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EFFECTS OF EXOGENOUS ESTROGEN AND TESTOSTERONE ON REPRODUCTIVE STRUCTURES AND SPERMATOGENESIS IN THE MALE RAT Ryan Olney 1, Anna Ballard 2, Cyndi Goberdhan 2, Ben Cooper 2, Debora Christensen (Mentor). Dept of Biology 1, Dept of BCMB 2, College of Arts & Sciences Results Methods Introduction Results (continued) Discussion Testosterone and estrogen are both important to the development and maintenance of male reproductive organs. Leydig cells produce testosterone, which causes development of secondary sex characteristics. Testosterone stimulates spermatogenesis by binding to the Sertoli cells that line the straight tubuli recti of the seminiferous tubules. Estrogen receptors are found in the seminiferous tubules. Estrogen is essential for normal spermatogenesis; however, elevated levels of estrogen can cause reproductive dysfunction in males. Bromodeoxyuridine (Brd-U) is used to visualize proliferating cells because of its ability to incorporate into newly synthesized DNA. Hypothalamus Sertoli Cells FSH LHInhibin Testosterone Anterior Pituitary GnRH Estradiol Inhibitory Stimulatory Leydig Cells O CH 3 OH HO CH 3 OH Aromatase TestosteroneEstradiol 12 reproductively mature male Sprague-Dawley rats were given one of three hormonal implants: empty (control, contained no hormone), estrogen (0.040mg), or testosterone (0.036mg) Implants were implanted into the subcutaneous tissue between the shoulder blades and remained in place for 3 weeks. 2 hours prior to sacrifice, rats were injected with Brd-U (1ml/100g). Rat testes were excised, lengths and weights recorded, and tissues fixed in formalin for immunohistochemistry. Testes were embedded in paraffin, sectioned (7.5m), and stained with H&E and Brd-U. Study Objective Investigate how exogenous estrogen and testosterone affect maintenance of reproductive organs in the adult male rat Investigate how estrogen and testosterone affect spermatogenesis in the adult male rat. Males with testosterone implants had gonads that were 50% lighter than controls. Estrogen-implanted male gonads were 75% lighter than controls. Elevated testosterone or estradiol decreased body size. Brd-U staining showed evidence of mitotic division, and hence spermatogenesis, in all groups. There was an overall reduction in spermatogenesis in hormone-treated rats when compared with controls. Both elevated estrogen and testosterone depressed normal spermatogenesis. Results indicate that rats might be more sensitive to estradiol than testosterone or that the testosterone:estrogen ratio may be more important than levels of either hormone individually. Rat #Implant Tx Weight 1 (g) Weight 2 (g) Gonad Weight (g) Length Gonad (mm) 6Empty4124344.202822.5 16Empty4004033.881123 8Estradiol3843300.661515 12Estradiol276.12821.258821 20Estradiol3133081.16817 1Testosterone305.53422.571219 13Testosterone283.83311.76120.5 19Testosterone298.43111.86418.5 Table 1. Gonads were significantly lighter in males that received estradiol implants (p=0.0015) when compared to controles. Males that received testosterone had lighter testes as well, but this difference was not significant (p=0.07). Figure 2. Average body weights before and after treatment with empty, estradiol, and testosterone implants. Rats with both steroid hormones were an average of 15% lighter than those receiving no additional hormone but not different from their pretreatment weights 3 weeks earlier. Acknowledgements Original experimental design, construction of hormonal implants, all rat surgeries, and testicular measurements were performed by Ryan Olney, Anna Ballard, and Cyndi Goberdhan. Liz Stucker for provided the rats. Thanks to Dr. Christensen for her dedication, mentoring, and support throughout the entire experiment. Figure 3. Histological sections of mature male Sprague-Dawley rat testis stained with Brd-U revealed that there an increase in spermatogenesis in the control males (far left) when compared to those receiving estrogen (middle) and testosterone (far right). Rats with additional steroid hormones had a visually-distinguished decreased seminiferous tubule size. Estradiol treated testis showed predominance of tubules with round spermatids and decreased cell height, whereas empty showed elongating spermatids that were more abundant throughout the tissue. Magnification x100. Figure 1. From left to right: Silastic implants. Ryan Olney performing rat surgery with Dr. Christensen. Rotary microtome used for sectioning tissues.