Effects of Vascular Endothelial Growth Factor A (VEGFA) Isoforms on the Ovarian Microenvironment in Androgen Excess Cows.
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.
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[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
[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
[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
[00:01:18.880]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.
[00:01:31.750]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,
[00:02:20.200]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
[00:02:47.350]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
[00:03:02.200]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
[00:03:45.820]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
[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.
[00:04:26.410]the hypothesis for my UCARE project
was that the high A4 cow ovarian
[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
[00:04:48.250]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
[00:05:50.710]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
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
[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
[00:07:21.410]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.
[00:08:07.570]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
[00:08:17.920]as well as UCARE for giving me the
opportunity to conduct research this
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