Feasibility of repeated testing for learning ability in juvenile primates for pediatric safety assessment.

Assessment of learning ability in nonhuman primate (NHP) models is sometimes requested by regulatory authorities. The double choice object discrimination task using a Wisconsin General Testing Apparatus (WGTA) approach is typically being applied. In this study, the WGTA approach was performed on 66 juvenile cynomolgus monkeys aged 8-9 months in the predose phase of juvenile toxicity assessment. In addition, reversal learning data of seven control animals/gender were obtained for the weeks 25 and 52 of dosing. Gender differences in the number of days required to pass the habituation, learning or reversal learning phases were statistically comparable, males and females may be combined for statistical analysis. At first instance, the habituation phase was passed on average after 6.4 days, and the learning test on average after 8.6 days with improvement to 2.0-2.6 days for habituation and 6.4-6.7 days for learning in weeks 52. Power analysis (α = 0.05, one-sided t-test) revealed a sample size of 8 and 41 to predict a 50% and 20% difference, respectively. In conclusion, examination for learning ability, but not for memory ability (during repeated testing) is feasible in juvenile NHPs using the WGTA approach.

Sensitivity of male reproductive endpoints in nonhuman primate toxicity studies: a statistical power analysis.

To determine the sensitivity of male reproductive toxicity endpoints in NHPs we performed a power analysis of routine and triggered endpoints using control data from sexually mature Asian and Mauritian NHPs. The power to detect a 50% change from control was 13-30% for male reproductive organ weights, ∼30% for testicular volume, 6-66% for seminal analyses and 10-78% for male hormones. Overall, male reproductive endpoints have poor power (less than 80%) to detect a 50% change from control with a group size of 3 monkeys. Confidently identifying adverse male reproductive effects with these endpoints would likely require specialized study designs with larger group sizes. Triggering of non-routine endpoints in cases where there is special concern for male reproductive toxicity is unlikely to increase sensitivity to detect adverse effects.

Functional assessment of sexual maturity in male macaques (Macaca fascicularis).

Selection of suitable criteria for assessing sexual maturity in the male long-tailed macaque (Macaca fascicularis) has yielded conflicting results. The present retrospective work investigates whether the sole presence of sperm in the baseline semen sample unequivocally (i.e. for every animal) hallmarks complete testicular maturation. For 956 animals providing the baseline semen sample, neither age, body weight nor testes volume unequivocally predicted the presence of sperm in that sample, and for 322 animals these parameters failed to predict testicular histology. In contrast, the presence of sperm in the baseline semen sample correlated with mature testis histology at study termination in every single animal (n=197/322). Surprisingly, for the 125/322 animals without sperm in the baseline semen sample, spermatogenesis was also mature in 95 animals. Thus, the mere provision of a semen sample without sperm--implying peripheral reproductive tract maturation--was associated with mature spermatogenesis in approx. 75% of animals. Interestingly, testicular maturation occurred approx. 2 years earlier in Mauritian compared to Asian mainland animals. In conclusion, a single semen sample that contains sperm provides unequivocal evidence for mature spermatogenesis and, thus, is suggested as a functional parameter for sexual maturity assessment in this species.

Quantified tracking and monitoring of diazepam treated socially housed cynomolgus monkeys.

Quantitative assessment of behavioural patterns is frequently used in rodent toxicity studies, however only limited approaches are available for monkeys. Often qualitative behavioural scoring using functional observation batteries (FOBs) is performed, with difficulties like poor reproducibility or lack of sensitivity. In this study, we investigated whether quantitative behavioural monitoring can be applied to group-housed cynomolgus monkeys. Video-tracking EthoVision® XT system and special analysis software were used to evaluate diazepam (i.v. 1mg/kg) related behavioural changes in group-housed animals. Recordings were made predose and at the anticipated time of maximum drug exposure (T(max)). General parameters such as distance travelled and velocity did not reveal the known sedative effects of diazepam. However, inspection of the automatically generated track images indicated that diazepam-treated animals had more a meandering movement pattern suggesting that diazepam induced a loss of balance which was regained by corrective movements. Therefore, parameters revealing specific aspects of the meandering movement pattern such as velocity profiles and turn angles have been analyzed and revealed an increase in the curvature and in the number of directional changes of the movement path.

High Definition Oscillometry: a novel technique for non-invasive blood pressure monitoring in the cynomolgus monkey (Macaca fascicularis).

BACKGROUND: Current approaches for accurate blood pressure determination rely predominantly on invasive techniques. High Definition Oscillometry (HDO) was evaluated as a potential non-invasive approach for accurate blood pressure recordings in cynomolgus monkeys. METHODS: In conscious animals, systolic, diastolic, mean arterial blood pressure (MAP) and pulse/minute were determined 15 times within approx. 9 minutes per individual. This session was performed during 3 consecutive days. Anaesthesia induced hypotension was controlled simultaneously with HDO and telemetry as reference. RESULTS: Repeated measurements were highly reproducible. After procedural habituation, mean MAP was 96.2 +/- 13.7 mmHg in males and 86.9 +/- 4.3 mmHg in females. Mean intraindividual coefficients of variation ranged between 10.8% and 2.4% depending on the session and parameter. Values determined by HDO corresponded to those reported for invasive techniques. CONCLUSION: Our results demonstrate, using telemetry as reference, the accuracy of HDO-based non-invasive blood pressure measurements in macaques to detect drug-related cardiovascular changes.

Gonadal protection from radiation by GnRH antagonist or recombinant human FSH: a controlled trial in a male nonhuman primate (Macaca fascicularis).

Chemotherapy and radiation often damage spermatogenesis irreversibly in oncological patients and various approaches to gonadal protection have been tested with equivocal results. In rats, hormonal protection of spermatogenesis can be achieved by blocking gonadotropin secretion. However, whether the same mechanisms can effect gonadal protection in primates remains questionable. To clarify this Issue we conducted a placebo-controlled trial in a preclinical animal model using macaques (Macaca fascicularis). Twenty adult male monkeys (five in each group) were randomized to receive either recombinant human FSH, GnRH antagonist or saline injections (two groups) for 36 days. On day 29 all groups except one saline-treated control group were exposed to a single testicular irradiation of 4 Gy. Every 2 weeks before, during and after the treatment, ejaculates, body weight, testicular Volume and hormones were analyzed until day 539. In addition, repeated testicular biopsies were performed. Testicular Volume and inhibin B decreased significantly in all irradiated groups compared with baseline and with the non-irradiated control group, followed by a gradual recovery of these parameters, which was, especially at the earlier time points, significantly better in the FSH-treated group compared with both other irradiated groups. Irradiation caused a drastic decrease of sperm parameters in all groups, followed by a partial recovery of sperm parameters, which was significantly slower in the early phases of recovery in the GnRH antagonist group compared with the vehicle group. Testicular histology showed a significant depletion on study day 261 in all irradiated animals. In conclusion, in clear contrast to rodent studies, GnRH antagonist treatment did not provide gonadal protection in this primate model. FSH treatment resulted in slightly better recovery of spermatogenesis, which appears to be of no or only little clinical relevance.

Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey mouse lemur, Microcebus murinus.

Germ cell production and organization of the testicular epithelium in a prosimian species, the grey mouse lemur, Microcebus murinus, was investigated to extend knowledge of comparative primate spermatogenesis. In addition, semen samples collected from adult male lemurs (body weight 53-92 g; n = 16) by rectal probe electroejaculation were evaluated using computer-assisted morphometric and kinematic analysis of spermatozoa. Epididymidal spermatozoa were collected from six animals after hemicastration; the testes were weighed and prepared for stereological analysis and flow cytometry. The relative testis mass (as a percentage of body weight) ranged between 1.17 and 5.6%. Twelve stages of testicular seminiferous epithelium as described for macaques were applied and only a single stage was observed in most of the seminiferous tubule cross-sections. On average (mean SD), a single testis contained 1870 +/- 829 x 10(6) germ cells and 35 +/- 12 x 10(6) Sertoli cells. Germ cell ratios (preleptotene:type B spermatogonia = 2, round spermatid:pachytene = 3; elongated spermatid:round spermatids = 1) indicated high spermatogenic efficacy. Sperm head dimensions and tail lengths of the ejaculated and epididymidal spermatozoa were similar. Percentages of defects (neck/mid-piece and tail) were low ( 10%) and similar for ejaculated and epididymidal spermatozoa. Spermatozoa were highly motile, characterized by extensive lateral head displacement, but relatively low progressive motility. In conclusion, the grey mouse lemur has unusually large testes with a highly efficient spermatogenic process and large sperm output. These features, together with the high proportion of morphologically normal and highly motile spermatozoa in the ejaculates, indicate that Microcebus murinus is a species in which sperm competition after ejaculation is likely to occur. The predominantly single spermatogenic stage system seems to be an ancestral feature among primates.

Germ cell transplantation into X-irradiated monkey testes.

BACKGROUND: An intense debate is ongoing regarding options for fertility protection in oncological patients. Germ cell transplantation has been applied to restore mouse spermatogenesis. Here, an attempt to apply autologous germ cell transplantation to a primate animal model is described. METHODS: Five adult male cynomolgus monkeys were biopsied to retrieve and cryopreserve germ cells before both testes were irradiated (dose 2 Gy). Six weeks later, each monkey received an infusion of its own cell suspension into the right testis, while the left testes were infused with saline. Testis size, sperm counts and serum concentrations of inhibin, FSH and testosterone were analysed weekly for 9 months. Spermatogenic recovery was determined histologically at the end of the study. RESULTS: In four monkeys, the germ cell-infused right testes showed a slight to moderate increase in the rate of regrowth in comparison with the left testes. In two monkeys the right testis proceeded to recover more prominently, resulting in larger right testis volumes and better or full spermatogenic recovery at the study end. Restoration of spermatogenesis occurred as an all-or-nothing event. Inhibin B concentrations increased, while FSH and testosterone concentrations decreased with testicular regrowth. Sperm counts did not recover. CONCLUSIONS: The present study demonstrates the immaturity and complexity of germ cell transplantation as a clinical approach.

cAMP response element modulator (CREM): an essential factor for spermatogenesis in primates?

CREM is a cAMP-related transcription factor and alternate promotor usage and splicing generate repressor and activator transcripts of CREM within the testis. CREM activators are highly expressed in post-meiotic haploid germ cells and are essential for spermatid maturation in the mouse model as revealed by gene-targeting studies. Analysis of testicular CREM expression in rodent and monkey species, and in men yielded a highly comparable pattern thus suggesting that CREM is of general importance for spermatid development in the mammalian testis. Also, many CREM target genes have been identified in haploid germ cells. Studies in men with spermatogenic disturbance and spermatid maturation arrest demonstrated abnormal CREM expression and altered splicing events. Collectively, the data strongly argue for an essential role of CREM during spermatid maturation in primates.

Atherosclerosis in apolipoprotein E-deficient mice is decreased by the suppression of endogenous sex hormones.

To elucidate the influence of gonadotropins, endogenous sex hormones and testosterone on atherosclerosis, 4-week-old male and female apoE-deficient mice received either 100 microg subcutaneous injections of the gonadotropin-releasing hormone (GnRH) antagonist Cetrorelix every 48 hours or a subcutaneous implantation of a permeable silastic tube with 35 mg of testosterone. Control mice received either subcutaneous injections of saline, a silastic implant with saline, or no treatment. The animals were sacrificed after eight weeks of treatment; blood was obtained by cardiac puncture and the aorta was taken out and prepared. The suppression of testosterone led to an increase in atherosclerosis in both the sinus aortae and the ascending aorta despite increases of cholesterol in male and decreases of HDL cholesterol in female mice. Treatment with testosterone led to small but significant increases of cholesterol levels and atherosclerotic lesions in male mice. Female mice showed no change in lipids and fewer atherosclerotic lesions. In conclusion, the suppression of gonadotropins appears to have a moderate anti-atherogenic effect. The effect of testosterone appears to be either neutral or opposed by gonadotropins.

Quantitative analysis of spermatogenesis and apoptosis in the common marmoset (Callithrix jacchus) reveals high rates of spermatogonial turnover and high spermatogenic efficiency.

Spermatogenesis is characterized by the succession in time and space of specific germ cell associations (stages). There can be a single stage (e.g., rodents and some macaques) or more than one stage (e.g., chimpanzee and human) per tubular cross section. We analyzed the organization of the seminiferous epithelium and quantified testicular germ cell production and apoptosis in a New World primate, the common marmoset (Callithrix jacchus). Tubule cross sections contained more than one stage, and the human six-stage system could be applied to marmoset spermatogenesis. Stereological (optical disector) analysis (n = 5) revealed high spermatogenic efficiency during meiosis and no loss of spermatids during spermiogenesis. The conversion of type A to type B spermatogonia was several-fold higher than that reported for other primates. Highest apoptotic rates were found for S-phase cells (20%) and 4C cells (15%) by flow cytometric analysis (n = 6 animals); histological analysis confirmed spermatogonial apoptosis. Haploid germ cell apoptosis was <2%. Marmoset spermatogenesis is very efficient and involves substantial spermatogonial proliferation. The prime determinants of germ cell production in primates appear to be proliferation and survival of spermatogonia rather than the efficiency of meiotic divisions. Based on the organizational similarities, common marmosets could provide a new animal model for experimental studies of human spermatogenesis.

Testosterone-induced inhibition of spermatogenesis is more closely related to suppression of FSH than to testicular androgen levels in the cynomolgus monkey model (Macaca fascicularis).

We have investigated the antigonadotropic and antispermatogenic effects of exposure to a long-acting testosterone ester in the cynomolgus monkey model. Groups of five adult animals were exposed either to vehicle or to 10 mg/kg or 20 mg/kg testosterone buciclate (TB) over a 26-week period with injections given in weeks 0, 11 and 18. In week 26, testicular biopsy tissue was collected. Serum testosterone levels were in the upper normal range with 10 mg/kg TB and were approximately twofold higher with 20 mg/kg TB. The estradiol pattern followed that of testosterone and body weights increased in a testosterone-dependent manner. TB completely abolished serum LH bioactivity. Serum concentrations of FSH and inhibin-alpha were suppressed in a TB dose-dependent manner. During weeks 4-8 after the first injection, a rebound of FSH and inhibin but not bioactive LH secretion occurred. This rebound was followed immediately by a restimulation of testis size and sperm numbers. After the next TB injections these parameters were once again suppressed. Nadir testis size was 30-40% of baseline and animals were severely oligozoospermic or transiently azoospermic. Consistent azoospermia was not achieved. Quantitation of serum inhibin B, proliferating cell-nuclear antigen staining and flow cytometric analysis of germ cell populations revealed pronounced suppression of spermatogenesis in both TB-treated groups whereas androgen receptor expression remained unchanged. Testicular androgens levels, determined in week 26, did not differ among all three groups and did not correlate with sperm numbers, histological and immunocytochemical findings. All suppressive effects were fully reversed during the recovery period. We have concluded that pronounced suppression of primate spermatogenesis seemingly requires inhibition of FSH rather than testicular androgen levels, at least in this preclinical non-human primate model. For the purpose of male contraception, FSH inhibition appears mandatory.

CREM activator and repressor isoforms in human testis: sequence variations and inaccurate splicing during impaired spermatogenesis.

cAMP responsive element modulator (CREM) activators are specifically expressed in haploid germ cells prior to cell elongation and are essential for spermatid development in mice. Recent studies indicate that CREM activators are also involved in the process of spermatid maturation in men. Unlike the activators, CREM repressors were suggested to be absent from adult mouse and dog testes. The present work investigates CREM transcripts in human testis with normal (n = 4) and impaired spermatogenesis (n = 2). Two activator transcripts could be identified corresponding to the tau2 isoform with and without exon gamma. Interestingly, four CREM repressors could be isolated from specimens with complete spermatogenesis. These were gamma-repressor (exons B, E, F, H, I(b)), CREM DeltaC-F, beta (exons B, G, H, I(b)), CREM DeltaC-G, beta (exons B, H, I(b)), and CREM DeltaC-G, alpha (exons B, H, I(a), I(b)). These isoforms were also present in cynomolgus monkey testes. A novel CREM splice variant (CREM DeltaC-H) was detected in a specimen with spermatid maturation defect. Beyond that, inaccurate CREM splicing, giving rise to inactive transcripts, was encountered in a specimen with impaired spermatogenesis. In conclusion, several CREM repressor transcripts are present in adult human testes, and altered transcript splicing is associated with impaired spermatogenesis.

Marmoset spermatogenesis: organizational similarities to the human.

The common marmoset (Callithrix jacchus), a small New World primate of high fecundity, is widely used in reproductive research. The aim of the present study was to determine the organization of the germ cells within the seminiferous epithelium, the duration of the spermatogenic cycle and the number of spermatogonial mitoses. Antibodies to cAMP response element modulator (CREM) and proliferating nuclear cell antigen (PCNA) and a cRNA directed against protamine P2 and morphological criteria were used to discriminate between stages of the spermatogenic cycle. Plastic sections were used to document the cell associations present in each of the nine stages of spermatogenesis. Up to five such stages could be observed within individual cross-sections of seminiferous tubules. Based on the pattern of incorporation of bromodeoxyuridine the length of the spermatogenic cycle was estimated to be 10 days and the duration of spermatogenesis to be 37 days. Four mitotic divisions were noted in spermatogonia. It is concluded that the organization of spermatogenesis in the marmoset has similarities to the human ('helical') and this makes the marmoset a suitable model for studies relevant to human testicular function.

Structure and expression of the mouse gene encoding the endozepine-like peptide from haploid male germ cells.

The endozepine-like peptide (ELP) represents a testis-specific isoform of the ubiquitous acyl-CoA binding protein (ACBP) and is highly expressed in late haploid stages of male germ cell development. The genomic sequence of the functional ELP gene as well as that of a pseudogene were analysed from independent bacteriophage clones of a 129sv mouse genomic library. Unlike the ACBP gene, which comprises four exons, the ELP gene has only a single intron within the region of the 5' untranslated region, suggesting that, like some other haploid expressed genes, the ELP gene might have evolved by retroposon-mediated gene duplication. Primer extension analysis was used to define the start site for transcription and hence the 5' promoter region. Electrophoretic mobility shift analysis was carried out on this region comparing nuclear extracts from adult mouse testis with those from mouse liver. Several testis-specific DNA-protein complexes were observed throughout 700 bp upstream of the transcription start site. One of these could be identified as corresponding to a steroidogenic factor-1 (SF-1) binding element. Further analysis using pure transcription factors showed that this element at position -340 was able to bind specifically to both SF-1 and to the germ cell nuclear factor (GCNF). Immunohistochemical analysis using an ELP-specific antibody showed that expression was very restricted within the testis to the postmeiotic germ cells, and in the ovary to interstitial/luteal cells, cell-types known to express GCNF and SF-1, respectively. Testes of CREM-tau knockout mice, lacking all spermatogenic stages later than round spermatids, were devoid of ELP immunoreactivity, whereas in RAD6 knockout mice the few remaining elongated spermatids were clearly defined by this excellent late haploid marker product. The ELP gene and its product thus offer an ideal system with which to investigate the differentiation of late haploid stages of spermatogenesis.

The rat endozepine-like peptide gene is highly expressed in late haploid stages of male germ cell development.

The structure of the endozepine-like peptide (ELP) gene is closely related to the intracellular acyl-CoA binding protein (ACBP), but unlike the generalized distribution of the latter, it is restricted to the male germ cells of the testis. In the present study, a combination of nonradioactive in situ mRNA hybridization and immunohistochemistry was used to precisely determine the cellular expression patterns of ELP mRNA and protein in control and methoxyacetic acid (MAA)-treated rat testes. ELP transcripts are first detectable in late stages (step 6) of round spermatids, with transcription increasing through late-elongating steps. Translation of the ELP mRNA is delayed, with first immunohistochemical staining occurring in elongated spermatids at step 16, and protein accumulating through step 19. ELP immunoreactivity proves to be an excellent marker for late spermatid stages and highlights the presumably clonal recovery of spermatids following MAA treatment.

Testosterone modulates growth hormone secretion at the hypothalamic but not at the hypophyseal level in the adult male rhesus monkey.

We investigated a possible modulation of growth hormone (GH) secretion by testosterone by measuring the growth hormone releasing hormone (GHRH)-stimulated and N-methyl-d,l-aspartic acid (NMA)-induced GH secretion in adult rhesus monkeys. Intact, orchidectomized and testosterone-substituted (testosterone enanthate 125 mg/week, i.m. for 5 weeks) orchidectomized monkeys (n=5) were used in the study. GHRH (25 microg/kg body weight) or NMA (15 mg/kg body weight) was infused through a Teflon cannula implanted in the saphenous vein. Sequential blood samples were collected 30-60 min before and 60 min after the injection of the neurohormone or the drug at 10-20-min intervals. All bleedings were carried out under ketamine hydrochloride anaesthesia (initial dose 5 mg/kg body weight i.m., followed by 2.5 mg/kg at 30-min intervals). The plasma concentrations of GH, testosterone and oestradiol (E(2)) were determined by using specific assay systems. Administration of GHRH elicited a significant increase in GH secretion in all three groups of animals. There was no significant difference in the responsiveness of pituitary somatotrophs to exogenous GHRH challenges between intact and orchidectomized monkeys and testosterone replacement in orchidectomized animals did not significantly alter the GHRH-induced GH response. The responsiveness of hypothalamic GHRH neurones apparently did undergo a qualitative change after orchidectomy, as GH response to NMA was less in orchidectomized animals than in intact monkeys. The responsiveness of GHRH neurones to exogenous NMA was restored and even potentiated when orchidectomized monkeys were treated with testosterone. Taken together, these findings suggest that testosterone does not affect the sensitivity of the pituitary somatotrophs to GHRH but stimulates the secretion of GH by modulation of the NMDA drive to GHRH neurones.

Cloning and expression analysis of testis-specific cyclic 3', 5'-adenosine monophosphate-responsive element modulator activators in the nonhuman primate (Macaca fascicularis): comparison with other primate and rodent species.

The cAMP-responsive element modulator (CREM) gene encodes a transcription factor that is essential for spermatogenesis. In mouse testis, several CREM repressors and activators have been identified. In contrast to the situation for the mouse, however, little is known about CREM isoforms in the primate testis. We analyzed CREM isoforms and mRNA expression in a clinically relevant primate model, the cynomolgus monkey (Macaca fascicularis). A cDNA library was generated from monkey testis; and two activator isoforms (tau2 with and without exon gamma) were identified, which displayed high sequence identity to mouse and human isoforms. The insertion of exon gamma was observed for the first time in the primate testis. CREM activator expression was confined to the testis, where it was seen in late pachytene spermatocytes and round spermatids in specific spermatogenic stages, as revealed by in situ hybridization. Comparison of the mRNA and the recently described protein expression indicated a lack of translational delay of CREM expression. Comparative analysis of testicular CREM expression by reverse transcription-polymerase chain reaction yielded several transcripts in the rat, mouse, hamster, and marmoset; two transcripts in cynomolgus and rhesus monkeys; and one transcript in men. These findings suggest an evolutionary trend from multiple activator isoforms to a single activator transcript in men.

Dazl protein expression in adult rat testis is up-regulated at meiosis and not hormonally regulated.

The Y-chromosomal DAZ (deleted in azoospermia) gene and the autosomal Dazl (deleted in azoospermia-like) gene are two crucial factors for the achievement and maintenance of spermatogenesis. Whereas Y-chromosomal DAZ is present in certain primates, it is lacking in rodents and other species. We have investigated the expression of Dazl protein during spermatogenesis in the adult rat testis using immunohistochemistry. Dazl immunoreactivity was found predominantly in the cytosol of primary pachytene spermatocytes. A weaker but clearly detectable signal was present in intermediate and B spermatogonia and in early spermatocytes from preleptotene to zygotene. The highest expression patterns were observed between stages IV and VIII during the spermatogenic cycle when spermatocytes prepare for the first meiotic division. Specific staining could also be observed in step 11-19 elongating spermatids in the acrosome region. Treatment for 42 days with a potent GnRH-antagonist abolished gonadotrophin secretion and led to a regressed testis, lacking most of the advanced germ cell types such as spermatids but still bearing spermatogonia and spermatocytes. No difference in staining pattern for Dazl protein was observed in GnRH antagonist-treated rats despite the lack of gonadotrophins and substantial impairment of the spermatogenic process, indicating that Dazl expression is clearly hormone-independent. The localization and level of Dazl expression suggests an important role in the regulation of the first meiotic stages of spermatogenesis. The hormone independent onset of expression points to an autonomous cell-cycle event in which Dazl seems to be essential for the entry into meiosis. The presence of Dazl in the acrosome region of elongating spermatids might reflect an unknown role of Dazl as a morphogenetic factor during spermiogenesis.