Kinnari Patel Brenna Hill BIOL 473, Section 005 Reproductive Endocrinology Lab INTRODUCTION The overall purpose

of this lab was to examine the effects of varying levels of reproductive hormones on the brain-ovarian axis. The variation of circulating hormone concentrations was achieved by performing surgeries to remove one or both of the hormone-secreting gonads. The groups included rats which underwent surgery to remove no gonads (control), one gonad (unilateral ovarectomy), or two gonads (bilateral ovarectomy) on female rat test subjects. After the effects of the surgeries were given time to enact their changes on the body and other organs of the rat, the rats were euthanized and autopsied in order to determine the weights of the uterus, pituitary, and any remaining ovaries. These values can be used to analyze the effects of the varying hormone levels on these other organs of the body. The brain-ovarian axis works to tightly and accurately regulate the function of the female reproductive system, namely the preparation of the uterus for implantation, ovulation, and negative feedback to control the timing of these events. Important hormones directing the function of this process include gonodotropin releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), estrogen, progesterone, and inhibin. GnRH is produced in the hypothalamus, which is stimulated by nerve endings in the brain. Upon stimulation, the hypothalamus releases GnRH, whose target is the anterior pituitary gland. After receiving the signal from GnRH, the anterior pituitary secretes both FSH and LH which travel to the ovary via the blood stream. The rising levels of FSH and LH at the ovary stimulate an increase in production of estrogen and subsequent ovulation. The surrounding cells of the estrogen-producing follicle remain on the ovary and develop into the corpus luteum. This structure secretes both estrogen and progesterone which maintain the uterine lining in case of implantation. Additionally, inhibin, which is a peptide hormone, is released

by the ovaries. Rising levels of this inhibin along with estrogen serve in a negative feedback loop to the anterior pituitary to inhibit further release of FSH and LH. In humans, this feedback mechanism prevents a second implantation while the first matures. (Silverthorn, 2007) Examining the effects of reproductive hormone levels on organs in rats can help scientists, researchers, and health professionals learn more about the mammalian response to abnormalities in hormonal pathways caused by disease or defect. Such insight has the potential to improve research efforts regarding the diagnosis and treatment of hormonal disorders in humans. It is hypothesized that rats that undergo unilateral ovarectomies will lose mass in the uterus but gain mass in the remaining ovary and pituitary when compared to control. It is also hypothesized that rats that undergo bilateral ovarectomies will also lose uterine mass and gain pituitary mass when compared to the control. Finally, it is hypothesized that the uterus of bilateral ovarectomy rats will weigh less than those of the unilateral ovarectomy rats and the pituitary will weigh more. METHODS Three groups of female rats (control, unilateral ovarectomy, and bilateral ovarectomy) underwent surgery, recovery,a and autopsy to collect the data for this experiment. An ovarectomy is defined as the surgical removal of the female gonad from the body. In the unilateral ovarectomy, only one of the ovaries of the rat was removed, while both ovaries were removed in the bilateral ovarectomy. TheseOvarian Mass in Control and the utmost care, using sterile procedures, and the rats surgeries were done with Figure 1: Mean were given both anesthetics and analgesics to minimize pain. After the surgeries, the rats were given
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Unilateral Ovarectom y

Figure 2: Mean Uterine Mass in Control, Unilateral, and Bilateral Ovaractomies

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about two weeks to recover and for the effects of the ovarectomy and subsequent changes in hormone
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levels to take place. The rats were then euthanized with carbon dioxide and autopsies were performed
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Mass (kg)

to determine the weight of the uterus, pituitary gland, and any remaining ovaries (Waters, 2003). The
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Control Unilateral Bilateral

weights of these organs were compared using statistical analysis. Unilateral

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RESULTS
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Figure 1: The average weights and standard error of a single ovary in female rats autopsied two weeks post-operation of sham and unilateral ovarectomy surgeries.

Figure 2: The average weights and standard error of the uterus in female rats autopsied two weeks post-operation of sham, unilateral, and bilateral ovarectomy surgeries.

Figure 3: Mean Pituitary Mass in Control, Unilateral, and Bilateral Ovarectomies

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Figure 3: The average weights and standard error of the pituitary gland in female rats autopsied two weeks post-operation of sham and unilateral ovarectomy surgeries.

It is demonstrated in Figure 1 that the mean ovarian mass of a female rat which has undergone a unilateral ovarectomy is significantly greater than the average ovarian mass of a control rat

(p=0.001371). Figure 2 shows that the uterine mass in a control about the same as that in a rat which has undergone a unilateral ovarectomy (p=0.445736). It also demonstrates that the uterine mass in a control is significantly greater than that in a rat which has undergone a bilateral ovarectomy (p=4.72029 x 10-8). Lastly, it shows that the uterine mass of the unilateral subjects is significantly greater than that of the bilateral subjects (p=2.2714 x 10-7). In Figure 3, it is demonstrated that the mass of the pituitary in a control rat is about the same as that in the unilateral group (p=0.944842) and the bilateral rat (p=0.2386537). Following the same trend, it is demonstrated that the pituitary mass in the rats which had undergone unilateral ovarectomy is about the same as that of those which underwent bilateral ovarectomy (p=0.4455189) All p-values were evaluated with 2 = 0.01, with values less than 0.01 being significant and values greater than 0.01 insignificant. DISCUSSION The overall purpose of this lab was to examine and analyze the effects of varying levels of reproductive hormones on the ovaries, uterus and pituitary gland in female rats which had undergone sham, unilateral ovarectomy, or bilateral ovarectomy surgeries. The data strongly supports the hypothesis that the average ovarian weight would significantly increase in the rats that underwent the unilateral ovarectomies when compared to rats that underwent the control surgery. Physiologically, when one ovary is removed, the other ovary attempts to compensate for its loss by producing and secreting more female sex hormones, becoming larger as it alone takes on the work of two ovaries. It was also hypothesized that the uterine weights of the rats that underwent the ovarectomies would be significantly less than the weight of the uterus of the control study rats, with the bilateral ovarectomy subjects having a lower mass when compared to the unilateral subjects. The data strongly supported the decrease in uterine mass of the bilateral ovarectomy subjects when compared to the control. It also showed a significant decrease in bilateral ovarectomy uterine mass when compared to the unilateral. The bulk of the uterus is the dense endometrial lining which is stimulated and

maintained by estrogen and progesterone which, in turn, are secreted by the ovaries. Losing both ovaries effectively eliminates this hormonal signal pathway, causing the uterus to atrophy and decrease in mass. Though it was predicted that the loss of a single ovary would be made apparent by a moderately smaller uterus, the data indicates no change. It is possible that the one remaining ovary is able to efficiently hypertrophy to compensate for the loss of the other, resulting in insignificant change in the uterine mass. Another reason behind the lack of change between control and unilateral could be that the rats were only given two weeks to recover from the surgery. Perhaps if more time was allotted for the system to be affected by the hormonal imbalance, the results would be altered. It was also hypothesized that the weight of the pituitary gland of the rats that underwent the ovarectomy treatments would be significantly greater than the weight of the pituitary gland of the control study rats. The pituitary gland is largely regulated by the negative feedback of estrogen and inhibin, which are both produced by the ovaries. When ovaries are eliminated from the system, the effect of this negative feedback mechanism is reduced. Thus, the pituitary gland does not receive any signals to slow down its production of hormones and continues to work, increasing in size and weight. However, statistical analysis of the results indicates that the differences were insignificant. This discrepancy may be justified by experimental error during the extraction of the pituitary gland during the autopsy. Removing and weighing the pituitary gland was an extremely difficult task to perform due to its guarded location under the brain and its small size. Errors in proper extraction could have contributed to the insignificant data obtained. The amount of blood on the extracted gland or potential breakage would be enough to offset the minute weights measured. Perhaps removing more of the brain and then carefully dissecting the pituitary away would lead to more accurate data. Additionally, because each trial involved living organisms, there is inherent variation among individuals in a species, which could have contributed to some outliers. A greater number of trials and more data points could help rectify these errors and obtain more accurate data.

Overall, the lab served to provide a thorough introduction to the physiological mechanisms of reproductive endocrinology topics with specific focus on the female brain-ovarian axis. The results discovered may be used by researchers and medical health professionals to better understand the mechanisms and functions of this pathway. Further studies like this could be used in the future to understand medical pathologies and develop treatments for patients whose hormonal balances are compromised by illness. REFERENCES Silverthorn, Dee Unglaub. Human Physiology: An Integrated Approach (Fourth Edition). San Francisco, California: Pearson Education, Inc., 2007. Waters, John R. Physiology Laboratory Manual with Tear-out Pages (Second Edition). The United States of America: The McGraw-Hill Companies, Inc., 2003.