Serum and Plasma Levels Of Total And Free Testosterone And Of Sex Hormone Binding Globulins In Rats Growing In The Below Sea Level Environment Of The Jordan Valley
F El-Migdadi, F Qaw
below sea level, jordan valley, sex hormone binding globulins, testosterone
F El-Migdadi, F Qaw. Serum and Plasma Levels Of Total And Free Testosterone And Of Sex Hormone Binding Globulins In Rats Growing In The Below Sea Level Environment Of The Jordan Valley. The Internet Journal of Endocrinology. 2008 Volume 5 Number 2.
This study was conducted to examine the effect of the below sea level environment of the Jordan Valley (JV) on serum and plasma levels of total and free testosterone (T) and those of sex hormone binding globulins (SHBG) in male and female rats. Two groups of male rats (total n=16) and another two groups of female rats (total n=16) were selected from litters prepared from housed and bred Sprague-Dawley rats. One group of 8 male and another of 8 female rats were kept at above sea level environment in Irbid City (IC) in the North of Jordan, while the rest 8 male and 8 female animals were transferred and kept to grow till maturation (124 days of age) in the below sea level environment in the JV. Serum and plasma levels of free and total T were higher in rats of both sexes growing in the JV compared to those maturing in IC. These data clearly suggest that environmental factors of the JV rather than the genetic background of the rats that play a significant role in T production and perhaps its clearance. Plasma levels of SHBG were similar among female rats of the two locations, while these levels in male rats were lower in rats growing in the JV. This study discusses the relationship between plasma levels of SHBG and serum T in rats and it also sheds more light onto the effects of the environmental factors of the below sea environment of JV on T homeostasis.
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
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
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
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
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
The author wishes to thank Ms. Heyam Kawasmeh and Mr. Muawiah Shatnawi for their assistance in sample preparation and hormonal measurements. The assistance of Dr. Yousef Qaoud on statistical analysis is appreciated. Funds from the Deanship of Scientific Research at Jordan University of Science and Technology were used to conduct these experiments and prepare this manuscript.