Deep Learning-Based Spermatogenic Staging in Tissue Sections of Cynomolgus Macaque Testes.

The indirect assessment of adverse effects on fertility in cynomolgus monkeys requires that tissue sections of the testis be microscopically evaluated with awareness of the stage of spermatogenesis that a particular cross-section of a seminiferous tubule is in. This difficult and subjective task could very much benefit from automation. Using digital whole slide images (WSIs) from tissue sections of testis, we have developed a deep learning model that can annotate the stage of each tubule with high sensitivity, precision, and accuracy. The model was validated on six WSI using a six-stage spermatogenic classification system. Whole slide images contained an average number of 4938 seminiferous tubule cross-sections. On average, 78% of these tubules were staged with 29% in stage I-IV, 12% in stage V-VI, 4% in stage VII, 19% in stage VIII-IX, 18% in stage X-XI, and 17% in stage XII. The deep learning model supports pathologists in conducting a stage-aware evaluation of the testis. It also allows derivation of a stage-frequency map. The diagnostic value of this stage-frequency map is still unclear, as further data on its variability and relevance need to be generated for testes with spermatogenic disturbances.

Intravitreal RTH258 (brolucizumab) demonstrates no effect on pregnancy, parturition, embryofetal or postnatal development in cynomolgus monkeys.

RTH258 (brolucizumab) is a humanized single chain antibody fragment, the smallest functional unit of an antibody designed to target vascular endothelial growth factor in angiogenic retinal disease. To further understand the safe use of RTH258, this study assessed the potential impact of intravitreal RTH258 on pre- and postnatal development in the offspring of cynomolgus monkeys following administration to the mother. Three groups of 16 pregnant females were included: a low dose group (RTH258 3 mg/50 µl [60 mg/ml]), a high dose group (RTH258 6 mg/50 µl [120 mg/ml]), and a control group. Maternal animals were administered a single injection of 50 µl in the right eye once every four weeks. Animals were observed daily and detailed observations were collected before and after the first dose, and then weekly thereafter. Following parturition, observations of infants included external, morphological, and ophthalmic examinations; neurobehavioral test battery; grip strength; and skeletal development. Blood samples for hematology, coagulation, and clinical chemistry were collected from non-fasted maternal and infant animals. No RTH258-related deaths occurred in maternal dams or infants. No RTH258-related clinical observations were noted in maternal animals or in surviving infants - there were no changes in gestation length; pregnancy loss; deaths; body weight/weight change; infant grip strength; infant external, morphological, or skeletal evaluations; ophthalmoscopy or neurobehavioral observations; or clinical pathology parameters. RTH258 had no impact on pregnancy or parturition; embryo-fetal development; or survival, growth, or postnatal development of offspring when administered via repeated intravitreal administration.

Effect of ofatumumab on pregnancy, parturition, and lactation in cynomolgus monkeys.

Knowledge of the impacts of the anti-CD20 monoclonal antibody ofatumumab on the developing immune system is limited. This study examined the effects of intravenous ofatumumab on pregnancy, parturition, and lactation, and on pre- and postnatal survival and development in cynomolgus monkeys, an established model for developmental toxicity assessment. Pregnant cynomolgus monkeys (n = 42) were randomized to receive vehicle only (control group; n = 14), low-dose ofatumumab (n = 14), or high-dose ofatumumab (n = 14). Survival, clinical outcomes, and clinical pathology investigations were evaluated regularly until lactation day (maternal animals) and postnatal day 180±1 (infants). Anatomic pathology was investigated in euthanized infants and unscheduled terminations of maternal animals and infants. Ofatumumab treatment was not associated with maternal toxicity or embryotoxicity and had no effect on the growth and development of offspring. As expected, B-cell depletion occurred in maternal animals and their offspring, with a reduced humoral immune response in infants of mothers on high-dose ofatumumab. Both effects were reversible. In the high-dose group, perinatal deaths of 3 infants were attributed to infections, potentially secondary to pharmacologically induced immunosuppression. The no-observed adverse-effect level for initial/maintenance ofatumumab doses was 100/20 mg, and 10/3 mg/kg for pharmacological effects in infant animals, which are associated with exposures significantly higher than those following therapeutic doses in humans. In this study with cynomolgus monkeys, ofatumumab treatment was not associated with maternal toxicity or embryotoxicity and had no effect on the growth and development of offspring.

Enhanced normograms and pregnancy outcome analysis in nonhuman primate developmental toxicity studies.

The incidence of spontaneous pregnancy/infant losses is highly variable in long-tailed macaques (cynomolgus monkey), making it potentially difficult to ascertain test item-related effects in developmental toxicity studies. Therefore, pregnancy normograms had been developed by Jarvis et al. [1] to aid in the distinction of normal (e.g. test facility background) versus non-normal pregnancy outcomes. These normograms were mostly derived from embryo-fetal development studies and from PPND studies with a postnatal phase limited to seven days. However, the enhanced pre- and postnatal developmental (ePPND) study paradigm has essentially replaced these former study types. This work aims at providing enhanced normograms (e-normograms) in the context of regulatory ePPND studies. Survival functions for the prenatal phase (286 control pregnancies) and the postnatal phase (222 live infants) were estimated using the Kaplan-Meier estimator. Normograms were generated from survival curves and pseudo-study simulations. Data were available from two test facilities with comparable EU-compliant animal husbandry. Pregnancy duration/outcome as well as survival functions did not differ significantly between test facilities indicating that this husbandry system yields comparable developmental observations across different test facilities, at least in this NHP species. These novel e-normograms were developed for pregnant long-tailed macaques and provide an extended postnatal period up to three months, a new concept of separate normograms for the prenatal and the postnatal period, specific information on the perinatal phase events, a prediction of expected number of live infants for group size management, and the option to evaluate effects on pregnancy duration through distinction of live births and infant losses.

Group size experiences with enhanced pre- and postnatal development studies in the long-tailed macaque (Macaca fascicularis).

Enhanced pre- and postnatal development (ePPND) studies have become the default developmental toxicity test for biopharmaceuticals if nonhuman primates represent the relevant species. Spontaneous pregnancy losses and infant deaths can be significant in macaques such as long-tailed macaques. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline S6(R1) states that pregnancy outcome can be judged also by the normogram-based variability of reference data according to a publication by Jarvis et al. (2010) defining a study as valid with six to eight live infants in the control group on postnatal day 7 (PND7). Since the release of ICH S6(R1) (2011), ePPND studies for biologics have replaced the former separate embryo-fetal and PPND study types. This work provides a retrospective analysis of pregnancy outcomes from 21 ePPND studies and group sizes of 14-24 animals per group. All studies reached the goal of at least six to eight infants on PND7, with overall losses ranging between 5 % and 45 %. Consistently, a group size of 14-24 maternal animals yielded more than six to eight infants on PND7. Therefore, it is suggested to reduce ePPND study group sizes from 20 to 14, yielding an animal number reduction of approx. 30 %.

Toxicologic Pathology Forum*: Opinion on Sexual Maturity and Fertility Assessment in Long-tailed Macaques ( Macaca fascicularis) in Nonclinical Safety Studies.

If nonhuman primates represent the only relevant species for nonclinical safety evaluation of biotechnology-derived products, male and female fertility effects can be assessed in repeat dose toxicity studies given that sexually mature monkeys are used. This opinion piece provides recommendations for determining sexual maturity and when/how fertility assessments should be conducted in the cynomolgus monkey. Male sexual maturity should be proven by presence of sperm in a semen sample, female sexual maturity by at least two consecutive menstrual bleedings. As per regulatory guidance, default parameters for an indirect assessment of fertility in both sexes are reproductive organ weight and histopathology. Beyond default parameters, daily vaginal swabs are recommended for females, and for males, it is recommended to include blood collections (for potential analysis of reproductive hormones), testis volume sonography, and collection of frozen testis samples at necropsy. Only if there is a cause for concern, blood collection for potential reproductive hormone analysis should be conducted in females and semen analysis in males. In principle, adverse reproductive effects can be detected within 4 weeks of test article administration, depending on study design and reproductive end point chosen. Therefore, there are options for addressing reproductive toxicity aspects with studies of less than 3 months dosing duration. *This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the authors. It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.

Experimental endocrine manipulation by contraceptive regimen in the male marmoset (Callithrix jacchus).

Marmosets are used as preclinical model in reproductive research. In contrast to other primates, they display short gestation times rendering this species valid for exploration of effects on fertility. However, their peculiar endocrine regulation differs from a those of macaques and humans. We subjected male marmosets to previously clinically tested hormonal regimens that are known to effectively suppress spermatogenesis. Beside a control group, seven groups (each n=6) were investigated for different periods of up to 42 months: regimen I, (four groups) received testosterone undecanoate (TU) and norethisterone enanthate (NETE); regimen II, (two groups) received TU and NETE followed by NETE only; and regimen III, (one group) received NETE only. Testicular volume, cell ploidy and histology, endocrine changes and fertility were monitored weekly. TU and NETE and initial TU and NETE treatment followed by NETE failed to suppress spermatogenesis and fertility. Testicular volumes dropped, although spermatogenesis was only mildly affected; however, testicular cellular composition remained stable. Serum testosterone dropped when NETE was given alone but the animals remained fertile. Compared with controls, no significant changes were observed in sperm motility and fertility. Administration of TU and NETE affected testicular function only mildly, indicating that the regulatory role of chorionic gonadotrophin and testosterone on spermatogenesis is obviously limited and testicular function is maintained, although the endocrine axis is affected by the treatment. In conclusion, marmosets showed a different response to regimens of male contraception from macaques or men and have to be considered as a problematic model for preclinical trials of male hormonal contraception.

Testosterone-induced prostate growth is blocked by co- and preadministration of norethisterone enanthate in castrated cynomolgus monkeys.

INTRODUCTION: Prostate size and function are regulated by testosterone. However, the progesterone receptor is expressed in the primate prostate. Progestins affect the prostate by endocrine suppression, but can also act directly. Examining the role of progestins, we studied the effects of norethisterone (NET) on testosterone undecanoate (TU)-induced prostate growth in castrated macaques. MATERIALS AND METHODS: Two groups (n = 6 for each group) received TU every 9 weeks. Using a crossover setting, group I received norethisterone enanthate (NETE) 3 times at 3-week intervals, while group II received placebo. After 9 weeks, placebo was administered to group I, and group II received NETE. RESULTS: In group II, the prostate grew under TU and placebo over the first period. In group I, coadministered with NETE, the increase was lower. After the crossover, prostates of animals previously treated with NETE did not increase to normal values under placebo. Prostates of animals treated with TU and placebo in the first period shrank following NETE administration after the crossover. The long half-life of NET can explain the lack of a TU effect on animals coadministered with NETE after the crossover. CONCLUSIONS: Pre- and coadministration of NET reduces testosterone-induced prostate growth with possible implications for the treatment of benign prostate hyperplasia and hormonal male contraception.

Male 41, XXY* mice as a model for klinefelter syndrome: hyperactivation of leydig cells.

Sex chromosome imbalance in males is linked to a supernumerary X chromosome, a condition resulting in Klinefelter syndrome (KS; 47, XXY). KS patients suffer from infertility, hypergonadotropic hypogonadism, and cognitive impairments. Mechanisms of KS pathophysiology are poorly understood and require further exploration using animal models. Therefore, we phenotypically characterized 41, XX(Y)* mice of different ages, evaluated observed germ cell loss, studied X-inactivation, and focused on the previously postulated impaired Leydig cell maturation and function as a possible cause of the underandrogenization seen in KS. Xist methylation analysis revealed normal X-chromosome inactivation similar to that seen in females. Germ cell loss was found to be complete and to occur during the peripubertal phase. Significantly elevated FSH and LH levels were persistent in 41, XX(Y)* mice of different ages. Although Leydig cell hyperplasia was prominent, isolated XX(Y)* Leydig cells showed a mature mRNA expression profile and a significantly higher transcriptional activity compared with controls. Stimulation of XX(Y)* Leydig cells in vitro by human chorionic gonadotropin indicated a mature LH receptor whose maximal response exceeded that of control Leydig cells. The hyperactivity of Leydig cells seen in XX(Y)* mice suggests that the changes in the endocrine milieu observed in KS is not due to impaired Leydig cell function. We suggest that the embedding of Leydig cells into the changed testicular environment in 41 XX(Y)* males as such influences their endocrine function.

Impaired recognition memory in male mice with a supernumerary X chromosome.

Several aberrant chromosomal constellations are known in men. Of these the karyotype XXY (Klinefelter syndrome, KS) is the most common chromosomal disorder with a prevalence of about one in 800 live-born boys. KS is associated with hypogonadism and is suspected to cause variable physical, physiological and cognitive abnormalities. As a supernumerary X chromosome is also associated with infertility, sound animal models for KS are difficult to obtain. In this study, male mice with two X chromosomes (XX(Y*)) were derived from fathers carrying a structurally rearranged Y chromosome (Y*) that resulted in physical attachment of a part of the Y chromosome to one X. These animals display certain physiological features that resemble closely those of human KS and can also be utilized to study X chromosomal imbalance and cognition. Therefore 15 XX(Y*) males and 15 XY* controls were subjected to a battery of behavioral tests, including a general health check, analysis of spontaneous exploration and locomotor activity, measures for anxiety-related behavior and the "novel object task" to test memory performance. Physiologically, XY* males did not differ from C57Bl/6 wild type mice carrying a normal Y chromosome, which provided a valid control group. All mice appeared healthy. XX(Y*) mice did not differ from their wild type littermates with respect to locomotion, exploration and anxiety-related behavior. XX(Y*) male mice, however, exhibited no significant recognition memory performance in contrast with wild type XY* males that readily fulfilled a given task. These findings support the hypothesis that the presence of a supernumerary X in male mice influences cognitive abilities. We suggest that the altered endocrine state and/or changes in the dosage of X-linked genes in the XX(Y*) mouse model affect brain function, in particular those regions responsible for cognition and learning behavior.

Complete spermatogenesis in orthotopic but not in ectopic transplants of autologously grafted marmoset testicular tissue.

Testicular grafting has the potential to become a method to preserve fertility in prepubertal boys undergoing cancer treatment. The possibility of successful germ cell maturation after autologous grafting should be proven preclinically in a nonhuman primate model. Therefore, in two experiments, we analyzed the potential of autologous testicular grafting in the marmoset model. A first experiment in immature and adult hemi-castrated monkeys addressed the question of whether full spermatogenesis in an ectopic graft could be achieved under a relatively normal endocrine milieu and whether the donor's age is of influence. A second experiment in castrated immature animals examined whether the transplantation site [ectopic (back skin) or orthotopic (scrotum)] influences spermatogenic progress and whether cryopreserved tissue can be successfully transplanted. Grafts were analyzed by histology, immunohistochemistry, and morphometry. Bioactive chorionic gonadotropin and serum testosterone were measured. In the adults, ectopic grafts degenerated, whereas in the immature animals, grafts survived at the spermatogonial level. In the castrates, none of the cryopreserved grafts survived, ectopic grafts were meiotically arrested, but orthotopic transplants completed spermatogenesis. Androgen and bioactive chorionic gonadotropin levels were not decisive for graft development. When ectopic and orthotopic transplantation sites were compared, the scrotum has a substantially lower temperature. Thus, the higher temperature at the ectopic transplantation site may contribute to spermatogenic arrest. Autologous grafting of nonhuman primate testicular tissues can result in complete spermatogenesis. Our findings indicate that transplantation site and developmental age of the tissue play a role more important than the endocrine milieu.

Coculture of spermatogonia with somatic cells in a novel three-dimensional soft-agar-culture-system.

Isolation and culture of spermatogonial stem cells (SSCs) has become an approach to study the milieu and the factors controlling their expansion and differentiation. Traditional conventional cell culture does not mimic the complex situation in the seminiferous epithelium providing a basal, intraepithelial, and adluminal compartment to the developing male germ cells. SSCs are located in specific stem cell niches whose features and functional parameters are thus far poorly understood. It was the aim of this study to isolate SSCs and to explore their expansion and differentiation potential in a novel three-dimensional Soft-Agar-Culture-System (SACS). This system provides three-dimensional structural support and multiple options for manipulations through the addition of factors, cells, or other changes. The system has revolutionized research on blood stem cells by providing a tool for clonal analysis of expanding and differentiating blood cell lineages. In our studies, SSCs are enriched using Gfralpha-1 as a specific surface marker and magnetic-activated cell sorting as a separation approach. At termination of the culture, we determined the type and number of germ cells obtained after the first 24 hours of culture. We also determined cell types and numbers in expanding cell clones of differentiating germ cells during the subsequent 15 days of culture. We analyzed a supportive effect of somatic cell lineages added to the solid part of the culture system. We conclude that our enrichment and culture approach is highly useful for exploration of SSC expansion and have found indications that the system supports differentiation up to the level of postmeiotic germ cells.

LHR splicing variants and gene expression in the marmoset monkey.

In the marmoset monkey, the LHR type II, lacking exon 10, is the native receptor type. We characterised the LHR splicing pattern in marmoset testes and the adrenals during puberty and in pre- and postpubertal ovaries and quantified mRNA LHR expression in the testis. We detected 11 LHR splicing variants expressed at similar levels and generated by exon skipping and/or usage of cryptic splice sites. No preferred splicing variant during pubertal maturation was observed in both sexes. Testicular and adrenal LHR expression levels did not significantly change with age. However, a significant increase during pubertal maturation for the serum testosterone/LHR ratio indicated that testosterone secretion increases in the presence of constant LHR mRNA expression levels. We conclude that LHR splicing in the marmoset displays a homogenous pattern and that the main function of the LHR is established in the testis, reaching its highest efficiency during pubertal maturation.

Chorionic gonadotropin beta-subunit gene expression in the marmoset pituitary is controlled by steroidogenic factor 1, early growth response protein 1, and pituitary homeobox factor 1.

In most mammals, the gonads are under the control of the pituitary gonadotropins LH and FSH. However, in the common marmoset monkey Callithrix jacchus, no LH is detectable in the pituitary but chorionic gonadotropin (CG) instead, normally produced in the placenta. This study investigated the mechanism of CGbeta subunit activation in the pituitary and why humans do not express CG in the pituitary. 5'-Rapid amplification of cDNA ends, EMSA, and promoter-driven luciferase assays performed with the gonadotropic LbetaT2 cells showed that marmoset monkey CGbeta is GnRH responsive and activated similar to human LHbeta by the transcription factors steroidogenic factor 1 (SF1), early growth response protein 1 (Egr1), and pituitary homeobox factor 1 (Pitx1) and displayed a transcriptional start site 7 bp upstream of exon 1. In contrast, the human CGbeta promoter displayed in the binding elements for pituitary homeobox factor 1 and early growth response protein 1 three consensus sequence mismatches, leading to very low activity that could be drastically increased by mutation to the consensus sequences. Vice versa, marmoset CGbeta promoter activity was reduced after introduction of the human CGbeta mismatches. An in vivo study in pregnant marmoset monkeys showed that during pregnancy, there is no significant decrease of pituitary CG production, contrasting human LH down-regulation. In conclusion, pituitary CG production is lacking in humans due to the absence of appropriate DNA-binding elements, which are present in marmosets, thereby enabling GnRH activation of expression. However, during pregnancy of marmosets, pituitary CG expression is not inhibited.

Male congenital hypothyroid Pax8-/- mice are infertile despite adequate treatment with thyroid hormone.

Severe forms of congenital hypothyroidism lead to serious clinical symptoms if thyroid hormone replacement therapy is not instituted immediately after birth. In this study, Pax8(-/-) mice that are born without a thyroid gland were used as an animal model to study the consequences of congenital hypothyroidism. As expected, adequate treatment of these animals with thyroxine restored the general deficits of congenital hypothyroidism; however, Pax8-deficient male mice were infertile. We report here that in these mice, the efferent ducts and epididymides are either absent or the efferent ducts exhibit a reduced lumen and extensive connective tissue, which appears to impair testicular drainage and subsequently leads to complete absence of spermatozoa from the epididymis. The results suggest that, starting with the onset of pubertal testicular fluid secretion, a backpressure is created in the testis by the absence of efferent ducts or constriction of their tubule lumen when present. This subsequently leads to secondary disorganization of the seminiferous epithelium that increases with age, resulting in mixed atrophy of the testis in the adult. Serum testosterone levels as well as mRNA expression of anterior pituitary hormones are in the normal range, indicating that the observed infertility is not due to hormonal imbalance, but rather to a developmental defect of the efferent ducts. The demonstration of Pax8 expression in the epithelia of the epididymis and the efferent ducts suggests a direct morphogenic role of Pax8 in the development of these organs. It remains to be elucidated whether congenital hypothyroid male patients with mutations in the Pax8 gene are similarly affected.

Tissue expression of the nuclear progesterone receptor in male non-human primates and men.

In females, progesterone is associated with reproductive functions. In males, its role and the expression of its genomic receptor are not very well understood. In attempts to achieve a hormonal male contraceptive method, gestagens are used to downregulate gonadotropin and sperm production. It is therefore essential to understand the mechanism of action of progesterone at the molecular level in males, especially in primates. This investigation was undertaken: (a) to determine whether the genomic progesterone receptor is expressed in males; and (b) to locate it in various organs that are potential targets of gestagens. Human tissues were obtained at surgery for benign prostatic hyperplasia or prostate cancer and at autopsy. Non-human primate tissues were obtained at autopsy. This study was performed by analyzing the genomic progesterone receptor by immunohistochemistry, Western blot and RT-PCR. The nuclear progesterone receptor was expressed in pituitary and hypothalamus of both monkeys and men. In the testis progesterone receptor expression was found in a few peritubular and interstitial cells, but not in germ cells. In addition, expression was detected in the epididymis, prostate and male mammary gland. Reverse transcriptase (RT)-PCR experiments indicated that progesterone receptor A and B are expressed in all tissues analyzed. These data exclude direct genomic effects of gestagens at the spermatogenic level but indicate that a male contraceptive based on gestagens might have some effects on other tissues, such as the epididymis, prostate and mammary gland.

Optimising workflow in andrology: a new electronic patient record and database.

AIM: To improve workflow and usability by introduction of a new electronic patient record (EPR) and database. METHODS: Establishment of an EPR based on open source technology (MySQL database and PHP scripting language) in a tertiary care andrology center at a university clinic. Workflow analysis, a benchmark comparing the two systems and a survey for usability and ergonomics were carried out. RESULTS: Workflow optimizations (electronic ordering of laboratory analysis, elimination of transcription steps and automated referral letters) and the decrease in time required for data entry per patient to 71%+/-27%, P<0.05, lead to a workload reduction. The benchmark showed a significant performance increase (highest with starting the respective system: 1.3+/-0.2 s vs. 11.1+/-0.2 s, mean+/-SD). In the survey, users rated the new system at least two ranks higher over its predecessor (P<0.01) in all sub-areas. CONCLUSION: With further improvements, today's EPR can evolve to substitute paper records, saving time (and possibly costs), supporting user satisfaction and expanding the basis for scientific evaluation when more data is electronically available. Newly introduced systems should be versatile, adaptable for users, and workflow-oriented to yield the highest benefit. If ready-made software is purchased, customization should be implemented during rollout.

Evolutionary comparison of the reproductive genes, DAZL and BOULE, in primates with and without DAZ.

Genes of the DAZ (Deleted in AZoospermia) gene family, DAZ, DAZL (DAZ-Like), and BOULE, encode closely related RNA-binding proteins that are required for fertility in numerous organisms, yet the genomes of different organisms possess different complements of DAZ family genes. Thus, invertebrates such as flies and worms contain just a single DAZ homolog, boule, while genomes of vertebrates, other than catarrhine primates (Old World monkeys and hominids), possess both Boule and Dazl genes. Finally, catarrhine primates possess BOULE, DAZL, and DAZ genes. Since the DAZ genes arose recently in evolution in the catarrhine lineage, we sought to examine how the sequences and expression of this gene family may have changed after the introduction of a new member, DAZ. Based on previous results, we hypothesized that the introduction of a new member of the DAZ gene family into catarrhines could reduce functional constraint on DAZL. Surprisingly, however, we found that platyrrhine DAZL demonstrated significantly more sequence divergence than catarrhine DAZL (p=0.0006 for nucleotide and p=0.05 for amino acid sequence); however, comparison of K (a)/K (s) ratios suggests that the DAZL and BOULE genes are under similar functional constraints regardless of lineage. Thus, our data are most consistent with the hypothesis that the introduction of DAZ did not affect the evolution of DAZL or BOULE, and that a higher neutral mutation rate in platyrrhines than in catarrhines, along with the greater tolerance of DAZL for variation relative to BOULE, may be the foundation for the observed differences in sequence divergence in this gene family.

Meiosis in autologous ectopic transplants of immature testicular tissue grafted to Callithrix jacchus.

Grafting of immature testicular tissue provides a tool to examine testicular development and may offer a perspective for preservation of fertility in prepubertal patients. Successful xenografting in mice, resulting in mature spermatids, has been performed in several species but has failed with testicular tissues from the common marmoset, Callithrix jacchus. Previous data indicate that the hormonal milieu provided by the mouse host might cause this failure. We conducted autologous ectopic transplantation of testicular fragments under the back skin in newborn marmoset monkeys. Seventeen months after transplantation, we found viable transplants in 2 out of the 4 grafted animals. In the transplants, tubules developed up to a state intermediate between the pregraft situation and adult controls. Dividing spermatogonia and primary spermatocytes were present. Boule-like positivity and CDC25A negativity indicated that spermatogenesis was arrested at early meiosis. Immunohistochemistry revealed normal maturation of Sertoli cells, Leydig cells, and peritubular cells. Serum testosterone values were not restored to the normal range and bioactive chorionic gonadotropin levels increased to castrate levels. Meiotic arrest could have occurred in the grafts because of lack of sufficient testosterone or because of hyperthermia caused by the ectopic position of the grafts. We conclude that autologous transplants of immature testicular tissues in the marmoset can mature up to meiosis but that normal serum testosterone levels are not restored. Further studies have to be performed to overcome the meiotic arrest to explore the model further and to develop therapeutic options.

GnRH-antagonist mediated down-regulation of the estrous cycle in marmosets.

BACKGROUND: Because of its small size and unproblematic captivity behavior the marmoset monkey is an attractive New World primate model for early developmental questions. However, superovulation protocols used in Old World monkeys and women are not successful in the female marmoset. A novel protocol is needed to utilize these New World monkeys as an efficient animal model for in vitro fertilization experiments or embryo stem cell research. METHODS: To create such a protocol we first examined the effects of long-term estrous cycle control, secondly, in a dose-finding study, we determined the length of a down-regulation protocol with a gonadotropic releasing hormone (GnRH)-antagonist. Twenty-nine female marmosets were grouped according to the number of estrous cycles, which had been controlled for a period of 12 months in which 88 cycles were monitored. Application of PGF2alpha in the mid-luteal phase led to immediate onset of the follicular phase. The blood progesterone concentration rapidly declined and increased again on day 9-11. RESULTS: The results show that the controlled ovarian cycle length and progesterone response are not altered by the number of PGF2alpha injections. The rapid decline was similar in all groups, indicating that all animals, independent of the number of controlled cycles, react equally to multiple PGF2alpha injections. To determine the proper dosage for a GnRH-antagonist (Cetrorelix), 12 animals in three groups of four female marmosets were treated with two different dosages and a sham dosage. Cetrorelix was applied in the mid-luteal phase, three times over 2 days. In both Cetrorelix-treated animal groups the early progesterone levels matched those in the controls. In the low-dose treatment group [0.01 mg/100 g body weight (BW)] the expected progesterone rise on day 10 was delayed between 9 and 15 days whereas in the high-dose treatment group (0.1 mg/100 g BW) the progesterone rise was delayed between 21 and 41 days. In the low-dose group the steepness of the slope from day 20 onwards was almost identical to that of the control group. This was reflected in the bioCG levels measured. CONCLUSIONS: Based on the GnRH-antagonist studies, complete ovarian down-regulation in female marmosets can be achieved by applying a low-dose regimen, and intrinsic gonadotropins would not interfere with an ovarian superstimulation protocol.