The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.

Clockwork blue: on the evolution of non-image-forming retinal photoreceptors in marine and terrestrial vertebrates.

This paper presents a hypothesis that could explain why blue light appears to dominate non-image-forming (NIF) ocular photoreception in marine as well as terrestrial vertebrates. Indeed, there is more and more evidence suggesting that 'novel' retinal photoreceptors, which are sensitive to blue light and were only discovered in the 1990s, could be a feature shared by all vertebrates. In our view, blue light photoreception evolved and persisted as NIF photoreception because it has been useful in the colonisation of extensive photo-dependent oceanic habitats and facilitated the move of vertebrates from an aquatic to a terrestrial environment. Because the available scattered evidence is compatible with the validity of our hypothesis, we hope that our rationale will be followed up. Indeed, it (1) involves testable predictions, (2) provides plausible explanations for previous observations, (3) unites phenomena not previously considered related to one another and (4) suggests tests that have not been carried out before. Overall, our approach not only embraces cross-disciplinary links; it, moreover, serves as a reminder of an all-embracing evolutionary history, especially with regard to a ubiquitous photoreceptive 'clockwork-blue' in marine and terrestrial vertebrates.

Exposure set-ups for in vivo experiments using radial waveguides.

Radial waveguide set-ups are introduced as exposure devices for long-term experiments with large numbers of non-restrained animals exposed simultaneously. Methods are presented to ensure well-defined exposure conditions even for potentially overmoded waveguides and for the exposure of large groups of animals per cage. The proposed methods are applied for a four-generation study being performed on up to 2500 mice exposed to a generic UMTS test signal at prescribed averaged whole body specific absorption rates (SARs). The variation of the whole body SAR due to the movement of the mice inside the cage is calculated by using the finite-difference time-domain method and detailed animal models for selected configurations of the mice inside the cage for all stages of the study.

Potentiation of antiproliferative effects of tamoxifen and ethanol on mouse hepatoma cells by melatonin: possible involvement of mitogen-activated protein kinase and induction of apoptosis.

Melatonin, the major secretory product of the pineal gland, is in focus of many research areas because of its ability to scavenge free oxygen radicals and thereby protect cells and tissues from radical damage. Some studies suggest melatonin may be a possible therapeutic agent with potential clinical applications against pathological states due to reactive oxygen species. Here, we investigated the effects of melatonin on the mouse hepatoma cell line HEPA 1-6, coincubated with ethanol, and tamoxifen, respectively. Cell proliferation rates were detected by the 3-[4,5 dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. A dose-dependent inhibition of the proliferative activity by melatonin was observed from 640 microM to 3 mM, which was significantly higher (P < 0.01) than with the solvent (ethanol) alone. Concentrations of 320 microM and less had no effect on cell proliferation. This antiproliferative effect might be because of the prolonged activation of mitogen-activated protein kinase which was activated by phosphorylation 15 min after the induction with melatonin. Furthermore, apoptosis was found to be enhanced by melatonin (75% more than with the solvent alone, P < 0.001). Finally, we show that the inhibitory effect of tamoxifen (25 microM) is markedly enhanced by the coincubation with melatonin (1.3 mM) up to 75% (P < 0.001). These data show that the antiproliferative effects of tamoxifen and ethanol, respectively, on mouse hepatoma cell line HEPA 1-6 are enhanced by melatonin. Although at the conditions described here the antiproliferative effects of melatonin occur at supraphysiological concentrations, these data may help to support clinical studies where melatonin is given simultaneously with tamoxifen or other standard chemotherapeutica.

Clearance of melatonin and 6-sulfatoxymelatonin by hemodialysis in patients with end-stage renal disease.

Patients with end-stage renal disease (ESRD) suffer from a number of related disorders. These include endocrine abnormalities, sleep disturbances, and depression. Melatonin is involved in the synchronization of exogenous zeitgebers with the endogenous rhythms, and it has effects on various psychological factors. As the concentrations of melatonin and the effects of dialysis have only occasionally been investigated in ESRD, we performed a study involving 35 patients, measuring the serum concentrations of melatonin, and of its major metabolite 6-sulfatoxymelatonin (aMT6s), before and after hemodialysis. Serum samples taken during morning hours from a control group (n=11) with intact kidneys served as controls. Patients were dialyzed for approximately 4 hr between 07:00 and 13:00 hr (S1), between 13:00 and 20:00 hr (S2), or between 18:30 and 22:30 hr (S3). Mean melatonin concentrations before hemodialysis were highly elevated when compared with the controls (40.6 vs. 6.7 pg/mL; P<0.001). Although melatonin levels were decreased to 20.3 pg/mL after dialysis, they were still well above the control levels. Likewise, aMT6s concentrations before dialysis were highly elevated in ESRD patients before dialysis when compared with controls (39.5 vs. 2.0 pg/mL; P<0.001), and also decreased by dialysis to levels still well above control levels (25.3 pg/mL). Clearance efficacy was better for melatonin (48.9%) than for aMT6s (36.6%; P<0.05). In ESRD patients, a diurnal rhythm for melatonin was observed (S1, 45.1 pg/mL; S2, 31.5 pg/mL; S3, 48.7 pg/mL; P<0.05), indicating that the normal synthesis rhythm is maintained. None of the following secondary disorders were correlated with melatonin concentrations: insomnia, delayed sleep onset, night-time arousals, and restless-leg syndrome. The reason for this observation is probably the melatonin concentrations, which were so high that no sub-classification could be identified. It is concluded that in ESRD patients, hemodialysis is unable to decrease elevated levels of melatonin and aMT6s to normal values. It is speculated that some of the secondary disorders in ESRD are caused by supraphysiological concentrations of melatonin.

Is inhibin B a pharmacodynamic parameter for FSH in normal men?

OBJECTIVE: This study aims to investigate the pharmacodynamic effect of FSH on inhibin B serum levels in normal men in order to elucidate the physiological regulation of inhibin B secretion in more detail. DESIGN AND METHODS: Injections of 3000 IU recombinant, human FSH (rhFSH) were followed by single-blinded injections of placebo, 1000 and 2000 IU rhFSH spaced by at least 28 days between injections. RESULTS: After injection of 3000 IU rhFSH, inhibin B values were significantly elevated above baseline for 24, 96 and 120 h (maximal increase after 96 h, mean +/- s.e.m. 303+/-18 pg/ml). Injection of 2000 IU rhFSH led to a significant increase in inhibin B (maximum mean +/- s.e.m. 318+/-20 pg/ml) from 24 to 120 h. Injection of 1000 IU rhFSH led to a significant increase in inhibin B after 96 h (maximum mean +/- s.e.m. 300+/-16 pg/ml). The inhibin B areas under the curve after injection of 2000 and 3000 IU rhFSH were significantly higher than those following the placebo and 1000 IU rhFSH. In the 12 fertile men investigated, at baseline a strong diurnal rhythm of inhibin B parallel to that of testosterone was observed. CONCLUSIONS: Serum inhibin B can be considered only a partial pharmacodynamic parameter of FSH in vivo, since the integrity of the spermatogenic process appears to be a second fundamental component in the regulation of its secretion from the testis.

Direct suppressive effects of weak magnetic fields (50 Hz and 16 2/3 Hz) on melatonin synthesis in the pineal gland of Djungarian hamsters (Phodopus sungorus).

In many investigations performed thus far on rodents, a suppression of melatonin synthesis was observed when animals were exposed to weak magnetic fields. However, among the several issues not yet resolved is the question of whether the observed changes are caused by direct effects on the pineal gland or by indirect effects, e.g., at the level of the eyes. We, therefore, performed a series of experiments in which direct effects of weak magnetic fields were studied in isolated pineal glands of Djungarian hamsters (Phodopus sungorus). After sacrifice of animals during morning hours, pineal glands were removed and placed individually into glass chambers that were perfused with oxygenated buffer. Experiments (n = 8) lasted for 8 hr. Magnetic fields (1,623 or 50 Hz at 86 microTesla) were generated by Helmholtz coils, and were present during the entire period. In each experiment, 12 exposed, and 12 sham-exposed, pineal glands were placed individually in glass chambers, which were surrounded by identical coils. Because of the design of the coils (bifilament), and the experimental setup (black box), the experimentator was not aware which coil was activated until the results were obtained. After 3.25 hr of adaptation, melatonin production was stimulated by isoproterenol (10(-7) M) for 30 min. Eluted fractions were collected, and melatonin concentrations were measured by radioimmunoassay. Maximum melatonin production was achieved after 5-7 hr. In all experiments, maximum melatonin concentrations were lower in the exposed groups compared with the sham-exposed controls. Statistical analyses for each frequency showed significant suppressive effects at 16(2/3) Hz (P < 0.01), and 50 Hz (P < 0.00). It is concluded that the suppressive effects of magnetic fields on the synthesis of melatonin are a result of primary mechanisms at the level of the pineal gland.

Estradiol and high-dose dihydrotestosterone treatment causes changes in cynomolgus monkey prostate volume and histology identical to those caused by testosterone alone.

To evaluate the effects of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2) on the regulation of prostate growth and tissue composition, the following study was conducted in a nonhuman primate model. Fifteen adult, long-term castrated cynomolgus monkeys were randomly assigned to receive implants filled with T (0.19 +/- 0.01 g), DHT alone (0.21 +/- 0.01 g), or (99%) DHT + (1%) E2 (0.21 +/- 0.01 g). Prior to and at 4-week intervals during the treatment phase of 252 days, prostate volumes (PV), body weight, ejaculate weight, hormone levels (of T, DHT, and E2), and red blood cell count were measured. Five adult, intact monkeys served as controls for prostate volume and histology. At the end of the study, histological analysis of an ultrasound-guided prostate biopsy was performed. T levels increased significantly in the T group compared with baseline (P < 0.01) and with the DHT and DHT + E2 groups (P < 0.05). Both groups receiving DHT showed higher DHT levels than did animals in the T group (P < 0.001). E2 levels in all groups increased over time (P < 0.05), although significant differences (P < 0.01) could only be detected between the DHT + E2 and the DHT group. Prostate volume in all groups increased (at baseline: T = 1.03 +/- 0.12 ml, DHT = 1.08 +/- 0.15 ml, DHT + E2 = 1.13 +/- 0.09; at day 252: T = 5.83 +/- 1.00, DHT = 4.72 +/- 0.9, DHT + E2 = 5.05 +/- 0.62) over time (P < 0.001), whereas no differences could be detected between the groups. Prostate biopsy could be performed successfully in 15 out of 20 monkeys. Prostate tissue evaluation between the treatment groups and the evaluated intact monkeys revealed no differences in the status of secretory epithelia, nuclear chromatin, excretory vacuoles, interstitial stroma, smooth muscles, and total functional status, whereas the prostate of a long-term castrated monkey showed severe atrophy. Thus, both androgens fully restored prostate volume and ejaculatory function. Highly supraphysiological DHT serum levels are not associated with abnormal volumetric or histological changes of the prostate. Comparing the DHT group with the DHT + E2 group, an additional stimulatory effect of normal or slightly elevated estrogens on the prostate cannot be found in the presence of highly supraphysiological DHT levels.

Sex ratios at birth and environmental temperatures.

The relationship between average monthly air temperature and sex ratios at birth (SRB) was analyzed for children born in Germany during the period 1946-1995. Both the absolute temperature and--more markedly--the monthly temperature deviations from the overall mean were significantly positively correlated with the SRB (P < 0.01) when temperatures were time-lagged against the SRB data by -10 or -11 months. It is concluded that the sex of the offspring is partially determined by environmental temperatures prior to conception.

Isolation method of Leydig cells from mature male Djungarian hamsters (Phodopus sungorus) and their steroidogenic activity in vitro.

For studies addressing the functions of Leydig cells, isolated cells are often better suited than intact animals. Here, the isolation procedure of Leydig cells from adult male Djungarian hamsters (Phodopus sungorus) is described. Cells were isolated using a procedure involving enzymatic dissociation and Percoll-gradient centrifugation. For each experiment, approximately 4.4 x 10(6) Leydig cells from six animals were obtained. The cells showed high steroidogenic responsiveness to physiological (ovine luteinizing hormone (oLH) and human chorionic gonadotropin (hCG)) and nonphysiological (forskolin) stimuli in vitro. Approximately 98% of cells were viable as assessed by trypan blue exclusion, and the purity varied from 80 to 95% as tested by 3 beta-hydroxysteroid dehydrogenase activity. Leydig cells were also identified by a bright yellow halo under phase-contrast microscopy. They contained numerous lipid droplets and showed round nuclei and prominent nucleoli. The cells responded to oLH, hCG and forskolin with an increased testosterone production in a dose-dependent manner. Dose-response curves in these studies suggest that Leydig cells of Djungarian hamsters undergo desensitization, probably due to down regulation of their LH/CG receptors.

Melatonin in epilepsy: first results of replacement therapy and first clinical results.

At a single evening dose of 5-10 mg, melatonin (MLT), the pineal gland hormone, can exert a positive effect on the frequency of epileptic attacks in children with sleep disturbances of various etiologies. We have shown that the sleep behavior can be normalized and an existing epilepsy can be favorably influenced. Pretherapeutic MLT secretion profiles can provide new information concerning the origin and treatment of these disturbances. In vitro experiments suggest that this effect might be the result of the interaction between MLT and MLT-specific receptors in the neocortex. Due to its favorable safety profile, MLT can be liberally administered in the specified doses and be considered as a useful antiepileptic drug.

The effects of pulsing magnetic fields on pineal melatonin synthesis in a teleost fish (brook trout, Salvelinus fontinalis).

Based on findings in various mammalian species, where exposure to electromagnetic fields decreased the nocturnal synthesis of the pineal secretory product melatonin, we investigated the effects of magnetic field (MF) exposure in a teleost fish, the brook trout (Salvelinus fontinalis). Fields were generated by Helmholtz coils (maximum flux density 40 microT, frequency 1 Hz, 200 ms on, 800 ms off). Melatonin concentrations were estimated by a specific radioimmunoassay. MF exposure significantly increased night-time pineal (P < 0.001) and serum (P < 0.01) melatonin levels, as compared with the controls. It is suggested that either the pineal glands are directly affected, i.e. by an increased Ca2+-influx into pineal photoreceptors, or that the responses are indirect since induced currents, caused by the rapid rise and decay of the generated MF, may have disturbed the sensory system for electric fields.

Exposure of female rats to a 100-microT 50 Hz magnetic field does not induce consistent changes in nocturnal levels of melatonin.

The hypothesis whereby alternating (50 or 60 Hz) magnetic fields such as those produced by electric power reduce the nocturnal production of melatonin in the pineal gland and thereby indirectly enhance development and growth of breast cancer has attracted a great deal of interest. In view of the potential importance of this hypothesis that there is a link between electric power and breast cancer, which is also known as the "melatonin hypothesis", we undertook various experiments in female Sprague-Dawley rats to evaluate whether 100-microT 50 Hz magnetic-field exposure, i.e. a flux density shown recently to exert a tumor (co)promoting effect in the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in Sprague-Dawley rats, consistently reduces melatonin levels and, if not, which factors may be involved in the inconsistent effects of magnetic-field exposure on production of melatonin. Long-term exposure of female Sprague-Dawley rats to magnetic fields for 13 weeks did not alter the nocturnal levels of melatonin in the pineal gland or serum (determined 5 h after the onset of darkness) significantly, irrespective of whether rats were treated with DMBA or not. In one experiment, when blood was sampled 3, 5 and 6 h after the onset of darkness after 2 weeks of magnetic-field or sham exposure, a significant decrease in melatonin was seen in magnetic-field-exposed rats at 6 h. However, the results could not be reproduced in two subsequent experiments in other groups of rats. Shorter (1 day, 1 week) or longer (4, 8, 13 weeks) exposure periods also did not result in any significant effects of the magnetic field on melatonin levels when blood sampling was performed either 5 or 6 h after onset of the dark phase. Various potential sources of variation in melatonin levels or in magnetic-field effects on melatonin levels were evaluated, but the reason(s) for the inconsistent effect of magnetic-field exposure remains unclear. Thus the present study failed to demonstrate a consistent effect of 100-microT 50 Hz magnetic-field exposure on melatonin levels in Sprague-Dawley rats.

Urinary 6-sulfatoxymelatonin profiles in male Djungarian hamsters (Phodopus sungorus) responding and not responding to short-day photoperiods: possible role of elevated daytime levels.

The lack of endocrine and physiological responses of some Djungarian hamsters (Phodopus sungorus) to the transition from long to short photoperiods (L:D 16:8 --> L:D 8:16) has been known for a long time but is not yet understood. We investigated the role of melatonin synthesis in this context because melatonin, as part of the circadian system, may play a role in non-responsiveness. In ten responding and ten non-responding male hamsters, the urinary 24 hr 6-sulfatoxymelatonin (aMT6s) profiles under L:D 8:16 and L:D 16:8 were measured. Both short day responding and non-responding hamsters showed diurnal aMT6s excretion rhythms. Whereas responders reacted to the transition L:D 16:8 --> L:D 8:16 with a marked elevation of aMT6s excretion, in non-responders no adjustment of the melatonin rhythm to the change of the photoperiod was seen. Furthermore, under L:D 16:8 the daytime levels of aMT6s were significantly (P<0.001) lower in responders compared to non-responders whereas under L:D 8:16 these levels were higher (P<0.01). It is speculated that high daytime levels of aMT6s under long-day photoperiods in non-responders result in down-regulation of melatonin receptors of the nucleus suprachiasmaticus, the pacemaker for the pineal gland, leading to a lack of response to the transition to short-day photoperiods.

Seasonality of sex ratio in Germany.

It was investigated whether there is a seasonal variation in sex ratio at birth in Germany. The analysis was based on records from the German Bureau for Statistics, covering the period from 1946 to 1995. A highly significant (P < or = 0.001), albeit low-amplitude rhythm was found with two peaks in May and December, and two nadirs in March and October. No correlations were found between sex ratios and seasonal birth rates during this period.

Acceleration of mammary tumorigenesis by exposure of 7,12-dimethylbenz[a]anthracene-treated female rats in a 50-Hz, 100-microT magnetic field: replication study.

In view of the methodological problems of epidemiological studies on associations between residential and occupational exposures to 50/60-Hz magnetic fields (MF) and increased incidence of cancers, laboratory studies are necessary to determine if 50/60-Hz MF can affect cancer development or growth. Recently, it was reported that alternating (50-Hz) MF of low flux density (100 microT) increase tumor growth and progression in a model of breast cancer in female rats in which mammary tumors were induced by the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The objective of the present study was to determine if a replicate experiment carried out in the same laboratory under the same experimental conditions yields a significant increase in tumor development and growth of similar magnitude. For the MF experiment, a group of 99 female Sprague-Dawley rats was exposed to a homogeneous horizontally polarized MF for 24 h/d (minus time for weighing, tumor palpation, cage cleaning, cage rotation), 7 d/wk; another group of 99 rats was sham exposed. DMBA was administered intragastrically at a dose of 5 mg/rat at the first day of exposure and at weekly intervals thereafter up to a total dose of 20 mg/rat. Duration of MF or sham exposure was 91 d. In both MF-exposed and sham-exposed rats, the first tumors could be recorded 6 wk after the initial DMBA application. At 9 wk after DMBA application, the group of MF-exposed rats exhibited significantly more animals with tumors than the sham-exposed group. This significant difference in the rate of tumor development was observed throughout the subsequent period of exposure. After autopsy, the incidence of macroscopically visible mammary tumors was 62% in controls, but 83% in MF-exposed rats, with the 35% difference between groups being statistically significant. Data substantiate that long-term exposure of DMBA-treated female Sprague-Dawley rats in an alternating MF of low flux density promotes the development and growth of mammary tumors, thus indicating that MF exposure exerts tumor-promoting and/or copromoting effects. Furthermore, the data show that the effects of MF exposure in the DMBA breast cancer model are reproducible if the same experiment is repeated in the same laboratory.