Effects of Vascular Endothelial Growth Factor A (VEGFA) Isoforms on the Ovarian Microenvironment in Androgen Excess Cows.

Brooke Bell Author

08/04/2021 Added

9 Plays

Description

Utilization of a cow model of androgen excess (High A4) to learn how to culture ovarian cortex and measure Anti-Mullerian hormone (AMH), cytokines and chemokines in cortex media which may be contributing to follicle arrest, inflammation and ultimately anovulation. Also VEGFA isoforms were utilized to reduce proinflammatory cytokines and may be used as a future therapeutic to enhance success of follicle development and ovulation.

Searchable Transcript

Search:

[00:00:02.590]Hello. My name is Brooke Bell and I'm a senior animal science major today.
[00:00:06.880]I'm happy to share with you my UCARE project,
[00:00:10.720]my research project focused on female infertility.
[00:00:14.200]A major female infertility disorder is anovulation,
[00:00:17.590]which means the egg doesn't ovulate. We utilize cows as our model system,
[00:00:23.110]Cows serve as an excellent model for themselves and for women because they share
[00:00:27.160]many of the same reproductive characteristics,
[00:00:30.310]such as similar ovary size. They ovulate one egg,
[00:00:34.900]they have similar endocrine hormones and the length of pregnancy is the same.
[00:00:40.450]We've identified a population of cows in our herd that have excess androstenedione
[00:00:44.620]in the dominant follicles as shown on the Y access in the top graph.
[00:00:50.050]The controls denoted by black dots had a concentration of less than 20
[00:00:54.700]nanograms per mil and high androstenedione cows,
[00:00:58.840]which we call high A4 cows are denoted by red dots had a concentration
[00:01:03.850]of greater than 40 nanograms per mil. Based on this information,
[00:01:08.620]studies were done looking at the follicular development of cows.
[00:01:12.790]Our bottom graph shows follicle diameter on the Y axis over days of
[00:01:17.650]estrous.
[00:01:18.880]The controls have steady waves of follicular development being indicated by the
[00:01:22.870]colored lines. Additionally, in our control cows,
[00:01:26.260]we see that ovulation denoted by the sun lines up with estrus.
[00:01:30.760]After ovulation,
[00:01:31.750]there was a rise in progesterone indicated by the light blue shading. In our
[00:01:36.550]high four cows.
[00:01:37.990]We don't follow the wave pattern as they do in our controls.
[00:01:42.160]There is a persistent follicle that develops and maintains its size for the
[00:01:45.760]duration of the estrous cycle and does not ovulate as a result.
[00:01:50.020]We don't see a rise in progesterone.
[00:01:54.130]Previous cortex cultures showed other differences between control and high
[00:01:58.150]A4 cows. Throughout my presentation on a bar graph control,
[00:02:02.930]cows will be indicated by white bars and black bars will be used to indicate
[00:02:07.210]high A4 cows.
[00:02:09.070]The top graph shows arrested follicular development in High A4 cows with
[00:02:13.960]increased early stage follicles, primordial and less later stage follicles,
[00:02:18.700]primary, secondary,
[00:02:20.200]and antral than controls. In the bottom left graph.
[00:02:24.160]We determined potential effects of the ovarian microenvironment by measuring A4
[00:02:28.690]concentrations in ovarian cortex,
[00:02:31.000]media daily and High A4 cows had consistently greater concentrations.
[00:02:36.250]The bottom right graph shows A4 concentrations over the ovarian cortex
[00:02:40.030]culture period and are 43 times greater in High A4 cows compared to the
[00:02:44.680]controls.
[00:02:47.350]Previous research investigated inflammation and ovarian cortex using Pico-Sirus
[00:02:52.210]red staining, which is a marker for inflammation and fibrosis.
[00:02:56.260]There were greater amounts in high A4 denoted by B. then it controls denoted
[00:03:01.120]by A,
[00:03:02.200]which is quantitated in C. Oxidative stress was measured using 4-HNE
[00:03:07.180]staining, which is a second indicator of inflammation.
[00:03:11.380]There is increased oxidative stress and high A4 cows indicated by G compared
[00:03:16.360]to controls and F and these were quantitated in H.
[00:03:22.990]Once an increase in fibrosis of high, A4 cows was discovered.
[00:03:26.890]Research was conducted to determine if VEGFA165 can
[00:03:31.540]rescue fibrosis and excess A4 production.
[00:03:35.440]We treated ovarian cortex cultures with VEGFA isoforms.
[00:03:40.510]We compared Pico-Sirus red slides from before culture in
[00:03:44.980]PBS,
[00:03:45.820]as well as ones treated with VEGFA 165, VEGFA,
[00:03:50.740]165B and the two isoforms in combination.
[00:03:55.300]It appears that in High A4 cows,
[00:03:57.670]the VEGFA165 decreases fibrosis. Pico-Sirus
[00:04:02.650]red staining is quantitated in the top right graph. Additionally,
[00:04:07.240]we see decreased levels of androstendione in high
[00:04:09.980]A4 cows treated with the VEGFA165 as shown in the
[00:04:14.740]bottom right graph.
[00:04:16.630]Thus VEGFA165 can rescue the inflammation and
[00:04:21.310]excess A4 in high A4 cows.
[00:04:25.840]Therefore,
[00:04:26.410]the hypothesis for my UCARE project was that the high A4 cow ovarian
[00:04:31.150]microenvironment,
[00:04:32.350]secretes intraovarian factors that contribute to follicular arrest
[00:04:36.550]and anovulation. In order to confirm my hypothesis,
[00:04:40.780]we conducted Elisa's for anti-Mullerian hormone or AMH measured
[00:04:45.190]pro-inflammatory cytokines and chemokines,
[00:04:48.250]and evaluated if VEGFA Isoform
[00:04:51.460]treatment can reduce cytokine production.
[00:04:56.410]For these experiments Cortex cultures were performed.
[00:04:59.680]I collected four pieces of ovarian cortex per well and cultured them and
[00:05:03.910]Waymouth media. For seven days,
[00:05:06.250]media was collected and new media was added. On the seventh day
[00:05:10.540]pieces were removed from culture. Using the cultured fixed pieces
[00:05:14.710]we were able to stain in for Pico-Sirus red.
[00:05:17.590]We can also use pooled media to perform AMH Elisa assays and Quantibody
[00:05:22.360]cytokine arrays. Earlier I mentioned,
[00:05:27.040]we were seeing arrested follicular development in our high A4 cows as shown in
[00:05:31.510]the top left graph.
[00:05:33.370]The increased concentration of the pro-inflammatory cytokines,
[00:05:36.790]may be one cause for this. Additionally anti-Mullerian hormone or
[00:05:41.140]AMH has been shown to cause follicular arrest.
[00:05:44.920]Previous studies showed an increase of AMH in blood plasma of High A4
[00:05:49.630]cows.
[00:05:50.710]We wanted to look within the dominant follicle as well as what was secreted in
[00:05:54.790]the ovarian microenvironment.
[00:05:56.830]We see in both the follicular fluid and the cortex cultures
[00:06:01.850]an increase in AMH in our high A4 cows.
[00:06:05.360]This could be why we see arrested follicular development,
[00:06:08.270]and may also contribute to anovulation.
[00:06:12.800]To determine if ovarian cortex from high A4 cows secretes increase
[00:06:17.220]proinflammatory cytokines and chemokines. We performed a cytokine array.
[00:06:22.430]We began by looking at cytokine production and high A4 cattle versus,
[00:06:26.660]our controls. Interferon-alpha Interleukin-13 and Tumor
[00:06:31.490]Necrosis factor-alpha were significantly higher.
[00:06:34.970]And CXCL10 and interferon-gamma had a tendency to be higher in the high
[00:06:40.050]A4 cows versus our controls.
[00:06:42.740]All of which are pro-inflammatory cytokines and chemokines,
[00:06:46.100]that are elevated in response to inflammation.
[00:06:49.250]Interleukin-13 is also known to cause fibrosis in tissues.
[00:06:54.770]Knowing that the VEGFA165 was shown to reduce fibrosis.
[00:06:59.930]We wanted to evaluate if VEGFA165 treatment
[00:07:04.370]reduces pro-inflammatory cytokines in a high A4 ovarian cortex cultures.
[00:07:10.010]These graphs show the concentration of individual cytokines on the Y axis with
[00:07:14.990]the treatments indicated on the X axis to further explore the effects of the
[00:07:19.640]VEGFA isoforms.
[00:07:21.410]We ran cytokines on cortex cultures that have been treated with VEGFA
[00:07:25.640]isoforms. In our High A4 cows
[00:07:28.670]VEGFA165 reduced a subset of pro-inflammatory cytokines
[00:07:33.710]as indicated by the graphs on the left. In the graph on the right.
[00:07:38.000]We see the Interferon-gamma was reduced by the VEGFA
[00:07:42.440]isoforms in both the control and the high A4 cows.
[00:07:51.160]In the ovarian microenvironment of high A4 cows,
[00:07:54.340]we had increased pro-inflammatory cytokines and increased AMH,
[00:07:58.540]which contribute to fibrosis and inflammation,
[00:08:01.480]which leads to persistent follicles anovulation and female infertility.
[00:08:06.550]Additionally,
[00:08:07.570]VEGFA isoforms may serve as a therapeutic treatment for these conditions.
[00:08:13.360]I would like to thank the following individuals and organizations for their
[00:08:16.810]contributions,
[00:08:17.920]as well as UCARE for giving me the opportunity to conduct research this
[00:08:21.400]summer.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

Effects of Vascular Endothelial Growth Factor A (VEGFA) Isoforms on the Ovarian Microenvironment in Androgen Excess Cows.

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments