Brooke Gushen Nathan Garvin, Biology 473, Section 3 Introduction

The Effects of Ovariectomy on Endocrinology March 30, 2012

The reproductive organs of any animal depend greatly on the hormones secreted by the endocrine system. The hypothalamus, anterior pituitary gland, and the ovaries are all important in the hormone regulation leading to reproduction in females. With the hypothalamus and anterior pituitary gland located in the brain and the ovaries located in the pelvic region, the communication from the brain is sent via three specific hormones. The signal begins in the hypothalamus, where gonadotrophin releasing hormone (GnRH) is produced. GnRH communicates with the anterior pituitary gland stimulating the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) into the blood stream. These two hormones then travel down to the ovaries where they trigger the production of estrogen, leading to the onset of ovulation. After ovulation occurs, a negative feedback loop circles back up to the brain, inhibiting further production of the three hormones. [1] These three hormones are part of the signal heading down from the brain, but once in the ovaries and uterus, estrogen, progesterone, and inhibin begin the second phase of signaling. The surge in estrogen, triggered by the presence of LH and FSH, leads to ovulation. Estrogen, as well as progesterone, are also important in preparing the uterus for the implantation of the fertilized egg. Inhibin comes into play post-ovulation and is responsible for the negative feedback loop mentioned earlier, halting the production of GnRH, FSH, and LH. [1] In this experiment, surgeries were performed on female rats, removing one or two ovaries. There were also sham (control) surgeries, where neither ovaries were removed, done to account for any results due to the physical trauma that comes with surgery. The removal of one ovary, a unilateral ovariectomy, means that there is one less location to receive input from LH and FSH and produce an output of estrogen. With one ovary gone and reduced estrogen levels, the other ovary may be affected. Perhaps the extra LH and FSH would be rerouted to the single ovary, leading to a higher production of estrogen than normally expected in a healthy female. Also, the level of inhibin may not be enough to produce enough inhibition

for two ovaries worth of LH and FSH. In the case that both ovaries are removed with a bilateral ovariectomy, the LH and FSH hormones would have no sites to act on. Estrogen production should be completely stopped in this pathway, allowing none to reach the uterine horn. There is also no way for inhibin to back regulate this production of GnRH, LH, and FSH. Perhaps this would lead to continued production and increased levels of LH and FSH in the bloodstream. This research may be important those scientists looking into the effects of ovariectomy performed in the case of ovarian cancer. Women with developing tumors or even those at risk are considering ovariectomies as a treatment or prevention plan for ovarian cancer. This surgery can greatly increase the chance of survival in ovarian cancer patients. [2] So this surgery is important to cancer treatment in humans, but the question is what are the physiological repercussions after this surgery is carried out on the patient? This experiment aims to gain a better understanding towards that answer. We expect that when comparing the control and unilateral surgeries, the ovary of the unilateral surgery will be heavier than one of the control ovaries due to the ovary receiving additional LH and FSH and compensating for the estrogen loss of the removed ovary. The uterine horn of the unilateral surgery should weigh less because there should be less available estrogen to maintain the uterine lining of both sides. The pituitary gland should weigh slightly less because there is less inhibin available for the negative feedback, therefore the anterior pituitary gland may continue producing LH and FSH. We expect that when comparing the control and bilateral surgeries, the uterine horn of the bilateral surgery should weigh less because there is no LH and FSH input to produce estrogen to maintain the uterine lining at all. The pituitary gland should weigh less because there is no inhibin available for the negative feedback, therefore the anterior pituitary gland may continue producing LH and FSH. We expect that when comparing the unilateral and bilateral surgeries, the uterine horn of the unilateral surgery should weigh more than that of the bilateral surgery because although there is less available estrogen, the bilateral should have little to no estrogen due to the lack of available LH and FSH. The pituitary gland of the bilateral should weigh less because again there is no inhibin coming from the ovaries for the negative feedback.

Method: In this lab, ovariectomies, the surgical removal of one or two ovaries, were performed on female rats. Xylazine, ketamine, and bupivacaine were used together for anesthesia during the operation. Sterile procedures were followed as discussed in the lab manual. Four weeks after the surgeries, autopsies were performed, removing and weighing the ovaries, uterine horn, and pituitary gland of each rat. The protocol presented in the lab manual was followed exactly. [2] Results: Graph 1: Mean Ovarian Weights of Control and Unilateral Surgeries
0.140 0.120 0.100 Weight (g) 0.080 Control 0.060 0.040 0.020 0.000 Surgery Type Unilateral

Results are based on calculated weights of the ovaries found in the rats four weeks after the surgeries. Above are the ovarian weights of the control and unilateral surgeries (bilateral is excluded because both ovaries had been removed). The error bars are included to show the range in the data. SEcontrol=0.007; SEunilateral=0.008. Statistical significance is determined at = 0.01 of a two-tailed t-test. No statistical significance found in ovarian weights.

Graph 2: Mean Uterine Horn Weights of Control, Unilateral, and Bilateral Surgeries
0.700 0.600 0.500 Weight (g) 0.400 0.300 0.200 0.100 0.000 Control Unilateral Bilateral

Surgery Type

Results are based on calculated weights of the uterine horns found in the rats four weeks after the surgeries. Above are the ovarian weights of all three surgeries. The error bars are included to show the range in the data. SEcontrol=0.045; SEunilateral=0.059; SEbilateral=0.033. Statistical significance is determined at = 0.01 of a two-tailed ttest. Statistical significance was found between control vs. bilateral and unilateral vs. bilateral uterine horn weights.

Graph 3: Mean Pituitary Gland Weights of Control, Unilateral, and Bilateral Surgeries
0.015 0.014 0.014 Weight (g) 0.013 0.013 0.012 0.012 0.011 0.011 Surgery Type Control Unilateral Bilateral

Results are based on calculated weights of the uterine horns found in the rats four weeks after the surgeries. Above are the ovarian weights of all three surgeries. The error bars are included to show the range in the data. SEcontrol=0.002; SEunilateral=0.001; SEbilateral=0.001. Statistical significance is determined at = 0.01 of a two-tailed ttest. No statistical significance found in pituitary gland weights.

In Graph 1, the mean ovarian weights of the control and unilateral surgeries are shown. The ovarian weight of the rats of the unilateral surgery showed an increase, not significant, from that of the control surgery (tobs=2.268, df=40, 2=0.01). In Graph 2, the mean uterine horn weights of the control, unilateral, and bilateral surgeries are shown. The uterine weight of the rats of the unilateral surgery showed a slight increase, not significant, from that of the control surgery (tobs=0.217, df=40, 2=0.01). The uterine weight of the rats of the bilateral surgery showed a significant decrease from that of the control surgery (tobs=5.928, df=27, 2=0.01). The uterine weight of the rats of the bilateral surgery showed a significant decrease from that of the unilateral surgery (tobs=4.649, df=29, 2=0.01). In Graph 3, the mean pituitary gland weights of the control, unilateral, and bilateral surgeries are shown. The pituitary gland weight of the rats of the unilateral surgery showed a slight decrease, not significant, from that of the control surgery (tobs=0.410, df=40, 2=0.01). The pituitary gland weight of the rats of the bilateral surgery showed a slightly greater decrease, not significant, from that of the control surgery (tobs=0.812, df=25, 2=0.01). The pituitary gland weight of the rats of the bilateral surgery showed a slight decrease, not significant, from that of the unilateral surgery (tobs=0.666, df=27, 2=0.01). There were only statistically significant changes seen in the weights of the uterine horns. Discussion: As shown in Graph 1, as expected there was an increase in ovarian weight in the unilateral treatment, however it was not significant. Since we were only expecting a change in hormones, a large change in weight may not occur, but a small change may which is what we observed. In Graph 2, there was little difference between the uterine horn weights of the unilateral and control treatment. In fact, there was a very slight increase in uterine weight in the unilateral condition compared to the control condition, which was not expected. This may be due to the increase in ovarian weight which led to an overabundance of estrogen in the one side of the uterine horn. This may have caused enough of a weight change while over-maintaining the uterine lining to show an increase in

overall weight. Comparing the uterine weight of the bilateral surgery to both the unilateral and control surgeries, there is a statistically significant decrease in uterine weight as expected. Since the unilateral was receiving at least some hormone, even if it had less of an area to process and further produce estrogen, it was still capable of producing estrogen. The significant decrease in the weight of uterine horn of the bilateral surgery suggests that removal of both ovaries completely cuts off the transmission of FSH and LH to the female reproductive organs. Without these hormones, the uterine horn is unable to produce estrogen and maintain its uterine lining. This should lead to a complete stop in ovulation and possible deterioration of the uterine lining, leading to the significant decrease in mass. Graph 3 shows the changes seen in the weights of the pituitary gland across the three surgery treatments. With each ovary removal, a very slight decrease in mass of the pituitary gland was seen, however it was not significant. Since this gland, like the ovary, is only seeing a change in hormones, unlike the uterine horn, which may actually losing uterine lining, there are only small decreases seen. The decreases in weight that were seen though were consistent with what we expected. Since there was less inhibin coming back from unilateral rats, there may have been less negative feedback as expected leading to an overproduction of LH and FSH. Then in the bilateral rats, there was no inhibin coming back from the reproductive organs, meaning there was no negative feedback leading to even more of an overproduction of LH and FSH and therefore a greater decrease in mass. These changes were not found to be significant though in comparison to the entire weight of the pituitary gland. I feel that since the ovaries and pituitary glands should only be varying in hormone concentrations, this change in weight is pretty insignificant compared to the weight of the entire organ. If the hormone levels could actually be measured, instead of monitoring them by weight, we may have been able to see the significant change between the different treatments. If there is a way of staining for the hormones and comparing the different organs in slides under a microscope, we may have been able to see increases in LH, FSH, and estrogen in the remaining ovary of the unilateral treatment, or we may have been able to see a decrease in the LH and FSH present in the pituitary gland and maybe even an absence of inhibin.

I feel that the results found are decently consistent with our hypotheses, however maybe not as significant as hoped for. Error may be due to the averaging of data across many sections calculated weights. As you can see in Graphs 1, 2, and 3, the error bars have a very wide range. This is due to having mice of varying sizes, therefore having varying sizes in endocrine and reproductive organs. If we could throw out the data of the outliers and focus on the rats within a narrower range, we may have been able to find greater significance in our results. From our findings, we found that in unilateral ovariectomies, there are some changes in endocrinology, however they are not too significant. The bilateral ovariectomies showed vast changes in the weight of the uterine horn in the female rats, expected to be due to a broken link in the pathway of hormone transmission from the hypothalamus to the reproductive organs. It is possible that similar results would be found in human ovarian cancer patients considering an ovariectomy surgery as a treatment. Due to the vast changes in hormones and the effects on the uterus observed in the bilateral ovariectomy, researchers and doctors may want to consider referring patients to the bilateral treatment only in extreme cases. References: [1] Overview of Reproductive Hormones. Department of Biology, The Pennsylvania State University 2012. [2] Kurian, A., Sigal, B., and Plevritis, S. Survival Analysis of Cancer Risk Reduction Strategies for BRCA1/2 Mutation Carriers. Journal of Clinical Oncology 28: 222-231, 2010. [3] Biol. 473 Rodent Survival Surgery Protocol Handout. Department of Biology, The Pennsylvania State University 2012.