Effect of Elimination of Vascular Endothelial Growth Factor A (VEGFA) in Granulosa cells on concentrations of Anti-Mullerian Hromone and follicle stages in mice

Katherine Hoffman Author

03/27/2021 Added

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This UCARE project included research that hypothesized that concentrations of AMH would be increased in VEGFA-granulosa conditional KO mice contributing to follicular arrest. Homozygous Vegfa-floxed mice were mated to mice whose genomic DNA genotyped positively for the Sry-Cre allele. We found that the KO mice overall had lower AMH levels compared to the control mice, and will stage follicles to see if there is further support for our findings that follicular development is slowed in the conditional KO mice.

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[00:00:00.608]Hi there, my name is Katherine Hoffman
[00:00:03.180]and I worked with Dr. Cupp this past year
[00:00:05.270]to look at the Effect of the Elimination
[00:00:07.390]of Vascular Endothelial Growth Factor A
[00:00:10.230]in Granulosa Cells on Concentrations
[00:00:12.590]of Anti-Mullerian Hormone and follicle stages within mice.
[00:00:17.404]A little bit of background on this particular study,
[00:00:19.890]is that vascular endothelial growth factor A,
[00:00:22.650]or what we call VEGFA,
[00:00:24.380]is important for regulating angiogenesis.
[00:00:27.150]And it's been found through previous research
[00:00:29.050]that inhibition of VEGFA is actually
[00:00:31.370]what we use to arrest a follicular development.
[00:00:34.200]We call follicular development folliculogenesis.
[00:00:36.761]It is commonly used to use follicle staging
[00:00:39.499]to classify and quantify follicles within the ovary.
[00:00:42.837]So for example,
[00:00:44.250]if you look to the right you can see figure one,
[00:00:46.140]which is our description of follicle staging
[00:00:48.100]that we used within this study.
[00:00:50.030]We used 5 different stages,
[00:00:51.670]ranging from stage 0 to stage 4.
[00:00:54.739]Previous research has demonstrated
[00:00:56.960]that eliminating VEGFA in granulosa cells
[00:00:59.551]leads to follicular arrest,
[00:01:01.311]reduced ovulation rate and reduced fertility.
[00:01:06.030]Anti-Mullerian Hormone, or AMH,
[00:01:07.880]is a glycoprotein hormone
[00:01:10.390]mainly expressed in the granulosa cells
[00:01:12.630]of growing ovarian follicles,
[00:01:14.330]including pre-antral and antral follicles.
[00:01:17.117]So if you look to the right, in figure 1
[00:01:19.560]and you look at stage 4,
[00:01:20.670]you can see the pre-antral follicles.
[00:01:22.810]Specifically, if you look at the cartoon diagram,
[00:01:25.260]you can see the O site in the middle,
[00:01:26.920]and surrounding the O site are multiple layers
[00:01:29.498]of cuboidal cells.
[00:01:31.025]Around the cuboidal cells, you have this flattened
[00:01:34.040]thin cell that are called the granulosa cells.
[00:01:36.476]These granulosa cells are what secrete AMH.
[00:01:40.208]In contrast to VEGFA,
[00:01:43.116]AMH actually arrests follicular development within ovaries.
[00:01:46.953]And PCOS, which is a common fertility disorder within women,
[00:01:52.137]have shown to have increased levels of AMH.
[00:01:57.456]In this particular study,
[00:01:59.393]we are interested in the AMH levels change
[00:02:02.750]over the reproductive lifespan of mice
[00:02:04.850]within VEGFA knockout mice
[00:02:06.505]compared to the wild-type mice, or control mice,
[00:02:09.831]that have the VEGFA present within granulosa cells.
[00:02:13.273]The hypothesis of this research is
[00:02:15.218]that the concentrations of AMH will be increased
[00:02:18.180]in the VEGFA knockout mice,
[00:02:19.779]and therefore, these increased AMH concentrations
[00:02:22.736]will lead to follicular arrest.
[00:02:25.139]Figure 2 is a schematic showing of reproductive lifespan
[00:02:27.775]of a mouse from birth to approximately 200 days of age.
[00:02:31.978]We collected mice, in this particular study,
[00:02:34.920]at ages day 15, day 30, and day 60.
[00:02:38.730]And you can see how these day ages
[00:02:41.310]fit into the key physiological processes taking place.
[00:02:48.190]Prior to my arrival to the lab,
[00:02:50.101]some of Dr. Cupp's previous grad students actually
[00:02:53.130]had ran these fertility trials,
[00:02:54.900]where they took homozygous VEGFA-floxed mice,
[00:02:57.990]and mated them to these mice
[00:03:00.110]that were genotyped positively for the Sry-Cre allele.
[00:03:03.810]The Sry-Cre allele is present,
[00:03:06.020]acts as though, kind of like scissors,
[00:03:08.130]and it can cut out the targeted gene,
[00:03:10.080]which in this case, is VEGFA.
[00:03:12.473]The resulting litters were then genotyped
[00:03:14.700]by myself and others as wildtype control, or knockout.
[00:03:18.200]The control mice are negative for the Sry-Cre allele,
[00:03:21.200]whereas the knockout mice are positive
[00:03:23.530]for the Sry-Cre allele
[00:03:24.960]and homozygous with the VEGFA-floxed.
[00:03:27.549]We then performed mouse dissections this past year,
[00:03:31.090]where we collected the blood plasma,
[00:03:32.750]along with organs from the day 15, day 30, and day 60
[00:03:36.830]control and knockout mice.
[00:03:38.491]With the blood serum that was collected
[00:03:40.890]during these mouse dissections,
[00:03:42.400]we were able to run a ELISA for AMH
[00:03:45.045]and we did this, again, with the same mice
[00:03:48.410]in order to find the levels of AMH concentration.
[00:03:51.628]We then took the ovaries collected
[00:03:54.590]from the mouse dissections
[00:03:55.984]that were fixed in Bouins' and then we went
[00:03:57.945]through the embedding section protocol,
[00:04:01.280]as well as H&E.
[00:04:03.180]With these slides that were then obtained
[00:04:05.080]following all of that,
[00:04:06.130]we will then go further
[00:04:07.730]and do staging of day 30 and day 60 mice.
[00:04:12.648]Figure 3 shows an example of what I would look at
[00:04:15.631]following gel electrophoresis
[00:04:17.720]when looking at the image of a gel.
[00:04:19.960]And the knockout mice, we were looking
[00:04:21.270]for homozygous VEGFA control so in figure 3A,
[00:04:25.422]you can see that's a single band.
[00:04:27.674]And additionally, in the knockout mice,
[00:04:30.080]we were looking for a Cre-positive mouse.
[00:04:32.720]So, in figure 3B, you can see
[00:04:35.200]that a positive Cre allele showed
[00:04:37.440]that there's a single band present.
[00:04:40.050]Figure 4 is created by Kevin and Mariah,
[00:04:44.350]who were Dr. Cupp's previous grad students,
[00:04:46.540]and they were the ones that did the fertility trial
[00:04:48.870]to generate and produce these knockout mice.
[00:04:54.212]Our results from the AMH ELISA found,
[00:04:56.760]for each genotype, that as the mouse ages,
[00:04:59.280]the AMH levels also increase.
[00:05:01.450]You can see this in figure 6A.
[00:05:03.810]This makes sense because as a mouse ages
[00:05:06.060]and gets closer to the age of puberty,
[00:05:08.150]they're gonna have more 3rd and Stage 4 follicles present.
[00:05:13.180]These stages of follicles are surrounded
[00:05:15.270]by granulosa cells which secrete AMH.
[00:05:18.720]Additionally, in figure 6A, you can also see
[00:05:21.360]there's less of a steep incline for the knockout mice,
[00:05:24.130]indicating their slower follicular growth
[00:05:26.170]compared to the control mice.
[00:05:28.480]Overall, you can see in figure 6B,
[00:05:31.030]that the knockout mice actually display
[00:05:32.870]less AMH concentration levels
[00:05:34.950]when compared to the control mice.
[00:05:37.000]This goes against our hypothesis.
[00:05:39.735]We found that the ovarian weight
[00:05:41.950]of the control mice, overall, was higher
[00:05:43.700]when compared to the knockout mice.
[00:05:45.700]This supports our findings
[00:05:46.940]that control mice have faster follicular growth
[00:05:49.380]because as follicles grow, they get larger
[00:05:51.680]and there's more follicular fluid present
[00:05:53.650]which, therefore, makes the ovary weigh more.
[00:05:56.715]You can see the knockout mice weight less
[00:05:58.940]when compared to the control mice,
[00:06:00.690]indicating that they do not have follicles
[00:06:02.690]that are as developed.
[00:06:05.100]We found that there is a significant difference
[00:06:07.290]between the weight of the uteri
[00:06:08.780]of the different genotypes,
[00:06:10.100]indicating that there is isolation of this effect
[00:06:12.850]of the VEGFA removal only on the ovary,
[00:06:16.820]which makes sense as the ovary is
[00:06:19.130]where the follicles are present.
[00:06:21.327]These are additional graphs
[00:06:23.060]which displayed the other weights that we took,
[00:06:25.040]the body, adrenal glands, and the kidneys
[00:06:27.730]where there is no significant difference among these,
[00:06:31.143]except for at day 15 mice, there is less kidney weight
[00:06:34.370]in the knockout mice compared to the control mice.
[00:06:38.850]With these results, we reject our hypothesis
[00:06:41.420]that the elimination of VEGFA in granulosa cells
[00:06:44.540]with the knockout mice did not result in
[00:06:46.240]increased production of AMH.
[00:06:48.170]Because as we saw in figure 6B,
[00:06:50.530]overall, the control mice showed higher levels of AMH.
[00:06:54.630]This reduction of AMH is potentially due
[00:06:57.050]to altered granulosa cell function and survival
[00:06:59.640]resulting in slower follicular progression
[00:07:02.742]to later stages of development.
[00:07:04.560]As we saw in the knockout mice,
[00:07:06.680]the ovaries of these mice weighed less
[00:07:10.172]compared to the ovaries of the controls.
[00:07:12.100]Additional data identifying follicle stages
[00:07:15.406]at day 30 and day 60 will allow us
[00:07:17.847]to further support these conclusions.
[00:07:20.004]The fertility data from these mice demonstrate
[00:07:22.167]that the knockout mice have reduced pups per litter,
[00:07:24.558]decreased numbers of parturitions
[00:07:26.598]due to increased parturition intervals,
[00:07:28.856]which effectively shorten their reproductive lifespan.
[00:07:31.994]Therefore, this points that the elimination of VEGFA
[00:07:35.156]in granulosa cells demonstrate that VEGFA
[00:07:37.995]is critical to granulosa cell function
[00:07:40.299]and female fertility.
[00:07:42.389]I would like to thank Dr. Cupp
[00:07:43.667]for welcoming me into the lab as a freshman
[00:07:45.585]and allowing me to work on this project
[00:07:47.204]over the last year.
[00:07:48.806]With her financial support, as well as the UCARE grant,
[00:07:51.306]this project was able to occur.
[00:07:53.659]I would also like to thank Scott, Courtney, and Alex.
[00:07:56.140]They were always more than willing
[00:07:58.524]to answer any questions I had while in the lab.
[00:08:01.271]Additionally, I would like to thank Mohamed
[00:08:02.880]for helping Dr. Cupp and I input our data into SAS
[00:08:07.040]and help us create the figures for this presentation.
[00:08:11.045]I also want to thank Kevin and Mariah
[00:08:13.007]who were Dr. Cupp's previous grad students
[00:08:15.588]who helped put together the fertility trials.
[00:08:19.482]These are the references I used
[00:08:21.049]for this specific presentation.
[00:08:22.969]I appreciate the time you took listening to this video
[00:08:25.341]and I hope you gained a better understanding
[00:08:27.179]of VEGFA and AMH and their impact on fertility.

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Effect of Elimination of Vascular Endothelial Growth Factor A (VEGFA) in Granulosa cells on concentrations of Anti-Mullerian Hromone and follicle stages in mice

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