INFLUENCE OF LEPTIN ON THE EXPRESSION OF BCl2 and BAX GENES IN CULTURED OVARIAN FOLLICLES IN SHEEP
Loading...
Date
2015-11
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502,A.P
Abstract
ABSTRACT:
To ascertain whether the support provided by Leptin to the PFs’ in culture
(Kamalamma et al., 2015) was due to its ability to modulate apoptosis related genes,
the present study is undertaken. Accordingly in the present study expression levels of
two test genes BCl2 (antiapoptotic), BAX (apoptotic) and three reference genes viz.,
18S rRNA, HPRT1, RPLPO were studied in the Cumulus cells and Oocytes during in
vivo and in vitro folliculogenesis in sheep.
Sheep ovaries were collected from the local slaughter house. The Cumulus
cells and Oocytes from different development stages of ovarian follicles i.e., preantral,
early antral, antral and large antral follicles were isolated. Preantral follicles (PFs’)
isolated from the ovaries were cultured in a medium containing 10 ng/ml of
recombinant human Leptin for 3 minutes, 2, 4, or 6 days to obtain cultured preantral,
early antral, antral and large antral follicles respectively. Furthur some COCs in in vivo
and in vitro grown large antral follicles were matured in vitro for 24h. The expression
pattern of the test and reference genes (18SrRNA, HPRT1, RPLPO) in the Cumulus
cells and Oocytes was studied separately at the different development stages mentioned
earlier by Quantitative Reverse transcription Real time PCR (qRT-PCR).
Quantitative expression of BCl2 gene in the Cumulus cells isolated from
different in vivo grown ovarian follicles was significantly lower in the pre antral and
early antral follicles than corresponding stages of follicles cultured in Leptin.
However, the expression was significantly higher in the in vivo grown antral, large
antral and COCs from large antral follicles matured in vitro for 24h. Quantitative
expression of BCl2 in the Oocytes isolated from the pre antral follicles and COCs in
large antral follicles matured in vitro for 24h was significantly lower than their
corresponding Leptin cultured stages. However, there was no significant difference in
the BCl2 expression in the Oocytes in early antral, antral and large antral follicles
compared to their corresponding Leptin cultured stages.
Quantitative expression of BAX in the Cumulus cells isolated from in vivo
grown ovarian follicles was significantly lower in the preantral follicles and Cumulus
cells from COCs after in vitro maturation for 24h than their corresponding Leptin
cultured stages. However, the Cumulus cells from in vivo antral follicles exhibited a
significantly higher level of expression than corresponding Leptin cultured follicles.
Quantitative expression of BAX in the Oocytes isolated from the in vivo grown antral
follicle and Oocytes from COCs after in vitro maturation for 24h was significantly
lower than the corresponding in vitro stage. However, Oocytes from large antral
follicles expressed BAX to a significantly higher level than corresponding in vitro
stage. The expression pattern of BAX in the Oocytes isolated from the different in vivo
grown ovarian follicles was similar to the corresponding in vitro grown stages in early
antral and large antral follicles.
From the present study it is concluded that supplementation of Leptin (10
ng/ml) in the culture medium induced a different quantitative expression pattern of
BCl2 in Cumulus cells and Oocytes than in vivo grown ovarian follicles. Leptin
inclusion in the culture medium was able to simulate the in vivo pattern of BAX gene
expression in the Cumulus cells and Oocytes at some but not all of the development
stages.
Description
Keywords
Sheep; Leptin; BCl2; antiapoptotic; BAX ; apoptotic; genes; folliculogenesis; 18S Rrna; HPRT1; RPLPO