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  
16  
J Infertil Reprod Biol, 2018, Volume 6, Issue 4, Pages: 16-18. https://doi.org/10.47277/JIRB/6(4)/16  
changes was assessed in 5 oocytes in each concentration under  
incubation. The apoptotic features appeared in the cells incubated  
beyond 40 hrs. One study showed that all the apoptotic cells as  
proved by TUNEL assay did not show cytoplasmic  
fragmentation (13). Another controversial result was given by a  
study that suggested a doubt on apoptosis occurring in atretic  
oocytes (14). Cells undergo continuous turnover by the process  
of apoptosis or programmed cell death (15). An apoptotic cell  
shows nuclear and cytoplasmic condensation and fragmentation  
along with formation of membrane bound apoptotic bodies.  
TNF-α is the mediator of extrinsic apoptotic pathway (15,16).  
Cytoplasmic fragmentation is the end point of apoptosis and the  
features observed in our study might be the initial changes  
produced by TNF-α. There have been various markers of  
apoptotic changes such as TUNEL, M30 immunostaining,  
annexin v staining etc. but the gold standard method remains the  
morphological criteria (17, 18). The clinical implications of  
excessive rate of cellular apoptosis and proliferation have been  
discussed in many studies (2, 19). The effect of excessive rate of  
oocyte apoptosis has been linked with ovarian disorders such as  
POF. The effect of cytokines has also been studied in recurrent  
pregnancy loss apart from POF and other autoimmune ovarian  
disorders (20).  
inverted microscope (light cytoplasmic granulations: 1; shrinkage  
of cytoplasm/ distorted shape/cytoplasmic oozing: 1; increased  
perivitelline space: 1; dark/densely granulated cytoplasm: 2).  
Results  
The morphological changes (cytoplasmic granulations,  
increased perivitelline space, cytoplasmic shrinkage, cytoplasmic  
oozing, distorted shape etc.) were observed with all the  
concentrations of TNF-α. By 8 hrs majority of oocytes in TNF-α  
showed degenerative changes. At 8 hrs, the morphological scores  
of 5 oocytes each at 0.1, 1, 10, 100 ng/ml concentrations of TNF-  
α were 13, 19, 20, 16 respectively (Table 1). The score was  
maximum at 10 ng/ml concentration of TNF-α and lesser at  
extreme lower and higher concentrations. Due to technical issues,  
the morphological observations were further continued with two  
concentrations only (1 and 100 ng/ml). Majority of oocytes in  
M2 media were normal till 8 hrs. Under TNF-α supplementation  
to media, majority of the oocytes showed all the degenerative  
st  
changes by 8 hrs. Within 1 hour, mild granulations were visible  
and by 12 hrs, all oocytes were completely atretic (Figure 1 and  
2
).  
Discussion  
Conclusion  
TNF-α induced morphological features have been studied on  
To the best of our knowledge TNF-α induced timeline  
morphological changes on mice oocytes have not been studied so  
far. Present study was done in smaller number of oocytes but  
could highlight the possible direct effect of TNF-α on mice  
oocytes. Results showed the degenerative effects of TNF-α on  
mice oocytes. Presence of cytoplasmic shrinkage and oozing of  
cytoplasm are the initial features of oocyte apoptosis. Still our  
study needs further confirmation of whether these changes are  
attributed to apoptosis or not. Similar larger studies should be  
planned for validation. Animal models of immune POF should be  
designed to see the effect of anti-TNF-α on oocyte growth. Our  
lab is planning to continue the study to confirm TNF-α induced  
morphological features as apoptotic by TUNEL or caspase  
staining and to find out the possible preventive or therapeutic  
options.  
ovarian granulosa and theca cells thereby affecting follicle  
number and oocyte status (11). Oocyte needs surrounding  
cumulus cells for its development. The oocyte atresia in the  
previous study has been interpreted indirectly via granulosa cells  
or follicular cell death. Results of our study showed the  
degenerative effects of TNF-α on mice oocyte morphology,  
maximum at 10 ng/ml as compared to lower and higher  
concentrations. This suggests that effects are produced only at  
optimum concentration. Similar result was shown in the previous  
study as well. The concentrations used in our study were based  
on that work with minor variations (11). Few studies have shown  
that apoptosis is the process of cell death in degenerative changes  
of unfertilized oocytes (12). This study showed that majority of  
unfertilized ova did not show the classical features of apoptosis  
(
cytoplasmic shrinkage and fragmentation) until 24 hrs. of  
Table 1: Morphological score of oocytes with different concentrations of TNF-α at 8 hrs  
Concentration  
of TNF-α (ng/ml)  
Morphological features at each concentration in 5 oocytes  
Total apoptotic score  
Dark granulated cytoplasm (2)  
Dark granulated cytoplasm (2)  
Dark Granulated Cytoplasm + Increased Perivitelline space (2+1)  
13  
0
.1  
Dark Granulated Cytoplasm + Increased Perivitelline space (2+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space (2+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space (2+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increase Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
Dark Granulated Cytoplasm + Increase Perivitelline space (2+1)  
19  
20  
16  
1
1
0
Dark Granulated Cytoplasm + Increase Perivitelline space (2+1)  
Dark Granulated Cytoplasm + Increase Perivitelline space (2+1)  
1
00  
Dark Granulated Cytoplasm + Increase Perivitelline space (2+1)  
Dark Granulated Cytoplasm + Increased Perivitelline space + Shrinkage of cytoplasm (2+1+1)  
17