Expression of Mitochondria-Associated Genes (PPARGC1A, NRF-1, BCL-2 and
BAX) in Follicular Development and Atresia of Goat Ovaries
G Zhang*, Y Wan*, Y Zhang, S Lan, R Jia, Z Wang, Y Fan and F Wang
Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China
Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via themitochondria-dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondriaassociated genes peroxisome proliferator-activated receptorgamma, coactivator1-alpha (PPARGC1A), nuclear respiratory factor-1 (NRF-1), B-cell CLL/lymphoma 2 (BCL-2) and BCL2associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria-associated proteins (PGC-1a, which are encoded by the PPARGC1A gene,
NRF-1, BCL-2 and BAX) mainly expressed in granulosa cells.
The mRNA and protein levels of PPARGC1A/PGC-1a and
NRF-1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF-1, increased BAX expression and increased ratio of BAX to BCL-2 expression.
These results suggested that changes of the mitochondriaassociated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.
Most follicles do not mature fully but undergo atresia during the developmental process (Quirk et al. 2004).
Previous researches indicated that follicular atresia was initiated or caused by apoptosis of granulosa cells in mammalian ovaries (Ortega-Camarillo et al. 2009; Sai et al. 2011). Apoptosis is a morphologically and biochemically distinct process that is inducted and executed via several signalling pathways, of which the mitochondria-dependent pathway is one of the most important (Choi et al. 2004). Mitochondrial functions are critical determinants of cell survival. Peroxisome proliferatoractivated receptor-gamma, coactivator1-alpha (PPARGC1A), a key regulator of mitochondrial biogenesis, is vital for cell survival (Puigserver and Spiegelman 2003). Won et al. (2010) found that PPARGC1A prevents apoptosis by enhancing ATP/ADP translocation activity. Nuclear respiratory factor-1 (NRF-1) is a downstream gene of PPARGC1A that plays a pivotal role in activating the expression of genes involved in mitochondrial biogenesis (Wu et al. 1999). Chan et al. (1998) reported that disruption of the NRF-1 gene leads to embryonic lethality and inhibits apoptosis through the c-Myc signalling pathway (Morrish et al. 2003). In addition, previous reports found that mitochondrial functions were critical to follicle formation and development (Yu and Yang 2010). Furthermore, our previous results showed that NRF-1 and PPARGC1A play roles in apoptosis during ovarian development in the goat (Zhou et al. 2012). However, the mechanisms underlying the mitochondria-associated genes leading to follicular atresia remain unclear.
The BCL-2 family proteins have either anti-apoptotic (BCL-2, B-cell lymphoma 2) or pro-apoptotic (BAX,
BCL2-associated X protein) activities, which regulate the mitochondrial apoptotic pathway by controlling permeabilization of the outer mitochondrial membrane (Brunelle and Letai 2009). Members of the BCL-2 protein family play significant roles in follicle growth and atresia by apoptosis of germ and somatic cells (Matsuda et al. 2012). Previous studies have shown that
BAX expression is greater in granulosa cells of atretic follicle compared with those of healthy follicles in human (Kugu et al., 1998) and porcine (Sai et al. 2011) ovaries, while overexpression of BCL-2 decreases apoptosis of granulosa cells in large antral follicles (Hsu et al. 1996). In addition, the ratio of BAX to BCL-2 expression is the critical determinant of the susceptibility to apoptosis (Korsmeyer et al. 1993). However, there are few reports on the presence and function of BCL-2 and
BAX in goat follicular development and atresia.
Therefore, the aim of this study was to investigate whether the PPARGC1A, NRF-1, BCL-2 and BAX genes were involved in follicular atresia through the mitochondria-dependent apoptotic pathway.
Materials and Methods
This research was conducted in accordance with procedures approved by the Guide for the Care and Use of
Laboratory Animals prepared by the Ethics Committee of Nanjing Agricultural University (SYXK2011-0036).
All antibodies were purchased from commercial suppliers (details are provided in Table 1). All other chemicals were obtained commercially and of reagent grade.
Yangtze River Delta White Goat, a Chinese indigenous goat, characterized by its broad use of meat, skin and*These authors contributed equally to this work.
Reprod Dom Anim 50, 465–473 (2015); doi: 10.1111/rda.12514
ISSN 0936–6768 © 2015 Blackwell Verlag GmbH hair, was used for our study. During the breeding season (October to March in the next year), the goat ovaries were collected at a local abattoir and transported to the laboratory in a thermos flask that contained sterilized physiological saline (30–35°C, including 100 IU/l penicillin and 50 mg/l streptomycin) within 2 h of collection. At the laboratory, the connective tissues and attached oviducts were removed after washing five times with physiological saline. Forty ovaries from twenty randomly selected goats were fixed in 4% formaldehyde for 48 h and embedded in paraffin for immunohistochemistry. The remaining three hundred and eighty ovaries were bisected, and antral follicles of different diameter (≤2, 2–5 and ≥5 mm) were carefully dissected from ovarian stroma. The isolated follicles were examined under a surgical dissecting microscope and classified as healthy or atretic according to morphological criteria described previously (Moor et al. 1978). Moreover, the isolated healthy and antral follicles were further confirmed by E2 : P4 ratio (Table S1). At least 120 small-sized (≤2 mm, healthy and atretic), 100 medium-sized (2–5 mm, healthy and atretic) or 80 large-sized (≥5 mm, healthy and atretic) follicles for each group (healthy or atretic follicle with different size) were then hemisected in 1 ml Dulbecco’s phosphatebuffered saline (DPBS) in the Petri dishes, respectively.