Linear somatic growth is essentially controlled by hormones. Current knowledge suggests that androgens stimulate skeletal growth both directly and indirectly (i.e., mediated by the growth hormone—insulin-like growth factor-I axis). These androgen mechanisms have been demonstrated in animal, and in vitro studies. Recently, evidence for the effect of oestrogens on skeletal growth and development in men through specific androgen receptors has appeared. As it is known that the human head grows in concert with the rest of the body, it has been suggested that sex steroids also have direct and/or indirect effects on facial and cranial-base growth.

In line with human studies, experimental rat studies have demonstrated the effects of sex steroids on craniofacial growth. We have preliminary evidence that both neonatal and prepubertal castration result in a modification (suppression) of craniofacial growth from puberty onward.

As for the effects of exogenous testosterone on general growth, Schoutens et al. (1984) demonstrated that postpubertal testosterone replacement was restorative in the rats tibia; it induced an increase in the bone apposition rate and reduced the number of osteoclasts. Jansson and Frohman (1987) demonstrated that giving testosterone to neonatally castrated rats in adulthood increased mean plasma growth hormone concentrations, and their findings support the assumption that androgen exposure in adults stimulates long-bone growth. Turner et al. (1990) showed that postpubertal castration of rats results in decreased bone formation and that androgen treatment stimulates bone formation.

Here, the effects of exogenous testosterone on craniofacial development in neonatally castrated rats were examined. Craniofacial growth, together with general growth, were evaluated in a cross-sectional cephalometric study from prepuberty to adulthood in male, neonatally castrated, Wistar rats given exogenous testosterone at day 57.

The main question was whether exogenous testosterone stimulates individual bony components of the craniofacial skeleton, and possibly restores the growth-suppressive effects induced by neonatal castration.

Pregnant Wistar rats were obtained from the animal laboratories of the University of Leuven. They were fed a diet adequate for their nutritional needs and kept in an air-conditioned and light-controlled room with an ambient temperature of 23°C. After birth, 50 male neonates were randomly divided into two groups, control (n=15) and experimental (n=35), the latter being castrated under light ether anaesthesia. After weaning, at day 21, they were randomly redistributed in cages containing five to six rats each. The protocol for animal use was reviewed and approved by the Ethics Committee of the Medical School, University of Leuven.

In half of the neonatally castrated rats (n=18), a 1.5 cm tube (0.40 inch inner diam. and 0.85 inch outer diam.) filled with testosterone was implanted subcutaneously in the back. The implantation was done at day 57, i.e., after the normally expected pubertal testosterone surge (around days 45–50; Piascek and Goodspeed, 1978), The length of the tube was based on the results obtained from the study of Vanderschueren et al. (1992) in which the release of testosterone from tubes was studied in vitro. For adult castrated rats with a body wt of 300 g, a tube length of 2.5 cm was used, which delivers 57.5 ?g testosterone/24 h. The release of testosterone in vitro was 23 ?g/cm Silastic daily. For our experimental rats of 57 days with a body wt of 150–200 g, a tube length of 1.5 cm was chosen. Constant release of testosterone could be expected from 2 days after the implantation.

At day 70 and day 110, five to nine rats were taken from each group. Sagittal radiographs of the skull were taken in a specially designed craniostat (occlusal film, Agfa Dentus M2 Comfort). Measures of general growth (body length and weight) were registered, under general anaesthesia with Nembutal. The dose of the anaesthetic was adapted to the weight of the rat (25–30 mg/kg). After decapitation, blood was collected from the Carotic artery. The cross-sectional approach of this experiment permitted an additional control of the effectiveness of the testosterone implant by weighing the prostate of the animals at the various time.

The cephalometric analysis included linear measurements from the analyses of Vilmann (1969), Engström et al. (1982), Persson, E.C., Engström, C. and Thilander, B., 1989. The effect of thyroxine on craniofacial morphology in the growing rat. Part I: a longitudinal cephalometric analysis. The choices of the craniofacial landmarks were made in accordance with the different growth types represented in the skull: (i) sutural growth between different bones of the skull vault given by measurements 2–3, 2–4, 2–5, and 1–6; (ii) enchondral growth of the synchondrosis of the cranial base given by 12–13, 13–14, and 14–15; (iii) upper-incisor growth given by 7–8 and lower-incisor growth given by 9–10; and (iv) mandibular growth given by 11–16. Photographic negatives were taken from the developed radiographic images and installed on a Kodak CD-ROM, to be analysed by the computer program NIH 1.57 (National Institute of Health) run on a Macintosh computer. The different craniofacial landmarks were digitized. The linear measurements were extracted from the data points using specially designed software. Replicate analyses showed no significant intra- and interobserver differences in the measurements.

Serum testosterone and growth hormone levels were measured by radio immuno assay. Serum testosterone was analysed with the ¹²⁵I assay kits provided by the Byk-Sangtec Diagnostica Gmbh and Co KG (von Hevesy-Strasse, D-63128 Dietzenbach). The kit consisted of testosterone antiserum from rabbits; testosterone ¹²⁵I tracer and testosterone standard dissolved in human serum, and goat antirabbit gammaglobulins in polyethyleneglycol solution. The detection limit for testosterone was 0.2 ng/ml. All experimental and control groups were assayed at the same time. The intra-assay coefficient of variation was between 5.8 and 6.3%. The inter-assay coefficient of variation was between 13.5% and 7.4%.

Radioimmunoassays for growth hormone were done in duplicate on all samples, using the kits provided by the National Institute of Diabetes and Digestive and Kidney Diseases (Rockville, MD., U.S.A.). The kit consisted of rat growth hormone I-7 for iodination, standard rat growth hormone RP-2 and monkey antirat growth hormone S-5. All treatment and control groups were assayed at the same time. The within-assay coefficient of variation was 1%.

A statistical procedure for analysing the data according to a mathematical linear model was used. Two-way ANOVA was employed, including the factors time and type of treatment as well as their interaction; significance was set at p<0.05. This analysis was combined with a multiple-comparison test. The effect of exogenous testosterone on the variables examined was determined by the combination of the significance and non-significance of the differences between these three groups.