J Infertil Reprod Biol, 2018, Volume 6, Issue 4, Pages: 16-18. https://doi.org/10.47277/JIRB/6(4)/16
A Timeline Morphological Study of TNF-α Induced
Changes in Mice Oocytes
Ranjana Rana, Mona Sharma*
Department of Reproductive Biology, AIIMS, New Delhi, India
Received: 06/09/2018
Accepted: 15/11/2018
Published: 20/12/2018
Abstract
Inflammatory cytokines induce cellular degenerative and apoptotic changes. Studies have shown increase in cytokine levels in
patients with premature ovarian aging or failure (POF) where excessive oocyte atresia is the major pathogenic feature. Enhanced
expression of tumour necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, and its receptors have been found in the oocytes of
immunized mice with experimental immune ovarian failure. The aim of the present work was to study the direct effects of TNF-α on
naked oocytes. Oocytes were isolated from superovulated Swiss Albino female mice and treated with different concentrations of TNF-α
(
0.1, 1, 10 and 100ng/ml). Morphological scoring was assessed based on shape, cytoplasmic and extra-cytoplasmic features of the
oocytes. It was found that TNF-α could induce morphological degenerative changes in mice oocytes at all the concentrations, maximum
being at 10 ng/ml, suggesting a direct effect of this inflammatory cytokine on mammalian oocytes. Present study was a small piece of
work that could highlight the possible direct effects of TNF-α on mice oocytes. Similar larger studies need to be planned in near future
for validation. Further studies should also be planned in animal models of immune POF or autoimmune ovarian disorders to see the effect
of anti-TNF-α treatment on ovarian physiology. Our lab is planning to continue the study further to confirm the morphological features as
apoptotic by TUNEL or caspase staining and to find out the possible preventive or therapeutic options.
Keywords: Cytokines, Tumor necrosis factor-alpha, Oocytes
Introduction
changes in the oocytes. Therefore, the aim of the present work
was to study the timeline of morphological changes induced by
TNF-α on mice oocytes in vitro.
The pool of primordial follicles that a woman born with
undergoes continuous atresia during reproductive years. It is a
well-known fact that the underlying mechanism of ovarian
atresia is apoptosis but the mode of death of atretic/degenerated
oocytes is yet to be confirmed (1). Apoptosis or programmed cell
death is an energy dependent biochemical event that is involved
in regulating the immune reactions happening in response to a
foreign agent. Apoptosis is a controlled process and is necessary
for the survival of an organism but when it becomes
uncontrollable, it can lead to various pathologies (2). Role of
cytokines have been very well studied in mediating apoptosis;
one such cytokine is TNF-α.
Methods
All animal experiments were approved by Central Animal
Ethics Committee of All India Institute of Medical Sciences,
New Delhi (53/IAEC-1/April 2018) and methods were carried
out in accordance with approved guidelines. 10 Swiss Albino
female mice, aged 6 to 8 weeks were issued from Central Animal
Facility, All India Institute of Medical Sciences, New Delhi.
Animals were housed in 12-hour light dark cycle provided ad
libitum food and water throughout the study. Chemicals were
obtained from Merck, India unless otherwise indicated. On Day
TNF-α is a 17.3kDa protein product that induces pleotropic
cellular responses and is chiefly produced by activated
macrophages. Numerous cell types such as osteoblasts,
hepatocytes, spleen cells contain TNF-α and have also been
identified in the ovaries of humans and mice. TNF-α can exist as
an insoluble trans-membrane protein and in a soluble form which
requires a proteolytic cleavage by a matrix metalloproteinase
known as TACE (TNF-α converting enzyme) (3). To exert its
biological activities, TNF-α has two distinct Type1 trans-
1, mice were given an intra-peritoneal injection of 10IU PMSG
(
Prospec, India). After 48 hours, 10 IU of hCG was injected
intra-peritoneally. After 12-14 hours of hCG injection, the
oviducts were dissected out. Oviducts were collected in a dish
containing 2 ml of M2 Media. Using a needle, the cumulus was
flushed out of the oviduct. To remove cumulus cells from the
oocyte, 5µL of hyaluronidase was added to 500µL of M2 media
in mineral oil and incubated at 37˚C for 5 minutes. After the
cumulus cells got separated, oocytes were then transferred to the
third dish with 100µL drop of M2 media in mineral oil.
For further experiments different concentrations of TNF-α
were used (0.1, 1, 10, 100 ng/ml) along with M2 media. The
experiments were conducted initially for standardization and
later the experiments were replicated for morphological
observations. The morphological changes were observed at
different hrs (1, 2, 4, 8, 12, 24, 48) and scoring was done based
on the degenerative features described earlier (10).
1 2
membrane receptors, TNFR and TNFR (4). Both receptors have
a similar cysteine rich extracellular domain and a unique
intracellular domain indicating different intracellular signalling
pathway. TNFR
function such as apoptosis (5). It has been reported that TNFR
necessary for cytotoxicity as it consists of an intracellular site,
Death Domain which is absent in TNFR but can stimulate NF-
B signalling and activation of various kinases (6-8). Enhanced
1
receptor has primarily pro-inflammatory
1
is
2
expression of TNF-α and its receptors are found in the oocytes of
immunized mice model of immune ovarian failure (9). Since it
has been proved that oocytes have membrane receptors for TNF-
α, we hypothesised that TNF-α can directly induce degenerative
An arbitrary morphological score of the degenerative
*
Corresponding author: Mona Sharma, Department of Reproductive Biology, 2 Floor, Teaching Block, AIIMS, New Delhi, India. E-
mail: dr.mona18sharma@gmail.com
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