Effects of delaying puberty on bone mineralization in female rats

Yardena Rakover1, Peggy Lu2, Julie N. Briody2, Chang Tao2, Ehud Weiner3, Antwan G.H. Ederveen4, Chris T. Cowell2 and Izhar Ben-Shlomo3,5

1 Pediatric Endocrine Unit, HaEmek Medical Center, Afula, Israel, 2 The Robert Vines Growth Research Center, The New Children's Hospital, Parramatta, NSW, Australia, 3 Department of Obstetrics and Gynecology, HaEmek Medical Central, Afula, 18101, Israel and 4 Department of Endocrinology, NV Organon, HB OssThe Netherlands


    Abstract
 Top
 Abstract
 fIntroduction
 Materials and methods
 Results
 Discussion
 References
 
The effect of delaying puberty on bone mineralization was studied using female rats as a model. Repeated injections of gonadotrophin-releasing hormone antagonist (GnRHa) were used to suppress the onset of puberty from the age of 6–10 weeks. A group of control female rats was given aqueous solution injections at the same age and for the same duration. The effect of delaying puberty on bone mineralization was examined using dual energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (QCT), both methods being adapted for small animals. Bone mineral parameters were measured at baseline and at the ages of 10, 17 and 24 weeks in total body, femur and spine. Compared to controls, bone mineral content (BMC) and bone mineral density (BMD), as measured by DXA, were significantly decreased in GnRHa-treated rats in total body and femur at 10 and 24 weeks of age (P < 0.05). The results were even more significant after adjusting for weight. After this adjustment, spine BMC and BMD at 10, 17 and 24 weeks were significantly lower in the treatment group (P < 0.05). Trabecular BMD at the distal femur in the GnRHa treated group as measured by peripheral QCT was significantly lower (P < 0.05). However, cortical bone in the mid-femur had higher BMD, concurrent with lower cortical thickness in the treatment group. In conclusion, a delay in the onset of sexual maturation may cause prolonged, possibly irreversible defect in bone mineralization.

Key words: bone mineralization/dual energy X-ray absorpiometry/gonadotrophin-releasing-hormone hormone antagonist/peripheral quantitative computerized tomography/puberty


    fIntroduction
 Top
 Abstract
 fIntroduction
 Materials and methods
 Results
 Discussion
 References
 
A high bone mass at skeletal maturity is a good predictor for lower rate of age-related fracture risk (Matrovic, 1992Go). During childhood, bone mineral content rises steadily with an acceleration of mineral accumulation at the time of puberty. Growth during puberty contributes about 50% of peak bone mass in women, whereas in men it contributes only about 15% (Gordon et al., 1991Go). It was previously believed that bone mineral density (BMD) increases until the third decade of life (Matrovic, 1992Go). Recent data suggests that peak BMD is already achieved at about 16 years in women and 17.5 years in men (Gordon et al., 1991Go; Lu et al., 1994bGo).

Although it is widely held that attainment of peak bone mass is dependent on timing of puberty, the evidence to support this hypothesis is fragmentary. In men with a history of constitutionally delayed puberty, radial and spinal BMD are decreased during the third decade of life (Finkelstein et al., 1992Go). In female athletes, late menarche may cause a failure to reach an adequate peak bone mass despite the intense physical activity (Constantini and Warren, 1994Go).

The aim of this study was to examine the effect of pharmacologically delayed puberty on peak bone mass in female rats, accepted as a good model for the human counterpart (Frost and Jee, 1992Go).


    Materials and methods
 Top
 Abstract
 fIntroduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Sixty-nine Sprague–Dawley rats were included in the study. Of those, 35 were in the treatment group and 34 served as controls. Rats were killed at 10, 17 and 24 weeks, in order to assess the treatment effect on bone mineral status from puberty to mid-adulthood. The food intake and the activity concentrations were the same in all groups. The Animal Ethics Committee of the Royal Alexandra Hospital approved the study protocol.

Treatment
Gonadotrophin-releasing hormone antagonist (Org. 30276; Organon, Oss, The Netherlands) was given by daily s.c. injection for 3 weeks, beginning at 6 weeks of age, corresponding to the onset of sexual maturation. The daily dose was 125 µg, dissolved in 0.2 ml of aqueous solution of gelatin (0.5%) and mannitol (5%). This dose was previously found effective in suppressing gonadotrophin secretion and block ovulation (Debeljuk and Schally, 1986Go). Vehicle solution in the same volume was given daily for the same period in the control group. Measurements of weight, body length (nose to the start of the tail) and total body length (nose to the end of the tail) were recorded at 6 weeks of age, and at the end of the study in all rats.

Bone mass assays
At the age of 6 weeks bone mineral content (BMC) and BMD of total body, femur and spine (L1-L5) were examined in each rat using dual energy X-ray absorptiometry (DXA; DPX, Small Animal Software Version 1.0; Lunar Corp., Madison, WI, USA). This measurement was used as a baseline measurement for each rat before giving any treatment. The technical details of this DXA software version for small animals were previously described (Lu et al., 1994aGo). The coefficient of variation (CV) was 0.8% for total body BMD (TBMD), 2% for femoral BMD (FBMD) and 5.5% for spine BMD (SBMD). Rats were anaesthetized with i.p. injections of pentobarbitone (35 mg/kg) prior to DXA scans in the prone position. All rats were scanned under identical parameters and the same investigator (J.N.B.) analysed all scan files, in order to minimize technical variation.

The method of peripheral quantitative computerized tomography (QCT) was used for measuring bone density in the dissected femora, after storing at –70°C. The distal part of the femur 5.5 mm from knee, which contains predominantly trabecular bone, and the mid part of the femur 13.5 mm from the knee, which is predominantly cortical bone, were assayed. The resolution of the peripheral QCT was set at 0.15x0.15 mm (Gasser, 1995Go).

Outcome measurements
The rats were killed using CO2 at the end of the study period. Blood samples were collected by cardiac puncture and stored at –20°C until determination of oestradiol, luteinizing hormone (LH) and follicle stimulating hormone (FSH). These measurements were performed to examine the efficiency of the treatment and to assess the hormonal status after its withdrawal. The second DXA scan was performed on the dead rats, either on the same day, or the day following killing.

Hormone assays
Oestradiol was measured using radioimmunoassay (Clinical Assays; Sorin Biomedica, France). The sensitivity of the assay was 17 pmol/l. LH and FSH were measured by radioimmunoassay specific for rats, as previously described (Crawford et al., 1994Go). The limit of assay detection was 0.125 ng/ml for FSH and 0.312 ng/ml for LH.

Statistical analysis
Statistical Analysis System (SAS) (Scholtzhauer and Littell, 1992Go) was used for data analysis. Student's t-test was used for variables with normal distribution and non-parametric test (Wilcoxon) was used for variables which were not normally distributed. Group differences were tested using analysis of variance (ANOVA) procedure. Associations between variables were examined using multiple linear regression. Since BMD and BMC are weight dependent, the relationships of the regression lines of BMD or BMC to weight in the treated animals and controls were examined for parallelism (interaction term for slopes). Further analyses on the influence of treatment and time factors on outcome variables were performed using both multiple linear regression and ANOVA procedures (two-way co-variance). Intra-group comparison was made using paired t-test. Significance level was set at 0.05.


    Results
 Top
 Abstract
 fIntroduction
 Materials and methods
 Results
 Discussion
 References
 
Baseline
At 6 weeks of age, there was a significant difference between the treatment and control groups in weight, body length, total body length, TBMD, total body BMC, femur BMC, FBMD, SBMD and spine BMC (Table IGo). Since BMD and BMC of total body and femur are weight dependent (r2 ranged 0.35–0.63, P < 0.0001 for all) we adjusted all the parameters for weight. After allowing for weight there was no difference in BMD or BMC of either site between treatment and control groups (P > 0.05 for slopes). Therefore the higher BMD and BMC found in the treated group were related to their larger size. The differences in weight between the subgroups were related to the variance between different litters as all the rats from one litter were included in the same group.


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Table I. Baseline characteristics (mean ± SD) of two groups of female rats tested for the effect of delaying puberty on bone mass parameters
 
Hormonal results
The mean concentrations of oestradiol, LH and FSH in each subgroup are shown in Figure 1Go. Comparison of oestradiol, LH and FSH concentrations between the subgroups at the age of 10 weeks showed significant differences between the treated group and the controls (P = 0.002, P < 0.0001, P = 0.0001 respectively). LH concentrations in the treated group at age 10 weeks were almost at the detection limit of the assay. No significant difference in oestradiol, LH and FSH concentrations were found between the treated groups and controls at the ages of 17 and 24 weeks, apart from lower serum LH in the treated group at 24 weeks (P = 0.0065).



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Figure 1. Serum LH, FSH and oestradiol concentrations in the GnRH antagonist-treated group and the controls (*P < 0.05).

 
Outcome measurements
As there were three subgroups with different study duration (10, 17 and 24 weeks) within the GnRH antagonist treated and the control groups, a comparison was made between subgroups for the same study period.

Ten weeks
The GnRH antagonist treated group had lower BMD and BMC compared to the controls (Table IIGo). Similar to those at baseline, the bone parameters were weight dependent (P < 0.05 for all). The curves of total body BMD and BMC, femur BMD and BMC, and spinal BMD and SBMC by weight were parallel between the treated and the control groups (P < 0.05 for all) but lower in the treated group after allowing for weight. We also measured the change between 10 weeks and baseline in each rat. Compared to baseline the increases in all the above parameters in the control group were significantly greater than in the treated group (P < 0.05 for all). This increase was related to the increase in weight and length for total body and femur BMC (P < 0.05), but not for the respective BMD (data not shown).


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Table II. Outcome measurements by weeks of age (mean ± SD) in two groups of female rats tested for the effect of delaying puberty on bone mass parameters
 
Seventeen weeks
The GnRH antagonist treated group was significantly heavier than the controls. However, there was no significant difference in any bone mineral parameter (Table IIGo). Since there was a weight difference between the GnRH antagonist treated group and the controls, multiple linear regression procedure was used to examine the group difference in bone mineral parameters. After adjusting for weight, BMC of total body and the femur were significantly higher in the controls, but no difference was seen in BMD at these two sites. Spinal BMC and BMD were both significantly higher in the controls. Similar to data at 10 weeks, the rise in bone mineral parameters was significantly higher in the control group after adjusting for the increase in weight.

Twenty-four weeks
The GnRH antagonist treated group was not heavier than the controls (Table IIGo). The bone mineral parameters were significantly higher in the control group compared to the treated group. This difference was even more prominent after adjusting for body weight.

Peripheral QCT measurements of the femora
At the distal femur significantly lower BMD was found in all the GnRH antagonist treated subgroups compared to the controls (P < 0.05; Figure 2Go). No difference was found in the area of the distal femur between the treated and control groups at the three developmental ages (data not shown). At the middle part of the femur significantly lower cortical bone density was found in the GnRH antagonist treated subgroup at 10 weeks compared to the related controls, however significantly higher cortical bone density was shown at 17 and 24 weeks in the treated groups compared to the controls (P < 0.05). No differences were found in the area of the middle part of the femur (data not shown), and the inner and outer diameter of the femur were not changed by treatment (data not shown). Lower cortical thickness was found at 10 and 24 weeks in the GnRH antagonist treated rats compared to the related controls (P < 0.05; Figure 2Go).



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Figure 2. Bone mineral density of the distal femur (trabecular bone), medial femur (cortical bone) and cortical thickness in the GnRH antagonist treated groups and the controls using peripheral quantitative computerized tomography (QCT) method (*P < 0.05).

 

    Discussion
 Top
 Abstract
 fIntroduction
 Materials and methods
 Results
 Discussion
 References
 
Much attention has been focused on the issue of adequate peak bone mass in early life as protection against bone loss later in life (Gordon et al., 1991Go; Matrovic, 1992Go; Johnston et al., 1992Go). It is believed that many factors affect the achievement of peak bone mass. Inheritance is the major factor determining peak bone mass (Slemenda et al., 1991Go; Matrovic, 1992Go). Sex, race, body size, physical activity, dietary calcium and gonadal steroid hormones are additional factors which influence the attainment of maximal peak bone mass (Krabbe et al., 1979Go; Kanders et al., 1988Go; Liel et al., 1988Go; Kelly et al., 1990Go; Ott, 1990Go).

Sex steroids have a major role in regulating bone mass and bone density. Adults with hypogonadism of any aetiology suffer from osteoporosis, the largest such group being post-menopausal women. In these, osteoporosis appears to be due to accelerated bone loss (Theintz et al., 1992Go). However, it appears that the increase in concentration of sex steroids in puberty has a major role in the rise of BMC and BMD during puberty (Ott, 1990Go). It has been shown that growth during puberty contributes about 50% of peak bone mass in females, while in males this contribution is about 15% (Gordon et al., 1991Go; Theintz et al., 1992Go).

The exact timing of attainment of peak bone mass is still debated. It has been assumed that peak bone mass is achieved by the age of 30 years (Matrovic, 1992Go). However, recent studies have shown an earlier peak at the end of the pubertal process (Gordon et al., 1991Go; Lu et al., 1994bGo). Furthermore, Finkelstein et al. (1992) have suggested that the timing of onset of puberty is critical for attainment of peak bone mass, those males with delayed puberty having low spine BMD as young adults.

It is arguable whether the rats are good models for studying human osteopenia. It has been shown that in rats, similar biological mechanisms control bone growth as in children. In female rats the skeleton does not grow continuously throughout life (Frost and Jee, 1992Go). In older rats, the osteopenia associated with oestrogen deprivation with ageing seems to mimic the human forms with respect to the anatomical patterns of bone loss and to the bone tissue dynamics. In addition, the effects of variable hormones on bone mass in rats are similar to their effects on the human skeleton. However, whereas the human pubertal growth spurt is modulated by a marked increase in the serum concentrations of insulin-like growth factor-I (IgF-I), which is increased at least partially by sex steroids, in male mice the IgF-I surge does not require androgens (Rosenfield and Furlanetto, 1985Go; Crawford et al., 1993Go).

In the present study we used Sprague–Dawley rats as a model to examine the effect of delaying puberty on bone mineral parameters. In a previous study in rats, Lu et al. (1994a) found that bone mass increased to a peak value and then declined gradually when the peak bone mass was achieved before 36 weeks of age. Kalu et al. (1989) demonstrated in female rats an increase in BMC and BMD until the age of 6 months. From the age of 5 months, there was no further change in femur density and femur calcium, and by 12 months, all bone parameters had reached plateau values in contrast to body weight, which was still increasing.

The conclusion of the pubertal change in female rats is shown by vaginal opening, which occurs at an average age of 6 weeks. We administered GnRH antagonist treatment from 6–10 weeks, representing an arrest of the onset of oestrous cyclicity for 4 more weeks into adulthood. The GnRH antagonist used here has the advantage of avoiding the initial rise in gonadotrophin release which occurs with GnRH-agonist therapy which is widely used in humans to inhibit puberty in girls with precocious puberty. The effectiveness of this GnRH antagonist preparation in rats has been previously proven (Nekola and Coy, 1985Go; Debeljuk and Schally, 1986Go; Meijs-Roelofs et al., 1990Go; Deckers et al., 1992Go). In our study we have shown suppression of oestradiol, LH and FSH at the age of 10 weeks, in treated compared to control rats. No further differences in hormonal concentrations between the treated and control groups were found at 17 and 24 weeks, apart from low LH concentrations in the treated group at the age of 24 weeks. This finding may indicate that some degree of gonadotrophin suppression exists after treatment withdrawal, possibly due to depot formation, but previous studies do not support this possibility (Deckers et al., 1992Go). Alternatively, different oestrus timing may have been induced in the treated group (Butcher et al., 1974Go).

In the present study we have shown significantly decreased total, femoral and spinal BMD and BMC in the GnRH treated groups compared with the controls. We also found a smaller increment of these parameters in the treated group. Adjusting for weight further strengthened these differences. Since BMC and BMD in rats are strongly affected by weight, the adjustment emphasizes the net effect of the therapy.

Peripheral QCT relates BMD to volume in contrast to DXA, which relates it to area. We used peripheral QCT to measure BMD in a predominantly trabecular site, the distal femur, and a predominantly cortical site, the mid-shaft of the femur. The treated group had significantly lower bone density at the trabecular site, whereas no consistent decrease was found at the cortical site. This would be consistent with oestrogen having its major anti-osteoporotic effect at trabecular sites. Data in human models of oestrogen deficiency such as post-menopausal women and the recently described male with an oestrogen receptor mutation (Smitth et al., 1994) demonstrate that the most significant decreases in BMD attributed to oestrogen are found at predominantly trabecular sites such as the spine and hip.

We found lower BMD in the treated group at 24 weeks of age, an age which represents mature female rats, indicating that there was no catch-up in bone mineralization after treatment withdrawal. However, it may be speculated that catch-up takes longer than 14 weeks and will occur later, up to the 36th week of life. In men with a history of delayed puberty, Finkelstein et al. (1996) did not find improvement with time in bone density after the mid-twenties age group. Another option to explain the decrease in bone density in the treated group is by bone loss caused by prolonged oestrogen deficiency induced by GnRH antagonist therapy, similar to the osteopenia seen in patients with hypogonadism. In this case the decrease in bone mineralization is caused by an increase in bone resorption. However, from previous studies using this GnRH antagonist (Org. 30276; Organon) in female rats, it was clearly shown that the recovery period is not longer than 4 weeks, whereas rats in our study were followed until 24 weeks of age, 14 weeks after discontinuation of the therapy.

In summary, bearing in mind that the processes of puberty and bone mineralization in rats are not identical to those in humans, the present study supports the notion that proper timing of puberty has a major role in allowing attainment of peak bone mass.


    Acknowledgments
 
The authors are grateful to John Leach from Organon, Australia and to Organon, OSS, The Netherlands, for providing the GnRH antagonist preparation (Org. 30276) and for performing the peripheral QCT studies. This study was funded by Pharmacia Upjohn (Sweden).


    Notes
 
5 To whom correspondence should be addressed at: E-mail: ibs{at}amiad.org.il Back


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Submitted on November 1, 1999; accepted on March 28, 2000.





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