Experiment and Testing of Testosterone Influence on Rats

Testosterone is an androgenic steroid hormone that is responsible for regulating normal physiological processes at all stages of an organism’s life. In females, testosterone is essential for healthy bone density, normal ovarian function, and fertility. It is produced in the ovaries, peripheral tissues and the adrenal gland (Yao, et al., 2019). In males, testosterone is produced in the testes at a much higher quantity and is essential for sex differentiation, post pubertal development, spermatogenesis, and fertility (Nassar & Leslie, 2019). This investigation focuses on testosterone produced and utilized by male lab rats. The effects of testosterones are first observed in embryonic development where it participates in the initiation of sexual differentiation of the penis, scrotum, seminal vesicles, and other male structures. After birth, testosterone secretion temporally spikes, then becomes quiescent until puberty (Hill, Wyse, & Anderson, 2016). The increase in testosterone during puberty regulates secondary male characteristics which may include change in hair patterns, vocal changes, growth spurts, and skeletal muscle growth (Nassar & Leslie, 2019). The production and secretion of testosterone is coordinated by the hypothalamic-pituitary-gonadal axis in which gonadotropin-releasing hormone (GnRH) is first released from the hypothalamus in pulses. GnRH then travels down the hypothalamo-hypophyseal portal system to stimulate the secretion of two gonadotropic hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH), from the anterior pituitary gland. LH and FSH circulate through the animal’s blood until they bind to receptors in the gonads. Receptors on Leydig cells located in the testes bind LH and stimulate the synthesis of testosterone from preexisting cholesterol. Receptors on Sertoli cells located in the seminiferous tubules bind FSH and interact with testosterone to stimulate spermatogenesis (Hill et al., 2016). Testosterone levels remain relatively consistent via negative feedback, with testosterone acting as an inhibitor if levels reach a certain threshold. This steady maintenance of testosterone slightly declines with old age; however, it still sustains the continuous production of sperm. The purpose of this experiment was to investigate the effects testosterone has on normalized organ mass in male lab rats treated with castration/sham implant (cast/sham), castration/testosterone implant (cast/test), or intact testis/testosterone implant (intact/test). The various organ masses under investigation included the heart, liver, kidneys, seminal vesicles, and the right gastrocnemius.

There is a copious amount of research devoted to the study of testosterone in order to better understand its physiological effects. Previous research has determined that testosterone plays an important role in the growth and size of several organs including the liver, kidneys, seminal vesicles, and skeletal muscles such as the gastrocnemius. Testosterone has been found to increase liver and kidney mass due to its role in stimulating hepatocyte proliferation and androgen-dependent renal hypertrophy (Wong, et al., 2016; Koya, Fujii, Yambe, & Tahara, 2015). The growth and development of seminal vesicles is also androgen dependent. The synthesis of proteins contributing to seminal plasma is regulated by the seminal vesicles and when there is a deficiency in testosterone, there is a decrease in seminal vesicle growth and activity. The opposite would be true if there is an increase in testosterone (Welsh, et al., 2010). Another study indicates that testosterone supplementation increases skeletal muscle mass and decreases fat mass by stimulating myogenic differentiation and inhibiting adipogenic differentiation (Singh, Artaza, Taylor, Gonzalez-Cadavid, & Bhasin, 2003).

The purpose of this experiment was to investigate the effects testosterone has on normalized organ mass in male lab rats treated with cast/sham, cast/test, or intact/test. The various organ masses recorded for each rat included the heart, liver, kidneys, seminal vesicles, and the right gastrocnemius. Based on previous research it was hypothesized that an increase in testosterone via implant, would lead to an increase in the normalized mass of the heart, liver, kidney, seminal vesicle, and gastrocnemius as well as decrease adipose fat deposition. The body mass percent change was not expected to be significantly different between the three treatment groups.

Materials and Methods

This experiment was carried out on seventeen male lab rats that were randomly divided into three treatment groups. Seven rats were included in the castration/sham implant (cast/sham) group, six in the castration/testosterone implant (cast/test) group, and four in the intact testes/testosterone implant (intact/test) group. A total of thirteen rats were castrated and all seventeen rats underwent surgery to receive an implant. Before operation, individual weights of each rat were recorded. To induce unconsciousness each rat was anesthetized with 5% isoflurane. 1-3% isoflurane was maintained for the duration of the surgery with 100% oxygen to obtain a surgical plane of anesthesia. 0.6 - 0.7 mg/kg of buprenorphine was then slowly administered via subcutaneous injection. Respirator rates was carefully monitored throughout the surgery to maintain a surgical level of anesthesia and proper aseptic techniques were implemented to avoid infection. Rats that were castrated and received an implant were prepped by shaving the scrotum and a 2 cm square portion on the back between the shoulder blades. Preparation of the surgical site for castration was then continued by applying three alternating layers of betadine and alcohol, starting and ending with betadine. A 1.5 cm midline incision was made along the ventral surface of the scrotal sac to expose the one of the testes. The protective tunica albuginea covering the teste was dissected to release the teste. The teste and epididymis were gently removed until the spermatic cord could be identified. Hemostats were used to gently squeeze the spermatic cord which was followed by firmly tying off the spermatic cord with 2-0 silk thread. The spermatic cord was cut distal to the tie removing the teste and epididymis from the surgical field. The severed spermatic cord was then tucked back into the hole of the tunica and the remaining teste was located and removed using the same procedural techniques. After both testes were removed, the scrotal incision was closed using square knot sutures. The surgical site was thoroughly cleaned with sterile saline and the testes were trimmed and weighed.

Each rat received either a sham or a testosterone implant. Rats that were castrated received the implant directly after castration and rats that were not castrated were anesthetized then shaved following the same procedure. The surgical field for implantation was sterilized using the same application of betadine and alcohol technique. A small 0.5 cm transverse incision was made just below the shoulder blades. Hemostats were then used to tunnel posteriorly under the skin to provide a space for the implant. Prior to surgery the implants were stored in BSA until one hour before surger where they were transferred to ethanol. The implant was rinsed in sterile saline then inserted into the surgical field. The incision was closed using square knot sutures, then sterilized.

Post-surgical care was monitored by the lab teaching assistants. The rats were euthanized, and the experiment was terminated three weeks after the operations. To induce unconsciousness each rat was anesthetized with 5% isoflurane and 100% oxygen. An intracardiac injection of 0.5 mL fatal plus was administered, then dissection was performed. Organs removed included the heart, liver, kidneys, seminal vesicles and the right gastrocnemius. The wet weight of each organ removed was recorded. Dry weights were obtained after placing the organs in an incubator for 24 hours at 50ºC. Data from each rat was then collected and analyzed using JMP statistical software.

Results

Data collected from this experiment indicates that testosterone has a significant impact on the normalized mass of wet seminal vesicles, dry kidneys, and dry seminal vesicles. Rats that received either cast/test or intact/test treatment had significantly higher normalized masses for wet seminal vesicles, dry kidneys, and dry seminal vesicles then rats that were treated with cast/sham. Normalized mass of wet kidneys, as well as, wet and dry heart, liver, and gastrocnemius resulted in no significant difference between treatment groups. Body mass percent change also showed no significant difference. Additional data collected and represented in Table 1 indicates that testosterone within in the implants did in fact diffuse through the course of the experiment, so it can be assumed that testosterone plays contributing role to these changes.