Introduction and Objectives

Our previous studies had suggested that there was a significant effect of the Jordan Valley (JV), which is located at about 280-400 meters below sea level, on serum levels of various sex hormones including testosterone (El-Migdadi et al. 1999; El-Migdadi et al. 2000; Banihani et al. 2001; El-Migdadi et al. 2004; El-Migdadi et al. 2005. However, since these studies were performed using human subjects, there was no concluding evidence on whether such an effect is due to the environmental factors of the JV, such as temperature, humidity, altitude, nutrition, socio-economic and cultural factors or to the genetic background of the inhabitants of the JV. The present work was designed to study the effect of the below sea level environment of the JV on serum levels of total and free testosterone (T) and of sex hormone binding globulins (SHBG) in male and female rats. By using rats in this study, the major environmental factors of temperature, humidity, food and drink can all be accounted for and were well controlled. The barometric pressure of the JV was not controlled.

Experimental Design and Methods

A group of Sprague Dawley rats, originally purchased from Harlan Sprague Dawley (Indianapolis, IN, U.S.A.), were used in this experiment. These rats were housed and bred at our Animal Facility in the Medical School of Jordan University of Science and Technology in the North of Jordan near Irbid city (IC). From all litters obtained, four litters, each having 4 male pups, and another four litters, each having 4 female pups, were selected. The selection process took place either on Day 1 or Day 2 after delivery of pups from their perspective pregnant mothers over a two-day period in November of 2000 (another similar experiment was performed in November of 2008; similar data were reproduced). From each of the selected litters, two pups were kept at our Animal Facility at the University (IC group), and the other two were transferred to our Animal Facility located in the JV (JV group). A total of sixteen rats, eight males and eight females formed the IC group of animals; another sixteen, eight males and eight females formed the JV group. To clarify this further, it is emphasized here that among male rats as well as among female rats, there were four siblings (pups from the same litter), two of which in IC group and another two in the JV group. Each rat was housed individually in a temperature-(22–28°C) and a light-controlled (12 h light, 12 h dark), specific-pathogen-free environment. It is emphasized here that the temperature in the JV group was closer to the upper allowed limit, while in the IC group it was closer to the lower allowed limit. This was evident from the repeated measurements of the atmospheric temperatures in the rooms housing the rats at both locations at various times on multiple days of the experiment (data is not shown). This small variation in the atmospheric temperature was due to the efficiency of the conditioning systems at both locations. This setup of temperatures at both locations was planned for in order to better simulate the real difference in the temperatures between IC and the JV. According to the National (Jordan) Geographic Society, the atmospheric temperatures during November in IC is about 4-6 °C lower than those temperatures in the JV.

Animals were fed standard rat chow ad libitum (Teklad, Madison, WI, U.S.A.) and water was available at all times. Body weight was measured for each rat at Day 30, Day 60, Day 124 (Table 1). The initial body weight measurements at Day 0 were not taken. The experimental protocols followed our Jordan University of Science and Technology (JUST) School of Medicine Animal Care and Use.

At Day 120, rats from the JV group were brought back to our Animal Facility at JUST. At Day 124, all rats from the two groups were killed by decapitation between 0900 and 1200 h. Serum and plasma from trunk blood were prepared and stored at -20°C until hormonal analyses took place. Citrate tubes were used for serum preparation. The DSL-4600 ACTIVE Coated-tube Immunoradiometric Assay (IRMA) kits were used for hormonal measurements. The Automatic Gamma Counter (LKB-Wallac Clini Gamma 1272-001) located at the Clinical Laboratories in Princess Basma Teaching Hospital in IC was the device used to measure the radioactivity of samples and to calculate their concentrations. Serum and plasma levels of SHBG were measured enzyme-linked immunosorbent assay (ELISA) (Lewis et al. 1999). The results were obtained in ng / ml for total T, pg / ml for free T, and nmole / l for SHBG.

Statistical analyses of hormonal and SHBG data were done by Wilcoxon Signed Ranks Test. In addition to the mean of data in a group, standard deviation was also calculated as seen in Tables 2 and 3, where raw data are shown. Tables 4 and 5 show the Z plus the Asymp Sig. (2-tailed), where numbers that are less than 0.02 were considered significant while those that are above 0.05 are not significant.

Results and Discussion

As seen in Table 1, rats growing in the JV had gained less body weight than those growing in IC. This is evident from the raw data at all 3 times of measurements, Day 30, Day 60 and Day 124. This difference in the body weight was observed in both sexes of the rats and it was significant when subjected to statistical analysis (Data not shown). It seems likely that the JV had a significant effect on the rat body growth of both sexes during development and maturation. However, it is not possible to relate this body weight gain to muscle mass growth or to adipose tissue growth. The sure thing is that there was more weight gain in those rats growing in the above sea level environment in IC compared to those in the JV. Further studies are needed to clarify this difference in body weight growth of rats between the two locations before one can make more and detailed conclusions.

As seen from Tables 2 and 4, serum levels of free and total T as well as those of serum SHBG in female rats were all lower than their corresponding levels at both locations above and below sea level environments (in IC and the JV). Plasma levels of free and total T were also lower in female rats than their corresponding levels at both IC and the JV (Tables 3 and 4). This tendency of low levels in the female animals compared to those in male rats was not seen for plasma levels of SHBG. Tables 3 and 4 show that plasma levels of SHBG were similar in both female and male rats growing in IC, while in the JV, plasma levels of SHBG were significantly higher in female rats compared to those in male animals. It is suggested here that measurements of SHBG and thus their levels seem to be affected by the blood coagulation that takes place following blood collection. In other words, serum preparation and removal of blood clotting factors have had a profound effect on SHBG and/or on some other factors and thus influencing SHBG stability and their levels. Sex hormone binding globulin (SHBG) has been shown to be a major determinant of testosterone clearance in the primate (Plymate et al. 1990). In adult men, there is about 45% of the total plasma testosterone that is bound with high affinity to sex hormone-binding globulin (SHBG), 50% is loosely bound to albumin, 1-2% to cortisol-binding globulin, and less than 4% is free (Winters 2004). It is clear why SHBG level in plasma is believed to be a strong predictor of the total testosterone concentration. Many factors have a significant effect on the level of SHBG and subsequently can similarly influence the total testosterone concentration. These factors include, but not limited to, ageing, estrogens and hepatitis, which are known to increase SHBG. On the other hand, obesity, glucocorticoids and androgens decrease SHBG. The lower body weight of rats, reported herein (Table 1), for those growing in the below sea level environment of the JV compared to those growing in IC at above sea level, supports this phenomenon of the effect of obesity on plasma SHBG in male rats. As seen from Table 5, plasma levels of SHBG in male rats growing in the JV were lower than those in male animals growing in IC, where rats had gained more body weight (392.13±11.667 versus 349.63±11.262). Clearly, data from serum determination of SHBG suggested different conclusion with rats growing in the JV have higher serum levels of SHBG compared to those growing in IC (Table 2). However, these data are not likely to be used for discussions and conclusions, since as stated above there is a profound loss of SHBG during preparation of serum from trunk blood of experimental animals. In addition, the loss of SHBG during this process of serum preparation seems to influence more the female rats than the males (Tables 2 and 5).

Serum and plasma levels of free T in both female and male rats growing in IC were lower than their corresponding levels in animals maturing in the below sea level environment in the JV (Tables 2, 3 and 5). In a similar fashion, all serum and plasma parameters for total T were also higher in rats growing in the JV. This clearly shows a profound effect of the environmental factors, perhaps the barometric pressure and/or the atmospheric temperature, on the these blood levels of rat T. These results indicate that the genetic determinants are of a lesser sound effect on the rat blood levels of T than the environmental factors. It had been suggested by Santner et al. (1998) that environmental factors rather than genetic factors are responsible for the differences between Caucasian and Chinese men in terms of their androgen production and the significant differences in total testosterone, free and weakly bound testosterone, sex hormone-binding globulin levels, and testosterone production rates. Our data are in line with these findings in man.

Raw data of free and total testosterone as well as those of SHBG at both locations above and below sea levels environments in IC and in the JV had also shown that there is a substantial variation in rat serum and plasma levels. Ring et al. (2005) had indicated that testosterone and SHBG concentrations have substantial genetic variation in human. Individual differences in serum sex hormone concentrations have a profound scientific and clinical interest. Variations in sex hormone concentrations have also been associated with obesity and bone density in both men and women (Morley et al. 1997; Riggs et al. 2002; Bray 1999. The reported herein data of testosterone and of SHBG had also indicated that there is a wide individual variation in their serum and plasma levels in rats and these levels seem to be influenced by, but not limited to, below sea level environment, body weight, temperature and barometric pressure.

Figure 1

Table 1. Raw Data of Body Weight (grams) of Male and Female Rats Growing in Both Irbid City (IC) and the Jordan Valley (JV)

Figure 2

Table 2. Raw Data of Serum Levels of Free and Total Testosterone (T) and of Serum Sex Hormone Binding Globulins (SHBG) in Female and Male Rats Growing in Irbid City (IC) and in those Growing in the Jordan Valley (JV)

Figure 3

Table 3. Raw Data of Plasma Levels of Free and Total Testosterone (T) and of Serum Sex Hormone Binding Globulins (SHBG) in Female and Male Rats Growing in Irbid City (IC) and in those Growing in the Jordan Valley (JV)

Figure 4

Table 4. Statistical Analyses of Data by Wilcoxon Signed Ranks Test

Figure 5

Table 5. Statistical Analyses of Data by Wilcoxon Signed Ranks Test

Acknowledgments