Identification of neurodevelopmental genes in the coral species

Acropora digitifera (Acroporidae)

Ingrid Camila Peñaloza Ortega

Supervisor: Clara Isabel Bermúdez Santana

Biology Deparment, Universidad Nacional de Colombia

July, 2022

ABSTRACT

Materials and methods

Cow oocyte retrieval and Maturation

Ovaries from different cow breeds (Limousin, German and Red Holstein) were obtained from
a local abattoirs. They were delivered in a thermo-flask within 3 hours after ovary collection,
keeping the temperature around 30°C. Ovaries with a temperature lower than 25°C were not
further processed. Oocyte isolation, in-vitro fertilization and embryo culture were carried out
following Bovine IVF protocol from IVF Bioscience, incorporating minor changes. Briefly, cow
ovaries were rinsed in pre-warmed saline solution (0.9% NaCl) four times. Follicular fluid
from follicles 12-18 mm in diameter were aspirated with a 21-gauge needle and transferred
to a 50 mL falcon tube. For oocyte pick-up, follicular fluid was incubated for 5 min at 38.5°C
to let the cumulus-oocyte complexes (COC) sediment. First and secondary pellets were
transferred to a petri dish with grid using a plastic pasteur pipette (~4 mL). COCs were
retrieved with a P200 pipette to preserve the association oocyte-cumulus cells. Finally, they
were washed through TCM199 media three times and equilibrated BO-IVM media before
final incubation in BO-IVM media at 38.8°C and 5.5-6.5% CO2 in humidified atmospheric air
(21% O2) for 20 h. Oocytes were denuded (removal of cumulus cells) in TCM199 media with
an overlay of Nidoil 15 h post incubation in BO-IVM media. COCs were gently devoided of
cumulus cells employing a 135 μm tip-size micropippete. Oocytes were then washed through
BO-IVM media and transferred back to the original IVM dish to complete maturation.

In vitro fertilization and embryo culture

Spermatozoa were prepared from a frozen bull semen straw –fertility proven beforehand by
evaluating motility and sperm count–. Semen straw was thawed in a water bath at 37°C and
immediately resuspended in 3 mL pre-warmed BO-SemenPrep media. After centrifugation at
300g for 5 min, pellet was resuspended in 2 mL BO-SemenPrep media and centrifuged
again under the same conditions. For sperm count, a sperm aliquot is further diluted in
distilled water (1:200 dilution factor) and counted in a Bürker chamber. Spermatozoa to a
final concentration of 1x106 cells/mL were placed in BO-IVF media along with denuded
oocytes, first washed through pre-warmed equilibrated BO-IVF media. In vitro fertilization
occurs at 38.8 °C and 5.5-6.5% CO2 in humidified atmospheric air (21% O2) for 20 h.
Zygotes were denuded as described above for COCs, removing remaining cumulus cells
and sperm. Embryo culture took place in pre-warmed equilibrated BO-IVC media at 38.8°C
(5% CO2 + 6% O2) for maximum 24 h. Embryo culture timing varies depending on drug
treatment and incubation time post drug release.

Drug treatment for zygote synchronization

Zygotes in BO-IVC media were treated with cell cycle inhibitors 24-26 h after in vitro
fertilization started. To arrest the zygotes at S/G1 boundary, cells were incubated with
thymidine (final concentration: 2 mM) for 16-18 h. Likewise, synchronization at G2/M phase
was achieved by incubating cells with R03306 (final concentration: 100 μM ) for 16-18 h.
Afterwards, zygotes were washed through BO-IVC media and transferred to fresh
pre-warmed equilibrated BO-IVC media for incubation post-release. Thymidine-treated cells
were incubated for 4 h, while R03306-treated cells were fixed at different time points after
incubation, from 1h 50 min to 3h 30min.

Immunofluorescence

Cells were briefly pre-permeabilized in 0.25% Triton X-100 solution at 4°C, and immediately
fixed in 1 M HEPES (pH 7.0), 0.5 M EGTA (pH 7.0), 1 M MgSO4, 10% Triton X-100 and 10%
formaldehyde at 37°C for 30 min. Fixed zygotes were extracted in PBS and 0.5% Triton
X-100 (PBT) at 4°C for 72 h. Then, cells were blocked in PBT and 5% BSA (blocking buffer)
at 4°C overnight. Prior to primary antibody incubation, optical clearing was performed as
described by So et al. (2019). Shortly, lipid droplets in zygotes were cleared with 4000 U/mL
lipase from Candida rugose (Sigma-Aldrich) in lipase buffer (50 mM Tris-HCl pH 7.2, 400
mM NaCl, 5 mM CaCl2, 0.2% sodium taurocholate (Sigma-Aldrich #86339), 1:20 mini
cOmplete EDTA free protease inhibitor (Roche #11836170001), 5% BSA) at 37 °C for 2 h.
Following lipase treatment, zygotes were washed in PBT and incubated overnight with
diluted primary antibodies (Table 1) in blocking buffer.

After Lipase treatment, the zygotes were washed in PBST-BSA and

incubated overnight at 4°C with primary antibodies in PBST-BSA at the
concentration listed in the following paragraph. Secondary antibody
incubations were performed in PBST-BSA for 1 hour at room temperature
at 20 μg/ml.

As secondary antibodies we used Alexa Fluor 405-, 488-, 568- or

647-conjugated anti-human IgG, anti-mouse IgG, anti-mouse IgM,
anti-rabbit IgG, or anti-rat IgG, all raised in donkey or goat (Thermo Fisher
Scientific). DNA was stained with DAPI at a final concentration of 20 μg/ml
(Thermo Fisher Scientific).

Confocal microscopy of bovine zygotes

method image adapted from

https://www.cambridge.org/core/journals/animal/article/review-recent-advances-in-bovine-in-vitro-embryo-produ
ction-reproductive-biotechnology-history-and-methods/4C4A7C008A6014ADBFDECCFED12FAE13

https://www.cell.com/cell/fulltext/S0092-8674(21)00492-X?_returnURL=https%3A%2F%2Flin
kinghub.elsevier.com%2Fretrieve%2Fpii%2FS009286742100492X%3Fshowall%3Dtrue#sec
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