) ratios were tabulated.RESULTS: The treatment and placebo groups were of similar gestational age (29-/+ 3 vs 31-/+ 3 weeks) and birth weight (1376 -/+ 491 vs 1404 -/+ 508 grams). They had similar Apgar scores and 24 hour SNAP scores. No deaths occurred during the first week of life among the treatment group while three deaths occurred in the placebo group. RhG-CSF treatment did not alter the serum concentrations of the cytokines measured (except for G-CSF). Serum G-CSF levels, blood leukocyte counts, absolute neutrophil counts, NSP percentages, and NSP/NPP ratios were higher in the treatment group 24 hours and 72 hours after dosing. The occurrence of a subsequent infection over the two week period following dosing was significantly lower in the treatment group (n=2) than in the placebo group (n=9; p<0.02, RR 0.19 [0.05-0.78]). The overall mortality rate during the entire hospitalization was not different between treatment and placebo groups.CONCLUSIONS: Administration of rhG-CSF to premature neonates with the clinical diagnosis of early-onset sepsis was associated with lower incidence of nosocomial infection over the ensuing three weeks period, but it did not change the overall mortality rate.

Estradiol-17beta stimulates the renewal of spermatogonial stem cells in males.

In this work, we examined the functions of the female hormone "estrogen" on spermatogenesis of the Japanese eel (Anguilla japonica). Estradiol-17beta (E(2)), a natural estrogen in vertebrates, was present in the serum and its receptor was expressed in the testis during the whole process of spermatogenesis. Spermatogonial stem cell renewal was promoted by E(2) implantation but was suppressed by tamoxifen (an antagonist of estrogen). In vitro, 10 pg/ml of E(2) was sufficient to induce spermatogonial stem cell division in cultured testicular tissue, therefore confirming the in vivo observations. These results clearly show that estrogen is an indispensable "male hormone" in the early spermatogenetic cycle.

Recombinant human insulin-like growth factor I stimulates all stages of 11-ketotestosterone-induced spermatogenesis in the Japanese eel, Anguilla japonica, in vitro.

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-ketotestosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type B spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.

cDNA cloning of a stage-specific gene expressed during HCG-induced spermatogenesis in the Japanese eel.

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)+ RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin betaB subunit, was expressed in the initial phase of spermatogenesis (1-6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames: 3-18 days (two clones), 6-18 days (one clone) and 9-18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full-length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12-15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. It has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG-induced spermatogenesis in the eel.

Two testicular cDNA clones suppressed by gonadotropin stimulation exhibit ZP2- and ZP3-like structures in Japanese eel.

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature eel testes consisting of premitotic spermatogonia. To understand the regulatory mechanisms of spermatogenesis, we have applied a subtractive hybridization method to identify genes in which changes in expression occur after HCG treatment in vivo. The subtraction was carried out 24 hours after HCG injection. Two up-regulated and six down-regulated cDNA clones by HCG stimulation were isolated, and named eel spermatogenesis-related substance (eSRS) 1 to 8. In this paper, down-regulated cDNA clones of eSRS3 and eSRS4 were sequenced. A homology search showed that eSRS3 and eSRS4 have amino acid sequences similar to those of the ZP-domains of zona pellucida sperm-binding protein (ZP)-2 and 3, respectively. Transcripts of eSRS3 and eSRS4 have been detected only in immature testes and ovaries. Both transcripts disappeared immediately after HCG injection and were not detected in testes throughout the experimental period. To determine whether HCG action on down-regulation of eSRS3 and eSRS4 transcription is direct or mediated through 11-ketotestosterone (11-KT), a spermatogenesis-inducing steroid in eel, we investigated the effect of HCG and 11-KT on testicular eSRS3 and eSRS4 mRNA transcription in vitro. Northern blot analysis using poly(A)+ RNA extracted from cultured testis showed that both HCG and 11-KT suppressed the mRNA transcription of both eSRS3 and eSRS4. We speculate that eSRS3 and eSRS4 may play important roles in the prevention of spermatogenesis in the eel.

Impaired spermatogenesis in the Japanese eel, Anguilla japonica: possibility of the existence of factors that regulate entry of germ cells into meiosis.

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG-injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11-ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 2(3) to 2(6) late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.

Effectiveness of concomitant setiptiline maleate (Tecipul) on negative symptoms of schizophrenia.

1. Setiptiline maleate was administered to schizophrenic patients with the object of improving their negative symptoms. 2. Moderate improvements were observed in 58% of the treated patients, thus usefulness of this drug was demonstrated. 3. There was no aggravation of symptoms, and side effects were minor. 4. Measurements of plasma monoamine metabolites showed a tendency of MHPG to decrease and a significant decrease in 5-HIAA, but no change in the level of HVA was observed, suggesting a relationship between the negative symptoms and noradrenaline and/or serotonin systems.

Intrapartum amnioinfusion in twin gestation. A preliminary report of three cases.

Amnioinfusion is an intrapartum technique that is not usually attempted in twin gestations. This report describes infusion of normal saline into the amniotic sacs of three twin gestations with oligohydramnios. All the twins were safely delivered vaginally. No untoward effects were noted. Ultrasound is advised following amnioinfusion to assess the increase in the amniotic fluid volume.

Effects of posture on flow-volume curves during normocapnia and hypercapnia in patients with obstructive sleep apnoea.

BACKGROUND: A high ratio of forced expiratory to forced inspiratory maximal flow at 50% of vital capacity (FEF50/FIF50) may identify upper airway dysfunction. Since hypercapnia increases the motor activity of airway dilating muscles its effects on the maximum expiratory and inspiratory flow-volume curves (MEIFV) in patients with obstructive sleep apnoea and in normal subjects in different postures was studied. METHODS: The effects of posture on the maximum expiratory and inspiratory flow-volume curves during the breathing of air and 7% carbon dioxide in 11 patients with obstructive sleep apnoea were compared with those in nine normal subjects. Measurements were made in the sitting, supine, and right lateral recumbent positions. Forced expiratory flow at 50% vital capacity (FEF50), forced inspiratory flow at 50% vital capacity (FIF50) and FEF50/FIF50 were determined. RESULTS: In the normal subjects FEF50, FIF50, and FEF50/FIF50 were not affected by change in posture or by breathing carbon dioxide. In the patients there was a fall in FIF50 and an increase in FEF50/FIF50 when breathing air in the supine position compared with values in the seated and lateral position. While they were breathing carbon dioxide there was a slight increase in FEF50 when patients were seated or in the lateral position compared with values during air breathing. Hypercapnia abolished the effects of posture on FEF50/FIF50. Values for FEF50/FIF50 in the supine position while they were breathing air correlated with the apnoeic index but not with other polysomnographic data. CONCLUSION: In patients with obstructive sleep apnoea the upper airway is prone to collapse during inspiration when the patient is supine, even when awake; this tendency can be reversed by breathing carbon dioxide.

[Relationship between bronchial asthma and mite].

We studied the relationship between bronchial asthma and mite as an antigen by reviewing previous reports. The incidence of the bronchial asthma has been increasing and it is estimated to be more than 2% of the total population. The main components of the antigen in the house dust (H, D.), which has been considered to be an important antigen of the perennial asthma, are Dermatophagoides farinae (D, f.) and Dermatophagoides pteronyssinus (D, p.) in H, D. The housing environment in recent years have become favorable for the reproduction of D, f. and D, p. It is considered that environmental pollution increases the incidence of asthma, especially infants.

Panic anxiety after abrupt discontinuation of mianserin.

We observed a case of withdrawal after abrupt discontinuation of mianserin. A 41-year-old woman was treated according to a diagnosis of depression, which was her 6th episode. Mianserin 30 mg/day, etizolam 1 mg/day and flunitrazepam 1 mg/day were administered. When the patient discontinued taking the drugs by herself because of subsiding of these symptoms, severe panic anxiety appeared. This panic anxiety was not relieved by taking etizolam and flunitrazepam again, but subsided rapidly by the re-administration of mianserin 30 mg/day, and because of that the depressive symptom also disappeared. From these experiences panic anxiety seemed to be a withdrawal symptom, and involvement of the noradrenergic system in panic anxiety as well as serotonergic system was suggested.

Parkinsonism manifesting depression as the first sign.

We have had experience in treating two patients with parkinsonism of which the first manifestation was depression. Case 1 was a 61-year-old woman. A diagnosis of depression had been made and repeated medication consisting mainly of antidepressants was given. However, the depressive state persisted and the signs of parkinsonism gradually became evident. The initial treatment with antidepressant drugs was replaced with one based on L-DOPA (400 mg a day). Subsequently, the symptoms of parkinsonism and depression diminished relatively rapidly. Case 2 was a 66-year-old man. His parkinsonism was suspected shortly after the onset, although the symptoms of depression were predominant. Similarly, the treatment based on L-DOPA (400 mg a day) relieved the symptoms of depression and parkinsonism rapidly. The present study described above indicates that parkinsonism should be suspected in cases of persistent depression and in patients who have depressive symptoms resistant to antidepressants, since parkinsonism may first manifest itself as depression.

Home sleep monitor for detecting apnea episodes by nasal flow and tracheal sound recordings.

We have developed a portable home sleep monitoring system using nasal airflow (NA), tracheal sound recordings (TSR), and electrocardiogram (ECG). NA was recorded by two thermisters. TSR was recorded by a microphone attached to the skin overlying the cervical trachea. Three kinds of signals were recorded with a cassette recorder. Thirty-seven outpatients who had sleep complaints were monitored during sleep at home using this recorder. Attachment of the pickups was performed by the patients themselves. Recordings were played back and analyzed by a personal computer to evaluate apnea episodes from TSR and R-R intervals beat by beat. This home monitoring system had labor-saving and cost-saving benefits and seemed to be a satisfactory technique for screening.