The common marmoset (
The common marmoset (
In primarily descriptive publications the pubertal onset in the common marmoset has been characterized by serum testosterone levels and a rise in testis volume (TV) during postnatal development (Abbot and Hearn, 1978; Jackson and Edmonds, 1984). Experimental manipulations were adopted to unravel the postnatal aspect of testicular development in immature common marmosets (Lunn et al., 1994, 1997; Sharpe et al., 2000, 2003a, b). Some features in marmosets are specific for New World monkeys. For example the pituitary gland in the common marmoset releases a chorionic gonadotropin (CG)-like molecule, having a much shorter serum half-life as compared with Luteinizing Hormone (LH; Muller et al., 2004). In the common marmoset the pubertal reactivation of hypothalamic pituitary gonadal axis, characterized by elevated levels of testosterone, is currently considered to occur between 6 and 12 months of age (Li et al., 2005). Keeping in mind aspects of the unique reproductive endocrinology of common marmoset, we have previously documented the pubertal development in the common marmoset employing a cross-sectional approach (Chandolia et al., 2006).
Here we aimed to investigate the pubertal growth in the common marmoset by observing immature common marmosets longitudinally for a period of 13 months. Our primary aims included (1) exploring the precise timing of pubertal onset in terms of hypothalamic pituitary gonadal axis activation and (2) characterizing the morphological features in the testis before, during and after pubertal onset. Our secondary aims were to correlate the somatic and reproductive growth pattern and to measure the time required by the immature testis to fully establish spermatogenesis qualitatively.
Immature common marmosets were observed for 13 months starting either from
birth or at an age of less than 12 months. Animals were housed in the
animal facility of the University Hospital Münster (UKM). Animals were
either housed with their families or were separated into groups of the same sex and age
(
The study was designed to longitudinally monitor animals for body weight, testicular volume and serum testosterone during the postnatal development. In order to document the testicular histomorphometric changes from neonatal phase to post-pubertal phase, groups of monkeys were sacrificed from birth to 12 months of age and during adulthood. Table 1 demonstrates the different age groups and the number of animals present in each age group.
Monthly body weight (balance) and testicular volume (caliper) were recorded
under manual restraint. At the same time blood samples were collected from
the femoral vein of the animals. The samples were collected between 08.00 and
10.30 a.m. The serum was separated from the blood samples and stored at
Testosterone (T) was assayed in serum samples using an in-house radioimmunoassay (RIA) method.
Serum testosterone was measured by a solid-phase, double-antibody RIA
technique, using a commercially available iodinated tracer
(testosterone-3-(0-carboxymethyl) oximino-2-[12T] iodohistamine; Amersham
International, Braunschweig, Germany) and an antiserum raised in rabbits
against testosterone-3(carboxymethyloxime)-BSA. The bound/free separation
was performed by addition of a solution of solid-phase antirabbit
immunoglobulins (Immunobead Second Antibody, Biorad, Munich, Germany). The
recovery after ether extraction was monitored by addition of trace amounts
of [l
Mean body weight in the common marmoset from birth until 100 weeks
of age (
Mean bi-testis volume in the common marmoset from birth until
100 weeks of age (
At the end of the study each animal was deeply anesthetized with ketamine, and body
weight and testicular volume were recorded. Subsequently the animals were
sacrificed by exsanguination. The weight of testis tissues were recorded
immediately after removal. Testis tissue was fixed in Bouin's solution
overnight and later routinely embedded in paraffin. Sections of 3
Slides were analyzed using an Olympus BX61 microscope (Melville, NY, USA)
with an attached Retiga 4000R camera (QImaging, Surrey, BC, Canada). All
images were acquired digitally using QCapture imaging software (QImaging,
Surrey, BC, Canada). Pictures were taken from five independent positions
from each section at 20
Details of the morphometry have been described previously (Schlatt et al., 1999). Volume density expressed as the relative proportion of testicular tissue was determined by point counting using a random systemic and blinded approach on six sections from each testis. A total of 120 points were scored for each testis. An ocular grid showing a square was used, and the four corners of the grid were analyzed to be located on interstitium, tubule epithelium or tubule lumen. Volume density of various components multiplied by testis weight (assuming a specific weight of testis tissue of 1) yielded the total weight of that component per testis. Sixty measurements were taken from each testis (10 measurements from each section). Round tubules were selected for absolute measurement of the tubule diameter.
All data were expressed as mean
Bi-testis volume of the individual common marmosets
(
Mean serum testosterone levels from birth until the 100th week of age in
the common marmoset (
The mean body weight in immature common marmosets showed robust growth
during the first 28 weeks (7 months) of age. The mean body weight at 7
months was significantly (
Mean serum testosterone levels depicted a sudden and significant (
Testis weights were determined in all 48 monkeys at the time of sacrifice. These values are relevant for comparison with the histological analysis. The absolute testis weights and volumes as well as relative testis weights in relation to body weight are shown in Figs. 8 and 9.
Description of advanced germ cell type, Sertoli cell
arrangement and the presence or absence of sperm in the epididymis of common
marmosets over the course of development (
Mean individual seminiferous tubule diameter from 16 weeks onwards
till 104 weeks in individual common marmosets (
Percentage of seminiferous tubules, interstitial area and
tubule lumen during development in individual common marmosets (
Calculated weight of seminiferous epithelium, interstitial
area and the tubule lumen during development in the individual common
marmosets (
Bi-testis weight and bi-testis volume of the sacrificed
individual common marmosets (
Relative percentage of the bi-testis weight to the body
weight in the sacrificed animals (
The seminiferous tubule diameter increased initially at 24 weeks and continued to rise until 68 weeks of age (Fig. 5). The presence of a lumen was first observed at 24 weeks (Figs. 6 and 7). With advancing age the relative proportions of the volume densities of the various compartments remain relatively constant; however, when expressed as absolute weights of each compartment the significant growth of the tubular compartment during population with differentiating germ cells is obvious (Fig. 7). Until 20 weeks Sertoli cell nuclei were randomly distributed in the cords. At 6 months of age the Sertoli cells are arranged towards the periphery of the seminiferous tubules in an epithelial-like fashion (Table 1). Gonocytes were no longer observed after 5 months of age, and A spermatogonia were present from around 6 months of age. B spermatogonia and few primary spermatocytes were first observed around 7 months of age, and round spermatids were first observed at 8 months of age, whereas sperm were first detected in the epididymis at 12 months of age (Table 1).
Testicular pubertal development in the common marmoset was examined in the present study. No experimental manipulation was performed in this study, and all data were obtained by recordings of animals in family breeding or same-sex group holding conditions. We assume that these conditions provide the environmental conditions for normal postnatal growth patterns in laboratory colonies. The primary aim of the present study was the identification of the initial age of pubertal onset, which was monitored by testis volume and serum testosterone levels. We observed that the initial pubertal activation of the hypothalamic pituitary gonadal axis in terms of high serum levels of testosterone in the common marmoset occurs after 6 months (24 weeks). This finding is in agreement with the observation by Abbot and Hearn (1978). We therefore see a slightly earlier initiation of puberty than reported by Lunn et al. (1994) and Kelnar et al. (2002).
First seminiferous lumen formation was observed after 6 months at a time
when high serum testosterone was first encountered. It can be speculated
that an expression of androgen receptors by Sertoli cells during this
developmental period window is causing the onset of Sertoli cell
Our data concerning the body weight gain depicted an important aspect of postnatal development in the common marmoset. The maximum body weight gain had been observed well within the first 6 months of age before the pubertal activation. After 6 months the animals showed a slower rise in the body weight. Whether the initial weight gain is a prerequisite for the pubertal activation remains to be explored. It seems that growth and sexual development are controlled independently in the common marmoset.
In conclusion pubertal testicular development in the common marmoset initiates at 6 months of age at the earliest and is visible by a parallel rise in testosterone and testicular growth. The pubertal onset in the common marmoset is preceded by a period of accelerated somatic growth. Qualitative completion of spermatogenesis occurs at 12 months of age at the earliest. The actual start and the kinetics of puberty changes are individually highly variable.
The work presented here was supported by a DAAD fellowship grant to Shahzad Irfan and by a DFG project (FOR 1041, project 3). We thank Martin Heuermann and Günter Stelke for excellent care and management of the marmoset breeding colony. We also like to thank Reinhild Sandhowe-Klaverkamp for skilled technical assistance with the testosterone radioimmunoassay and Jutta Salzig for support on histological procedures. Edited by: E. Fuchs Reviewed by: two anonymous referees