**1. Introduction**

184 Zoology

Servos, M.R. (1999). Review of the aquatic toxicity, estrogenic responses and

Shephard, K.L. (1994). Functions for fish mucus. Reviews in Fish Biology and Fisheries. Vol.

Soto, A.M., Chung, K.L. & Sonnenschein, C. (1994). The pesticides endosulfan, toxaphene

Soto, A.M., Lin, T.M., Justicia, H., Silvia, R.M. & Sonnenenschein, C. (1992). An 'in culture'

Srivastava, S.K., Tiwari, P.R. & Srivastav, A.K. (1990). Effects of chlorpyrifos on the kidney

Talmage, S.S. (1994). Environmental and Human Safety of Major Surfactants: Alcohol Ethoxylates and Alkylphenol Ethoxylates. Lewis Publishers, Boca Raton, FL. Toomey, B.H., Monteverdi, G.H. & Di Giulia, R.T. (1999). Octylphenol induces vitellogenin

Uguz, C., Iscan, M., Ergu, A., Belgin I.V. & Togan, I. (2003). The bioaccumulation of

Wendelaar Bonga, S.E. (1997). The stress response in Fish. Physiological Reviews. Vol. 77:

Sayed, A.H., Mekkawy, I.A. & Mahmoud, U.M. (2011). Histopathological alterations in some

Mekkawy, I.A., Mahmoud, U.M. & Sayed, A.H. (2011). Effects of 4-nonylphenol on blood

Mahmoud, U.M., Sayed, A.H. & Mekkawy, I.A. (2011). Biochemical changes of African

Yokota, H., Seki, M., Maeda, M., Oshima, Y., Tadokoro, H., Honjo, T. & Kobayashi, K.

Kang, I.J., Yokota, H., Oshima, Y., Tsuruda, Y., Hano, T., Maeda, M., Imada, N., Tadokoro,

Seki, M., Yokota, H., Maeda, M., Tadokoro, H. & Kobayashi, K. (2003). Effect of 4-

Environmental Toxicology and Chemistry. Vol. 20: 2552-2560.

Research Journal of Canada. Vol. 31: 123–177.

Environmental Health Perspectives. Vol. 102: 380–385.

Contamination and Toxicology. Vol. 45: 748–751.

*mykiss*). Environmental Research. Vol. 92: 262–270.

4: 401-429.

pp. 295–309.

591-625.

Egypt.

2445.

Vol. 22:1507-1516.

Chemistry. Vol. 18: 734–739.

doi:10.1016/j.tice.2011.03.006

Biochemistery,11-053 (Accepted)

bioaccumulation of alkylphenols and alkylphenol polyethoxylates. Water Qualality

and dieldrin have estrogenic effects on human estrogen sensitive cells.

bioassay to assess the estrogenicity of xenobiotics (E-screen), In: Colburn, T.C. (Eds.), Chemically Induced Alterations in Sexual and Functional Development; The Wildlife-human Connection. Princeton Scientific Publishing, Princeton, NJ, USA,

of freshwater catfish. *Heteropneustes fossilis*. Bulletin of Environmental

production and cell death in fish hepatocytes. Environmental Toxicology and

nonyphenol and its adverse effect on the liver of rainbowtrout (*Onchorynchus* 

organs of adults of *Clarias gariepinus* (Burchell, 1822) exposed to 4-nonylphenol. The 19th Conference of Egyptian-German Scocity of Zoology, Bin Sueif University,

cells of the African catfish *Clarias gariepinus* (Burchell, 1822) Tissue Cell,

catfish *Clarias gariepinus* exposed to 4-nonylphenol. African Journal of

(2001). Life-cycle toxicity of 4-nonylphenol to medaka (*Oryzias latipes*),

H. & Honjo, T. (2003). Effect of 4-nonylphenol on the reproduction of Japanese medaka, Oryzias latipes, Environmental Toxicology and Chemistery. Vol. 22: 2438-

nonylphenol and 4- tert octylphenol on sex differentiation and vitellogenin induction in medaka (*Oryzias Latipes*), Environmental Toxicology and Chemistery. The reproductive performance of any mammalian species can be enhanced by enriching nutrition, regulating environmental factors like photoperiod, temperature, humidity etc., through selective breeding, endocrine manipulations and better management practices (Sharma, 2000; Maillet *et al.,* 2002; Bussiere *et al.,* 2002; Iwata *et al.,* 2004, 2005; Rudolf, 2007; Bhardwaj and Sharma, 2011). The endocrine regulation despite being the most complicated is very effective in improving reproductive output of the species. In females, follicular growth and estrous cyclicity are intricately linked (Craig *et al.,* 2007; Sharma and Batra, 2008). Of the thousands of oocytes and primordial follicles present in neonatal ovary only a small fraction i.e. approximately 0.001% are ovulated throughout the active reproductive life span of mammals (Tabarowski *et al.,* 2005; Sharma and Bhardwaj, 2009). Follicular atresia is a wide spread phenomenon that limits the number of ovulations and thus restricting the full reproductive potential of a species. It results in extensive loss of germ cells during development, prenatal, neonatal, prepubertal, pubertal, estrous cycle, pregnancy, lactation and post reproductive life of mammals (Guraya, 1997, 1998; Sharma, 2000; Manabe *et al*., 2003, 2004; Bhardwaj and Sharma, 2011). Follicular atresia is a natural fertility regulatory mechanism that can best be exploited for increasing or decreasing the fertility of the species by decreasing or increasing the frequency of atresia. It is, therefore a lot of research papers have been published on morphology, histochemistry, biochemistry and endocrinology of follicular atresia (Williams and Smith, 1993; Guraya, 1998, 1999; Monniaux, 2002; Sharma, 2003; Sharma and Batra, 2008; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma, 2011). However, the mechanism of atresia still needs to be further explored and analysed for its effective implementation in fertility regulation programme. The molecular mechanism of atresia can be best explained on the basis of apoptosis (Palumbo and Yeh, 1994; Sharma, 2000; Yu *et al*., 2004; Sharma and Bhardwaj, 2009). Apoptosis, a type of physiological cell death is the antithesis of mitosis involved in the regulation of tissue homeostasis (Collins and Lopez, 1993; Schwartzman and Cidlowski, 1993). It affects the single scattered cells in the midest of living tissues without eliciting an inflammatory response and is influenced by growth factors and hormones. Many studies on the morphological changes that occur in granulosa cells and theca-interstitial cells of follicles progressing through atresia have documented that apoptosis is, in all likelihood, the primary mechanism by which cell loss is mediated during follicle degeneration (Tsafriri and Braw, 1984; Hirshfield, 1991; Tilly, 1996). The earliest descriptions of apoptosis, the physiological cell death, in the ovary was made in

Apoptosis and Ovarian Follicular Atresia in Mammals 187

different developmental stages, follicles behave differently in response to factors promoting follicular cell proliferation, growth, differentiation and apoptosis and very few reach upto

A large number of follicles undergo atresia in the late pre-antral to early antral stages. During this hormone dependent growth phase, a selection operates whether to allow the growth of the follicles upto the pre-ovulatory stage or not (Durlinger *et al*., 2000). Gonadotropins are survival factors that prevent atresia (Uilenbroek *et al*., 1980; Chun *et al*., 1994). In addition, the growth factors like epidermal growth factor, transforming growth factor-α, basic fibroblast growth factor (Tilly *et al*., 1992), insulin like growth factor (Chun *et al*., 1994); and cytokine interleukin (IL)-1β (Chun *et al*., 1995), also check onset of atresia. The activity of catalase in follicular fluid and granulosa cells exhibited a declining trend from healthy to atretic follicles. Catalase is an ubiquitous enzyme found in all known organisms. It catalyzes the breakdown of hydrogen peroxide to water and molecular oxygen (Aebi, 1984 and Vohra, 2002). The activity of enzyme superoxide dismutase shows a declining trend from healthy to slightly atretic to atretic follicles both in the follicular fluid and granulosa cells. The superoxide dismutase (SOD) family of metalloenzymes are antioxidants that protect cells from the deleterious effects of the oxygen free radical superoxide (Fridovich, 1975, 1986). SOD catalyzes the conversion of superoxide anion in the oxygen and hydrogen peroxide. Hydrogen peroxide is further converted to oxygen and water by catalase and peroxidases. Trace elements have also traditionally been known to play an important role in cellular homeostasis which entails the tight regulation of cell death. Although zinc known as microelement prevents or suppresses apoptosis, several published reports have clearly demonstrated that zinc may actively induce cell injury and cell death (both apoptosis and necrosis) in malignant as well as normal cells (Iitaka *et al*., 2001; Bae *et al*., 2006; Wiseman *et al*., 2006; Rudolf, 2007; Bhardwaj and Sharma, 2011). Zinc has been shown to induce apoptosis as well as necrosis by altering calcium homeostasis via the generation of oxidative stress, with subsequent activation of mitogen activated protein kinases (MAPKs), via p53 – dependent and p53 independent signaling or through its interaction with the cytoskeleton (Feng *et al*., 2002; Chen *et al*., 2003). The role of reactive oxygen species (ROS) and antioxidants in relation to female reproductive function has, in contrast, received relatively little attention, although there are evidence of both physiological and pathological effects (Guerin *et al*., 2001; Chaudhary *et al*., 2004).. Yang *et al*., (1998) have found high levels of hydrogen peroxide in fragmented embryos and unfertilized oocytes, whilst Paszkowski and Clarke (1996) have reported increased antioxidant consumption (suggesting increased ROS activity) during incubation of poor quality embryos. Recent studies indicate that apoptosis of granulosa cells shows a close relationship between estradiol and progesterone titre in the follicular fluid (Yu *et al*., 2004; Sharma and Batra, 2008; Sharma *et al*., 2008). The level of insulin like growth factor-1 IGF-1 but not insulin like growth factor-II IGF-II is the crucial factor in deciding whether a follicle will mature or undergo atresia (Yu *et al*., 2004). The role of trace elements as pro-apoptotic or anti-apoptotic factors is not well known till date. The cascade of morphological and biochemical alterations need to be studied to rescue the germ

A large population of ovarian follicles in mammals is lost through a wide spread phenomenon of follicular atresia that limits the number of ovulations thus restricting the full reproductive potential of a species (Sharma, 2000; Yu *et al.,* 2004; Tabarowski *et al.*, 2005;

ovulation (Tilly *et al*., 1991; Jiang *et al*., 2003; Craig *et al*., 2007).

cells or somatic cell losses.

1885 on the morphological analysis of granulosa cells during the degeneration of antral follicles in the rabbit ovary (Flemming, 1885). A process referred to as 'Chromatolysis' was proposed as a mechanism by which granulosa cell loss was mediated, and in retrospect these observations closely matched all of the morphological criteria now known to be the hallmarks of apoptosis (Kerr *et al.,* 1972, 1994). In the ovary, the mechanisms underlying decisions of life and death involve cross dialogue between pro-apoptotic and pro-survival molecules (Hussein, 2005). Apoptosis operates in ovarian follicles throughout fetal and adult life. During fetal life, apoptosis is restricted to the oocytes, whereas in adult life, this phenomenon is frequently observed in granulosa cells of secondary and antral follicles (Hussein, 2005).

Apoptosis, a genetically regulated cell suicide is an energy requiring process observed commonly during early embryonic development. Apoptosis permits the safe disposal of cells at the point in time when they have fulfilled their biological functions (Hussein, 2005). The ovary represents the paradigm of effective apoptosis during active reproductive period of the animal. Apoptosis is central to many aspects of the ovary and executed by several molecular pathways. Bcl-2 (B-cell lymphoma 2) family, TNF(Tumor necrosis factor), caspases and Transforming growth factor-β TGF- β proteins constitute well established mechanisms of apoptosis (Hussein, 2005). Morphologically, apoptosis is characterized by cell shrinkage, membrane blebbing and cytoplasmic fragmentation in oocytes (Wu *et al*., 2000; Chaube *et al*., 2005), granulosa cells (Sharma and Sawhney, 1999; Sharma, 2003; Sharma and Bhardwaj, 2007, 2009) and corpus luteum (Sharma and Batra, 2005). A unique biochemical event in apoptosis is the activation of a Ca+2/Mg+2 dependent endogenous endonuclease. This enzyme cleaves genomic DNA at internucleosomal regions, resulting in chopping of DNA in to fragments of 180-200 base pairs (bp). These DNA fragments can be visualized as a distinct ladder pattern by agarose gel electrophoresis. The presence of this DNA pattern in cells is considered a hallmark of apoptosis (Schwartzman and Cidlowski, 1993; Hsueh *et al*., 1994; Yang and Rajamahendran, 2000). In addition to the detection of oligonucleosomes in isolated DNA, the occurrence of apoptosis is also inferred from the characteristic morphological appearance of degenerating cells, together with the detection of fragmented DNA in single cells *in situ* through the use of the 3' end-labeling technique (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] (Gavrieli *et al*., 1992; Palumbo and Yeh, 1994; Sharma and Bhardwaj, 2009). The early development of mammalian ovarian follicles is poorly understood. Although follicle stimulating harmone FSH and luteinizing harmone LH are essential for follicular development from the secondary stage onwards, but the onset of growth of primordial follicles is independent of gonadotrophin hormones (Tilly, 1996). Factors initiating the transition of quiescent primordial follicles to the pool of growing follicles are still unknown. Studies on animals have indicated that several locally produced growth factors are involved in the multifactorial regulation of the growth of primary and secondary follicles (Durlinger, 2000). According to recent findings, the loss of follicles by atresia would be counteracted by the formation of new primordial follicles from germ line stem cells (Johnson *et al*., 2004), a condition still controversial and is yet to be confirmed (Bristol Gould *et al*., 2006; Liu *et al*., 2007). Throughout active reproductive life, follicles leave the resting repository pool to enter the growing pool on a regular basis and pass through subsequent developmental stages under the influence of stage specific subset of intra-ovarian regulators and endocrine factors like growth factors, cytokines and gonadal steroids (Gilchrist *et al*., 2004, Pangas, 2007). At

1885 on the morphological analysis of granulosa cells during the degeneration of antral follicles in the rabbit ovary (Flemming, 1885). A process referred to as 'Chromatolysis' was proposed as a mechanism by which granulosa cell loss was mediated, and in retrospect these observations closely matched all of the morphological criteria now known to be the hallmarks of apoptosis (Kerr *et al.,* 1972, 1994). In the ovary, the mechanisms underlying decisions of life and death involve cross dialogue between pro-apoptotic and pro-survival molecules (Hussein, 2005). Apoptosis operates in ovarian follicles throughout fetal and adult life. During fetal life, apoptosis is restricted to the oocytes, whereas in adult life, this phenomenon is frequently observed in granulosa cells of secondary and antral follicles

Apoptosis, a genetically regulated cell suicide is an energy requiring process observed commonly during early embryonic development. Apoptosis permits the safe disposal of cells at the point in time when they have fulfilled their biological functions (Hussein, 2005). The ovary represents the paradigm of effective apoptosis during active reproductive period of the animal. Apoptosis is central to many aspects of the ovary and executed by several molecular pathways. Bcl-2 (B-cell lymphoma 2) family, TNF(Tumor necrosis factor), caspases and Transforming growth factor-β TGF- β proteins constitute well established mechanisms of apoptosis (Hussein, 2005). Morphologically, apoptosis is characterized by cell shrinkage, membrane blebbing and cytoplasmic fragmentation in oocytes (Wu *et al*., 2000; Chaube *et al*., 2005), granulosa cells (Sharma and Sawhney, 1999; Sharma, 2003; Sharma and Bhardwaj, 2007, 2009) and corpus luteum (Sharma and Batra, 2005). A unique biochemical event in apoptosis is the activation of a Ca+2/Mg+2 dependent endogenous endonuclease. This enzyme cleaves genomic DNA at internucleosomal regions, resulting in chopping of DNA in to fragments of 180-200 base pairs (bp). These DNA fragments can be visualized as a distinct ladder pattern by agarose gel electrophoresis. The presence of this DNA pattern in cells is considered a hallmark of apoptosis (Schwartzman and Cidlowski, 1993; Hsueh *et al*., 1994; Yang and Rajamahendran, 2000). In addition to the detection of oligonucleosomes in isolated DNA, the occurrence of apoptosis is also inferred from the characteristic morphological appearance of degenerating cells, together with the detection of fragmented DNA in single cells *in situ* through the use of the 3' end-labeling technique (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] (Gavrieli *et al*., 1992; Palumbo and Yeh, 1994; Sharma and Bhardwaj, 2009). The early development of mammalian ovarian follicles is poorly understood. Although follicle stimulating harmone FSH and luteinizing harmone LH are essential for follicular development from the secondary stage onwards, but the onset of growth of primordial follicles is independent of gonadotrophin hormones (Tilly, 1996). Factors initiating the transition of quiescent primordial follicles to the pool of growing follicles are still unknown. Studies on animals have indicated that several locally produced growth factors are involved in the multifactorial regulation of the growth of primary and secondary follicles (Durlinger, 2000). According to recent findings, the loss of follicles by atresia would be counteracted by the formation of new primordial follicles from germ line stem cells (Johnson *et al*., 2004), a condition still controversial and is yet to be confirmed (Bristol Gould *et al*., 2006; Liu *et al*., 2007). Throughout active reproductive life, follicles leave the resting repository pool to enter the growing pool on a regular basis and pass through subsequent developmental stages under the influence of stage specific subset of intra-ovarian regulators and endocrine factors like growth factors, cytokines and gonadal steroids (Gilchrist *et al*., 2004, Pangas, 2007). At

(Hussein, 2005).

different developmental stages, follicles behave differently in response to factors promoting follicular cell proliferation, growth, differentiation and apoptosis and very few reach upto ovulation (Tilly *et al*., 1991; Jiang *et al*., 2003; Craig *et al*., 2007).

A large number of follicles undergo atresia in the late pre-antral to early antral stages. During this hormone dependent growth phase, a selection operates whether to allow the growth of the follicles upto the pre-ovulatory stage or not (Durlinger *et al*., 2000). Gonadotropins are survival factors that prevent atresia (Uilenbroek *et al*., 1980; Chun *et al*., 1994). In addition, the growth factors like epidermal growth factor, transforming growth factor-α, basic fibroblast growth factor (Tilly *et al*., 1992), insulin like growth factor (Chun *et al*., 1994); and cytokine interleukin (IL)-1β (Chun *et al*., 1995), also check onset of atresia. The activity of catalase in follicular fluid and granulosa cells exhibited a declining trend from healthy to atretic follicles. Catalase is an ubiquitous enzyme found in all known organisms. It catalyzes the breakdown of hydrogen peroxide to water and molecular oxygen (Aebi, 1984 and Vohra, 2002). The activity of enzyme superoxide dismutase shows a declining trend from healthy to slightly atretic to atretic follicles both in the follicular fluid and granulosa cells. The superoxide dismutase (SOD) family of metalloenzymes are antioxidants that protect cells from the deleterious effects of the oxygen free radical superoxide (Fridovich, 1975, 1986). SOD catalyzes the conversion of superoxide anion in the oxygen and hydrogen peroxide. Hydrogen peroxide is further converted to oxygen and water by catalase and peroxidases. Trace elements have also traditionally been known to play an important role in cellular homeostasis which entails the tight regulation of cell death. Although zinc known as microelement prevents or suppresses apoptosis, several published reports have clearly demonstrated that zinc may actively induce cell injury and cell death (both apoptosis and necrosis) in malignant as well as normal cells (Iitaka *et al*., 2001; Bae *et al*., 2006; Wiseman *et al*., 2006; Rudolf, 2007; Bhardwaj and Sharma, 2011). Zinc has been shown to induce apoptosis as well as necrosis by altering calcium homeostasis via the generation of oxidative stress, with subsequent activation of mitogen activated protein kinases (MAPKs), via p53 – dependent and p53 independent signaling or through its interaction with the cytoskeleton (Feng *et al*., 2002; Chen *et al*., 2003). The role of reactive oxygen species (ROS) and antioxidants in relation to female reproductive function has, in contrast, received relatively little attention, although there are evidence of both physiological and pathological effects (Guerin *et al*., 2001; Chaudhary *et al*., 2004).. Yang *et al*., (1998) have found high levels of hydrogen peroxide in fragmented embryos and unfertilized oocytes, whilst Paszkowski and Clarke (1996) have reported increased antioxidant consumption (suggesting increased ROS activity) during incubation of poor quality embryos. Recent studies indicate that apoptosis of granulosa cells shows a close relationship between estradiol and progesterone titre in the follicular fluid (Yu *et al*., 2004; Sharma and Batra, 2008; Sharma *et al*., 2008). The level of insulin like growth factor-1 IGF-1 but not insulin like growth factor-II IGF-II is the crucial factor in deciding whether a follicle will mature or undergo atresia (Yu *et al*., 2004). The role of trace elements as pro-apoptotic or anti-apoptotic factors is not well known till date. The cascade of morphological and biochemical alterations need to be studied to rescue the germ cells or somatic cell losses.

A large population of ovarian follicles in mammals is lost through a wide spread phenomenon of follicular atresia that limits the number of ovulations thus restricting the full reproductive potential of a species (Sharma, 2000; Yu *et al.,* 2004; Tabarowski *et al.*, 2005;

Apoptosis and Ovarian Follicular Atresia in Mammals 189

type IV collagen, proteoglycans, insulin like growth factor II/mannose 6 phosphate receptors, and matrix metaloproteinases 2 and -9 increased whereas 450 aromatase and connexin 43 decreased within the wall of granulosa cells during follicular atresia in sheep (Huet *et al*., 1997, 1998, Sharma and Guraya 1998a, b). Guraya *et al*., (1994) have demonstrated that the degenerative signs also appear in cumulus cells of antral atretic follicles, whereas atresia proceeds gradually, membrana granulosa thins off and theca hypertrophy enhances in antral atretic follicles (Sharma *et al*., 1992; Guraya *et al*., 1994). The appearance of pyknotic granulosa cells as the first sign of atresia in goat strongly advocate the concept that phenomenon of atresia in bovine species is similar (Zimmerman *et al*., 1987; Wezel *et al.*, 1999; Hastie and Haresign, 2006). The chromophilic pyknotic granules observed in goat atretic follicles were similar to that of DNA - positive masses observed in sheep (Hay *et al.,* 1976, Hay and Cran 1978; Zhou and Zhang, 2005). However such granular material is negligible in cow (Guraya, 1997). The delamination of mural granulosa cells from the basal lamina leads to disruption causing a decline in oxygen carrying blood transudate that decreases the metabolic pace and results in onset of atresia (Guraya, 1985, 1998; Sharma and Guraya, 1998 b, c). However, Hay *et al*., (1976) suggested that it is because of decline in estrogen synthesis and release. In advance stage of atresia, there is a tendency towards accumulation and storage of sudanophilic lipids in the atretic oocytes (Guraya, 1973 a). The mural layers acquire more lipids as compared to cumulus cells or peripheral granulosa cells. Similar trends were reported in hamster and rabbit (Guraya and Greenwald, 1964), thus indicating granulosa heterogeneity in morphology and physiology (Parshad and Guraya 1983; Sharma and Guraya, 1990). The NADH and NADPH-dependent tetrazolium reductase activity in goat further endorse this concept. The theca interna of atretic antral follicles undergo hypertrophy and develop lipid droplets largely comprised of phospholipids which are associated with steroidogenesis in mammals (Guraya, 1973 b, 1974 a, b, 1977, 1978 a,b; Nicosia, 1980). This hypertrophied theca interna in atretic follicles of goat finally constitute conspicuous masses of interstitial gland tissue which show strong 3-β-HSDH activity (Guraya, 1971, 1973; Guraya *et al*., 1994; Sharma and Batra, 2008). These lipid droplets are largely phospholipids and triglycerides which change to cholesterol, its esters and phospholipids, and remain associated with theca type interstitial gland tissue. Various carbohydrates, proteins and enzyme histochemistry were used as an index to indicate whether atresia has initiated or not (Guraya, 1984, 1985; Sharma and Guraya, 1992, 1997; Burke *et al.,* 2005). The follicles undergoing atresia disintegrate oocytes and granulosa cells are reabsorbed while theca interna hypertrophy and acquire morphological and

histochemical characteristics of a steroidogenic tissue (Sharma and Batra, 2008).

Intrafollicular paracrine steroid interactions are dependent on FSH and LH which regulate follicular development and oestrogen synthesis and release (Guraya, 1985, 1998; Ireland, 1987; Richards *et al.,* 1993; Gore-Langton and Armstrong, 1994; Hillier, 1994; Guthrie and Cooper, 1996; Maillet *et al.,* 2002; Burke *et al.,* 2005). Alterations in the production of steroid in the antral fluid are the first biochemical manifestations of atresia. (Guraya, 1985, 1998; Hillier, 1985; Greenwald and Terranova, 1988; Westhof *et al.,* 1991; Gore-Langton and Armstrong, 1994; Greenwald and Roy, 1994; Quirk *et al.,* 2006). Variations in normal and atretic follicular fluid concentration of androgens and that of progesterone are not significantly different (Hillier, 1985; Westhof *et al.,* 1991; Moor, 1977; Gore-Langton and

**3. Structure and ultrastructure of the process** 

Slomczynska *et al.*, 2006; Sharma and Bhardwaj, 2009). Follicle atresia is a wide spread degenerative phenomenon by which follicles lose their structural integrity and oocytes are lost from the ovaries other than the process of ovulation (Guraya, 1997; Sharma, 2000). Follicular atresia decreases the number of ovulations and the follicle wall components transmutate into a steroidogenically functional interstitial gland tissue (Sharma and Guraya, 1992; Manabe *et al*., 2003) that helps in the endocrine regulation of ovarian physiology. Atresia affects the follicles at all stages of development but extensive loss of germ cells occurs during early development, prenatal, neonatal, prepubertal, pubertal, estrous cycle, pregnancy, lactation and post reproductive life of mammals (Guraya, 1998; Sharma, 2000; Tabarowski *et al*., 2005). During early development primordial follicle population is the most affected whereas in the active reproductive phase the frequency of atresia is maximum in antral follicles (Danell, 1987; Guraya *et al.*, 1994, Guraya, 2000; Sharma, 2000) The modulation of frequency of atresia can regulate the fertility of the animal (Sharma, 2000). Follicle atresia is characterized by the appearance of pyknotic granulosa cells in intact membrana granulosa; free floating large sized granulosa cells with pyknotic nuclei; hyalinization of granulosa cells; chromolysis of the granulosa cell nuclei and their enucleation; loosening of the intercellular matrix; delamination of intercellular matrix; colloidal, opaque and cloudy follicular fluid; detachment of cumulus-oophorous complex from mural granulosa cells; appearance of RBCs in the follicle; invasion of connective tissue fibres within the follicle and huge accumulation of follicular fluid resulting in the cyst formation (Sharma, 2000; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma, 2011).
