**2. Apoptosis in normal cycling mature ovary**

In prepubertal and mature mammalian ovaries the preantral atretic follicles, the atretic oocyte is characterized by shrinked ooplasmic contents surrounded by a peripheral zone of lipoidal components which is strongly sudanophilic. This zone in a few atretic oocytes is so dense that it occuludens the central mass. (Sharma *et al.,* 1992; Guraya *et al.,* 1994). Initially, pyknosis in granulosa cells was restricted only to specific area which consequently prolonged and large zone of atretic pyknotic granulosa cells was formed, which results in loosening of membrana granulosa (Sharma *et al.,* 1992). These pyknotic granulosa cells give positive test for lipids and 3-β-HSDH, indicating their active role in steroidogenesis (Guraya *et al.,* 1994). Pyknosis of granulosa cells as the first sign of atresia is evocative of the general plan and path of atretogenic changes in mammals (Guraya *et al.,* 1987; Tilly *et al.*, 1996; Burke *et al*., 2005; Tatone *et al.*, 2008; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma, 2011). The various nuclear and ooplasmic contents are drastically affected in atretic preantral follicles. The organelles showing abnormal morphology and distribution increase in number and proceed towards disintegration and then lead to an accumulation of lipids in the oocytes and degenerating granulosa cells because of liberation of cytosolic membrane bound lipids (Byskov, 1978). In the atretic preantral follicles the granulosa cells develop histochemical characteristics of steroidogenic cells as evidenced by weak 3-β-HSDH activity (Sharma, 2000). In antral follicles, apoptosis is characterized by the presence of pyknotic nucleus in the membrana granulosa, appearance of spaces and release of fragments of pyknotic nuclei in the peripheral zone of antrum in type 1b (Sharma *et al.,* 1992; Guraya *et al.,* 1994; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma,2011). The granulosa cell glycoconjugates are changed during follicular atresia in the pig and rat ovaries as shown with lectins (Sharma and Guraya 1992; Kimura *et al.,* 1999). The levels of fibronectin, laminin,

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).

In prepubertal and mature mammalian ovaries the preantral atretic follicles, the atretic oocyte is characterized by shrinked ooplasmic contents surrounded by a peripheral zone of lipoidal components which is strongly sudanophilic. This zone in a few atretic oocytes is so dense that it occuludens the central mass. (Sharma *et al.,* 1992; Guraya *et al.,* 1994). Initially, pyknosis in granulosa cells was restricted only to specific area which consequently prolonged and large zone of atretic pyknotic granulosa cells was formed, which results in loosening of membrana granulosa (Sharma *et al.,* 1992). These pyknotic granulosa cells give positive test for lipids and 3-β-HSDH, indicating their active role in steroidogenesis (Guraya *et al.,* 1994). Pyknosis of granulosa cells as the first sign of atresia is evocative of the general plan and path of atretogenic changes in mammals (Guraya *et al.,* 1987; Tilly *et al.*, 1996; Burke *et al*., 2005; Tatone *et al.*, 2008; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma, 2011). The various nuclear and ooplasmic contents are drastically affected in atretic preantral follicles. The organelles showing abnormal morphology and distribution increase in number and proceed towards disintegration and then lead to an accumulation of lipids in the oocytes and degenerating granulosa cells because of liberation of cytosolic membrane bound lipids (Byskov, 1978). In the atretic preantral follicles the granulosa cells develop histochemical characteristics of steroidogenic cells as evidenced by weak 3-β-HSDH activity (Sharma, 2000). In antral follicles, apoptosis is characterized by the presence of pyknotic nucleus in the membrana granulosa, appearance of spaces and release of fragments of pyknotic nuclei in the peripheral zone of antrum in type 1b (Sharma *et al.,* 1992; Guraya *et al.,* 1994; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma,2011). The granulosa cell glycoconjugates are changed during follicular atresia in the pig and rat ovaries as shown with lectins (Sharma and Guraya 1992; Kimura *et al.,* 1999). The levels of fibronectin, laminin,

**2. Apoptosis in normal cycling mature ovary**

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).

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

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

Apoptosis and Ovarian Follicular Atresia in Mammals 191

clumping of chromatin, swelling and dissolution of organelles, rupture of the cell membrane, and finally disintegration of all cellular components. Ultrastructural changes in degenerating granulosa cells of goat ovaries strongly support the morphological hallmarks of apoptotic cells. First, the nucleus and cytoplasm became condensed, then the condensed cells were fragmented but retained the integrity of organelles, i.e. mitochondria, and nuclear and plasma membrane. Since cell debris in atretic follicles increased in number following the appearance of apoptotic bodies and had condensed nuclei similar to apoptotic bodies, the debris is possibly comprised of degraded apoptotic bodies. The investigations on fine morphology of granulosa cells *in vivo* showed similar characteristic features as observed after gonadotrophins and steroid treatment in rat *in vitro*, thus, confirming the role of hormones in structural modification of granulosa cells at different phases of the cycle (Balboni and Zecchi, 1981; Yu *et al*., 2004; Sharma *et al*., 2008; Sharma and Batra, 2008).

Fig. 1. Electron micrograph of apoptotic granulosa cells showing increased indentation (arrow) and vacuoles (star) of different shapes within condensed chromatin material.

Withdrawal of typical intercellular microvilli or interacting surfaces with the advancement of atresia, the appearance of lysosomal vacuoles and lipid droplets of varied dimensions observed during atresia in goat, further support the histological and histochemical characteristics of follicular atresia in mammals (Motta, 1972; Parshad and Guraya, 1983; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma,2011). The presence of lysosomal vacuoles in the degenerating granulosa cells of goat during advanced stage of atresia further support the findings of Sharma and Guraya (1997) who have reported that lysosomal

Armstrong, 1994; Slomczynska *et al.,* 2006). Hormonal profile of normal and atretic follicles depends largely on the stage of advancement of atresia. Normal1y it is oestrogen to progesterone ratio which determines whether a follicle will mature or undergo atresia (Moor *et al.,* 1978; Ireland and Roche, 1982, 1983 a, b; Guraya, 1997; Yu *et al.,* 2004; Sharma *et al.,* 2008). The estrogen level is highest in the preovulatory healthy follicles of pig, sheep, cow, and goat as compared to atretic ones (Eiler and Nalbandov, 1977; Moor *et al.,* 1978; Carlson *et al.,* 1981; Fortune *et al.,* 1988; Burke *et al.,* 2005; Sharma *et al.,* 2008). Proliferation, migration, differentiation and cellular death constitute the most important stages in the development and growth ovarian follicles and is actively involved in turnover of ovarian tissues. The cellular death basically involves two pathways: necrosis or apoptosis (Wyllie *et al.,* 1980; Kressel and Groscurth, 1994). Necrosis is induced as the result of injuries or environmental pathological influences and produces a series of cellular alterations that begin with the changes in cellular membrane permeability with consequent disruption of cytoplasmic structures and ensuing nuclear degeneration (Pol De *et al.,* 1997). On the other hand, apoptosis is the process of cellular self destruction which also involves active process of intracellular synthesis (Wyllie *et al.,* 1980; Cohen and Duke, 1984) and is controlled by cellular genes. In granulosa cell apoptosis, the primary events observed were the nuclear compaction, the chromatin collapses in to large irregular masses surrounded by nuclear envelope, and plasma membrane introflexes forming deep incisions that confer to the cell a very irregular appearance (Sharma and Bhardwaj 2009; Bhardwaj and Sharma, 2011). Despite this, the cellular organelles maintain their morpho-functional integrity. Subsequently the cell fragments into spheroidal subunits surrounded by membranes (apoptotic bodies) that contain portions of cytoplasm and nucleus that are finally phagocytosed by neighbouring cells or by macrophages. The cellular death in granulosa cells therefore is distinguishable on accounts of histological and ultrastructural morphology, and pattern of changes in cell organelles and the chromatin. In necrosis, chromatin was altered at the end in a disorderly fashion, whereas during apoptosis it was precociously excised in a regular sequence. The apoptotic changes observed in the degenerating granulosa cells from goat ovarian follicles revealed diminished size, withered surface morphology and pyknosis (Hay and Cran, 1978; Hirshfield, 1983; Guraya, 1985; Kaur and Guraya, 1987; Tilly, 1996; Manabe *et al*., 2003, 2004; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma, 2011). The goat granulsoa cells resemble rat atretic granulosa cells in terms of nuclear condensation and cytoplasmic shrinkage as well as presence of apoptotic bodies (Hurwitz *et al.,* 1996). The undulations of nuclear membrane and pinching off of the apoptotic bodies strongly advocate the concept that the apoptosis is the basic mechanism involved in the phenomenon of atresia of granulosa cells in mammals. The vacuolization of condensed chromatin material within the nucleus was the specific positive indicator of apoptosis (Figs. 1, 2). Various studies, conducted during atresia of ovine and caprine follicles, cumulatively indicate that degenerative changes are restricted only to membrana granulosa layers that lie adjacent to the antral cavity during early phases of atresia (Sharma *et al.,* 1992). The detachment or delamination of mural granulosa cells from the basal lamina in the initial phases induce atresia due to disruption of oxygen and nutritional milieu carying blood transudate to the cells (Guraya, 1985, 1998; Sharma and Guraya, 1998 b, c; Sharma, 2000, 2003). Ultrastructurally, typical apoptotic granulosa cells from the antral follicle of goat show compaction and segregation of chromatin, condensation of the cytoplasm maintaining the integrity of organelles, and subsequent fragmentation in to membrane bounded apoptotic bodies (Figs.3, 4). Necrotic cells are characterized by irregular

Armstrong, 1994; Slomczynska *et al.,* 2006). Hormonal profile of normal and atretic follicles depends largely on the stage of advancement of atresia. Normal1y it is oestrogen to progesterone ratio which determines whether a follicle will mature or undergo atresia (Moor *et al.,* 1978; Ireland and Roche, 1982, 1983 a, b; Guraya, 1997; Yu *et al.,* 2004; Sharma *et al.,* 2008). The estrogen level is highest in the preovulatory healthy follicles of pig, sheep, cow, and goat as compared to atretic ones (Eiler and Nalbandov, 1977; Moor *et al.,* 1978; Carlson *et al.,* 1981; Fortune *et al.,* 1988; Burke *et al.,* 2005; Sharma *et al.,* 2008). Proliferation, migration, differentiation and cellular death constitute the most important stages in the development and growth ovarian follicles and is actively involved in turnover of ovarian tissues. The cellular death basically involves two pathways: necrosis or apoptosis (Wyllie *et al.,* 1980; Kressel and Groscurth, 1994). Necrosis is induced as the result of injuries or environmental pathological influences and produces a series of cellular alterations that begin with the changes in cellular membrane permeability with consequent disruption of cytoplasmic structures and ensuing nuclear degeneration (Pol De *et al.,* 1997). On the other hand, apoptosis is the process of cellular self destruction which also involves active process of intracellular synthesis (Wyllie *et al.,* 1980; Cohen and Duke, 1984) and is controlled by cellular genes. In granulosa cell apoptosis, the primary events observed were the nuclear compaction, the chromatin collapses in to large irregular masses surrounded by nuclear envelope, and plasma membrane introflexes forming deep incisions that confer to the cell a very irregular appearance (Sharma and Bhardwaj 2009; Bhardwaj and Sharma, 2011). Despite this, the cellular organelles maintain their morpho-functional integrity. Subsequently the cell fragments into spheroidal subunits surrounded by membranes (apoptotic bodies) that contain portions of cytoplasm and nucleus that are finally phagocytosed by neighbouring cells or by macrophages. The cellular death in granulosa cells therefore is distinguishable on accounts of histological and ultrastructural morphology, and pattern of changes in cell organelles and the chromatin. In necrosis, chromatin was altered at the end in a disorderly fashion, whereas during apoptosis it was precociously excised in a regular sequence. The apoptotic changes observed in the degenerating granulosa cells from goat ovarian follicles revealed diminished size, withered surface morphology and pyknosis (Hay and Cran, 1978; Hirshfield, 1983; Guraya, 1985; Kaur and Guraya, 1987; Tilly, 1996; Manabe *et al*., 2003, 2004; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma, 2011). The goat granulsoa cells resemble rat atretic granulosa cells in terms of nuclear condensation and cytoplasmic shrinkage as well as presence of apoptotic bodies (Hurwitz *et al.,* 1996). The undulations of nuclear membrane and pinching off of the apoptotic bodies strongly advocate the concept that the apoptosis is the basic mechanism involved in the phenomenon of atresia of granulosa cells in mammals. The vacuolization of condensed chromatin material within the nucleus was the specific positive indicator of apoptosis (Figs. 1, 2). Various studies, conducted during atresia of ovine and caprine follicles, cumulatively indicate that degenerative changes are restricted only to membrana granulosa layers that lie adjacent to the antral cavity during early phases of atresia (Sharma *et al.,* 1992). The detachment or delamination of mural granulosa cells from the basal lamina in the initial phases induce atresia due to disruption of oxygen and nutritional milieu carying blood transudate to the cells (Guraya, 1985, 1998; Sharma and Guraya, 1998 b, c; Sharma, 2000, 2003). Ultrastructurally, typical apoptotic granulosa cells from the antral follicle of goat show compaction and segregation of chromatin, condensation of the cytoplasm maintaining the integrity of organelles, and subsequent fragmentation in to membrane bounded apoptotic bodies (Figs.3, 4). Necrotic cells are characterized by irregular clumping of chromatin, swelling and dissolution of organelles, rupture of the cell membrane, and finally disintegration of all cellular components. Ultrastructural changes in degenerating granulosa cells of goat ovaries strongly support the morphological hallmarks of apoptotic cells. First, the nucleus and cytoplasm became condensed, then the condensed cells were fragmented but retained the integrity of organelles, i.e. mitochondria, and nuclear and plasma membrane. Since cell debris in atretic follicles increased in number following the appearance of apoptotic bodies and had condensed nuclei similar to apoptotic bodies, the debris is possibly comprised of degraded apoptotic bodies. The investigations on fine morphology of granulosa cells *in vivo* showed similar characteristic features as observed after gonadotrophins and steroid treatment in rat *in vitro*, thus, confirming the role of hormones in structural modification of granulosa cells at different phases of the cycle (Balboni and Zecchi, 1981; Yu *et al*., 2004; Sharma *et al*., 2008; Sharma and Batra, 2008).

Fig. 1. Electron micrograph of apoptotic granulosa cells showing increased indentation (arrow) and vacuoles (star) of different shapes within condensed chromatin material.

Withdrawal of typical intercellular microvilli or interacting surfaces with the advancement of atresia, the appearance of lysosomal vacuoles and lipid droplets of varied dimensions observed during atresia in goat, further support the histological and histochemical characteristics of follicular atresia in mammals (Motta, 1972; Parshad and Guraya, 1983; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma,2011). The presence of lysosomal vacuoles in the degenerating granulosa cells of goat during advanced stage of atresia further support the findings of Sharma and Guraya (1997) who have reported that lysosomal

Apoptosis and Ovarian Follicular Atresia in Mammals 193

(1992) have reported changes in glycoconjugates and carbohydrates of atretic granulosa cells in rat and have postulated that changes in histochemical mapping of negatively charged moieties induces uncoupling of membrane interactions subsequently leading to impairment of membrane permeability characteristics that finally lead to atresia or cell death due to apoptosis. The alterations in acidic phospholipids phosphatidyl serine content that acts as apoptosis inducing agent (Krishnamurthy *et al.,* 2000), modulates the membrane chemistry leading to a change in its permeability to water molecules. The cell becomes larger and hyaline if the permeability is enhanced whereas the contents become pyknotic and electron dense if the permeability decreases. Recent studies have demonstrated that apoptosis involves cleaving of DNA in several animal species. Internucleosomal DNA fragmentation has been considered to be characteristic of apoptosis and is one of the earliest event (Schwartzman and Cidlowski, 1993; Okamura *et al*., 2001; Hastie and Haresign, 2006).

Fig. 3. Electron micrograph of apoptotic granulosa cells showing the pyknotic chromatin material adhering to the periphery of nuclear membrane (arrow) and vacuoles (v) of different shapes and sizes were observed within condensed chromatin material.

**v**

In addition to the detection of oligonucleosomes in extracted DNA, the occurrence of apoptosis may also be inferred from the characteristic morphological appearance of degenerating cells, together with the detection of fragment DNA in single cells *in situ* using TUNEL (Gavrieli *et al.,* 1992; Palumbo and Yeh, 1994; Bristol and Gould *et al*., 2006; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma,2011). Using *in situ* 3' end labeling (TUNEL), which can detect apoptosis precisely at the single cell level without disruption of the tissue

vacuoles appear in the cytoplasm which subsequently destabilize plasma membrane completely leading to the extrusion of interacellular contents making it more hyaline.

Fig. 2. Electron micrograph of apoptotic granulosa cells showing uneven wavy nuclear envelope (arrow) and increased vacuolization (star) of cytoplasm.

The presence of hyaline granulosa cells in the mural and antral layers of membrana granulosa and free floating in follicular fluid were observed in goat ovary (Sharma and Bhardwaj, 2007). The similar distribution of cells were also observed in primates and other mammals (Byskov, 1974; Balboni and Zecchi, 1981; Bill and Greenwald, 1981; Sharma and Guraya, 1992, 1997). The increased undulation and indentations of the nuclear membrane and pinching off of the nuclear fragments suggests that apoptosis is involved in initiation and execuation of cell death during atresia in goat. The increase in the frequency and dimensions of nuclear pores as well as flattening of the nuclear membrane observed in goat follicles are similar to the earlier findings on ultrastructure of apoptotic granulosa cells in rat and cow (Coucouvanis *et al.,* 1993; Grotowski *et al.,* 1997; Isobe and Yoshimura, 2000; Yang and Rajamahendran, 2000; Inoue *et al*., 2003), thereby suggesting a common plan of apoptosis in bovine species. The membrane bound pyknotic chromatin material carrying apoptotic vesicles were observed lying within the cytoplasm during the advanced stages of atresia which endorse the concept that apoptotic bodies are formed from condensed chromatin material packed in small vacuoles limited by the nuclear membrane. In a few cells, the presence of condensed cytoplasm in contrast to hyaline one was possibly due to the differential functional impairment of the cytoplasmic membrane. Sharma and Guraya

vacuoles appear in the cytoplasm which subsequently destabilize plasma membrane completely leading to the extrusion of interacellular contents making it more hyaline.

Fig. 2. Electron micrograph of apoptotic granulosa cells showing uneven wavy nuclear

The presence of hyaline granulosa cells in the mural and antral layers of membrana granulosa and free floating in follicular fluid were observed in goat ovary (Sharma and Bhardwaj, 2007). The similar distribution of cells were also observed in primates and other mammals (Byskov, 1974; Balboni and Zecchi, 1981; Bill and Greenwald, 1981; Sharma and Guraya, 1992, 1997). The increased undulation and indentations of the nuclear membrane and pinching off of the nuclear fragments suggests that apoptosis is involved in initiation and execuation of cell death during atresia in goat. The increase in the frequency and dimensions of nuclear pores as well as flattening of the nuclear membrane observed in goat follicles are similar to the earlier findings on ultrastructure of apoptotic granulosa cells in rat and cow (Coucouvanis *et al.,* 1993; Grotowski *et al.,* 1997; Isobe and Yoshimura, 2000; Yang and Rajamahendran, 2000; Inoue *et al*., 2003), thereby suggesting a common plan of apoptosis in bovine species. The membrane bound pyknotic chromatin material carrying apoptotic vesicles were observed lying within the cytoplasm during the advanced stages of atresia which endorse the concept that apoptotic bodies are formed from condensed chromatin material packed in small vacuoles limited by the nuclear membrane. In a few cells, the presence of condensed cytoplasm in contrast to hyaline one was possibly due to the differential functional impairment of the cytoplasmic membrane. Sharma and Guraya

envelope (arrow) and increased vacuolization (star) of cytoplasm.

(1992) have reported changes in glycoconjugates and carbohydrates of atretic granulosa cells in rat and have postulated that changes in histochemical mapping of negatively charged moieties induces uncoupling of membrane interactions subsequently leading to impairment of membrane permeability characteristics that finally lead to atresia or cell death due to apoptosis. The alterations in acidic phospholipids phosphatidyl serine content that acts as apoptosis inducing agent (Krishnamurthy *et al.,* 2000), modulates the membrane chemistry leading to a change in its permeability to water molecules. The cell becomes larger and hyaline if the permeability is enhanced whereas the contents become pyknotic and electron dense if the permeability decreases. Recent studies have demonstrated that apoptosis involves cleaving of DNA in several animal species. Internucleosomal DNA fragmentation has been considered to be characteristic of apoptosis and is one of the earliest event (Schwartzman and Cidlowski, 1993; Okamura *et al*., 2001; Hastie and Haresign, 2006).

Fig. 3. Electron micrograph of apoptotic granulosa cells showing the pyknotic chromatin material adhering to the periphery of nuclear membrane (arrow) and vacuoles (v) of different shapes and sizes were observed within condensed chromatin material.

In addition to the detection of oligonucleosomes in extracted DNA, the occurrence of apoptosis may also be inferred from the characteristic morphological appearance of degenerating cells, together with the detection of fragment DNA in single cells *in situ* using TUNEL (Gavrieli *et al.,* 1992; Palumbo and Yeh, 1994; Bristol and Gould *et al*., 2006; Sharma and Bhardwaj, 2009; Bhardwaj and Sharma,2011). Using *in situ* 3' end labeling (TUNEL), which can detect apoptosis precisely at the single cell level without disruption of the tissue

Apoptosis and Ovarian Follicular Atresia in Mammals 195

histochemical mapping of alkaline phsophatase activity opining the possible role of alkaline phsophatase in active transport of nutrients and secretary material across the membrane (Verma and Guraya, 1968; Sangha and Guraya, 1988/89; Sharma, 2000). The association of alkaline phosphatase positive sites with theca interna indicates the involvement of this enzyme in steroid metabolism and transport (Britenecker *et al.,* 1978; Gilchrist *et al*., 2004). The decline in levels of alkaline phosphatase in follicular fluid and granulosa cells of atretic follicles may be tangibly due to increased vascularity and changed morphology and biochemistry of granulosa cells for steroid hormone synthesis (Guiseppe, 1983; Gilchrist *et al*., 2004; Pangas, 2007; Tatone *et al*., 2008). Acid phosphatase activity in follicular fluid and granulosa cells exhibited an increasing trend from healthy to slightly atretic to atretic follicles. The increase in Golgi complex and lysosomes in atretic follicles/cells is possibly attributable to the rise in acid phosphatase activity. The ultrastructural modifications associated with the lysosomal accumulation during atresia which is further increased due to luteinization wherein chief protein synthesizing cells transmutate to steroidogenic cells may be attributable to the increase in acid phosphatase activity in the granulosa cells (Dorrington *et al*. 1975; Armstrong and Dorrington, 1976; Sangha and Guraya, 1988/89). The degenerative/transformative changes involved in reshaping of ovarian subcellular components that facilitate differentiation during follicular development while bringing about lysis and formation of apoptotic vesicles may be responsible for the rise in lysosomal activity (Sangha and Guraya, 1988/89; Sharma, 2000). The increase in acid phosphatase enzyme activity observed in the follicular fluid and granulosa cells of atretic follicles may also be related to some mechanism for the secretion of steroids (Sawyer *et al.,* 1979; Dimino and Elfont, 1980; Pangas, 2007). It has been reported that acid phosphatase activity is higher in active and regressing corpora lutea, provides a lurking possibility that during follicular atresia the rise in acid phosphatase activity may not be associated exclusively with regression but also with the formation of interstitial gland tissue. Catalase is generally associated with superoxide dismutase, constituting a reciprocally protective set, while catalase is inhibited by oxyradicals, and SOD is inhibited by H2O2 (Lapluye, 1990). If H2O2 produced by SOD, action on oxyradicals is not removed immediately it will react with super oxide radicals (Haber-Weiss reaction) giving rise to highly reactive hydroxyl radicals (Michiels *et al.,* 1994). However, with the increase in H2O2 concentration, the catalase contribution for its degradation concomitantly increased (Verkek and Jond Kind, 1992). Michiels *et al.,* (1991) reported a 30 percent increase in survival when catalase was injected in combination with SOD, whereas the survival was only 21 percent and 4 percent for SOD and catalase respectively, when independently injected in human fibroblasts. Singh and Pandey (1994) reported an increased catalase activity in the ovary of metaoestrous rats *Pari pasu* with a decline in H2O2 production in the mitochondria and microsomal fraction. In addition to its effects on oxygen free radical metabolism, SOD has been shown to influence cell functions by increasing the levels of the second messenger cGMP (Ignarro *et al*., 1987; Schmidt *et al*., 1993; Burke *et al*., 2005). There are three known forms of SOD with specific subcellular and extracellular distributions (Ravindranath and Fridovich, 1975; Crouch *et al*., 1984; Redmond *et al*., 1984; Tibell *et al*., 1987). The manganese-associated form of SOD is localized in mitochondria of cells, whereas the copper/zinc associated form is found in the cytoplasm. Furthermore, there is an extracellular form of SOD that is secreted from cells. All three forms of SOD are expressed in the ovary (Laloraya *et al*., 1988; Shiotani *et al*., 1991; Sato *et al*., 1992; Hesla *et al*., 1992, Tilly and Tilly, 1995) and the pattern of expression appears to

morphology (Gavrieli *et al.,* 1992; Palumbo and Yeh, 1994; Liu *et al*., 2007; Sharma and Bhardwaj, 2009), the specific morphological features of granulosa cell death in follicular atresia (nuclear pyknosis, karyorrhexis, and formation of apoptotic bodies) can be related to the physiological process of apoptosis. The relationship is supported by a combination of biochemical, classic histological evidences, and *in situ* histochemical localization of DNA fragmentation. Different cellular details were observed in atretic and healthy follicles classified by morphological criteria, including cells with a single shrunken and dense nucleus (pyknotic appearance) and cells with marginated chromatin and/or nuclear fragmentation. According to Lussier *et al.,* (1987), non atretic follicles should have intact and normal granulosa layers with the mean pyknotic index per class varying from 0.13 percent to 0.67 percent. However, in another study in cows (Ireland and Roche,1983), pyknotic cells were observed in the granulosa cell layer in 30-60 percent of estrogen-active large follicles.

Fig. 4. Electron micrograph of granulosa cells revealing vacuolated cytoplasm (star) and mitochondria (arrow).

Thus, the mere presence of pyknotic cells in the granulosa cell layer does not imply that they are atretic. However, the morphological and biochemical results strongly indicate that apoptosis may occur to a certain level during normal follicle growth and development and that apoptotic death of granulosa cells may be detectable before other morphological and biochemical signs of degeneration in goats. Alkaline phosphatase activity in follicular fluid and granulosa cells exhibited a declining trend from healthy to slightly atretic and atretic follicles. The biochemical estimation of alkaline phosphatase endorse the earlier

morphology (Gavrieli *et al.,* 1992; Palumbo and Yeh, 1994; Liu *et al*., 2007; Sharma and Bhardwaj, 2009), the specific morphological features of granulosa cell death in follicular atresia (nuclear pyknosis, karyorrhexis, and formation of apoptotic bodies) can be related to the physiological process of apoptosis. The relationship is supported by a combination of biochemical, classic histological evidences, and *in situ* histochemical localization of DNA fragmentation. Different cellular details were observed in atretic and healthy follicles classified by morphological criteria, including cells with a single shrunken and dense nucleus (pyknotic appearance) and cells with marginated chromatin and/or nuclear fragmentation. According to Lussier *et al.,* (1987), non atretic follicles should have intact and normal granulosa layers with the mean pyknotic index per class varying from 0.13 percent to 0.67 percent. However, in another study in cows (Ireland and Roche,1983), pyknotic cells were observed in the granulosa cell layer in 30-60 percent of estrogen-active large follicles.

Fig. 4. Electron micrograph of granulosa cells revealing vacuolated cytoplasm (star) and

Thus, the mere presence of pyknotic cells in the granulosa cell layer does not imply that they are atretic. However, the morphological and biochemical results strongly indicate that apoptosis may occur to a certain level during normal follicle growth and development and that apoptotic death of granulosa cells may be detectable before other morphological and biochemical signs of degeneration in goats. Alkaline phosphatase activity in follicular fluid and granulosa cells exhibited a declining trend from healthy to slightly atretic and atretic follicles. The biochemical estimation of alkaline phosphatase endorse the earlier

mitochondria (arrow).

histochemical mapping of alkaline phsophatase activity opining the possible role of alkaline phsophatase in active transport of nutrients and secretary material across the membrane (Verma and Guraya, 1968; Sangha and Guraya, 1988/89; Sharma, 2000). The association of alkaline phosphatase positive sites with theca interna indicates the involvement of this enzyme in steroid metabolism and transport (Britenecker *et al.,* 1978; Gilchrist *et al*., 2004). The decline in levels of alkaline phosphatase in follicular fluid and granulosa cells of atretic follicles may be tangibly due to increased vascularity and changed morphology and biochemistry of granulosa cells for steroid hormone synthesis (Guiseppe, 1983; Gilchrist *et al*., 2004; Pangas, 2007; Tatone *et al*., 2008). Acid phosphatase activity in follicular fluid and granulosa cells exhibited an increasing trend from healthy to slightly atretic to atretic follicles. The increase in Golgi complex and lysosomes in atretic follicles/cells is possibly attributable to the rise in acid phosphatase activity. The ultrastructural modifications associated with the lysosomal accumulation during atresia which is further increased due to luteinization wherein chief protein synthesizing cells transmutate to steroidogenic cells may be attributable to the increase in acid phosphatase activity in the granulosa cells (Dorrington *et al*. 1975; Armstrong and Dorrington, 1976; Sangha and Guraya, 1988/89). The degenerative/transformative changes involved in reshaping of ovarian subcellular components that facilitate differentiation during follicular development while bringing about lysis and formation of apoptotic vesicles may be responsible for the rise in lysosomal activity (Sangha and Guraya, 1988/89; Sharma, 2000). The increase in acid phosphatase enzyme activity observed in the follicular fluid and granulosa cells of atretic follicles may also be related to some mechanism for the secretion of steroids (Sawyer *et al.,* 1979; Dimino and Elfont, 1980; Pangas, 2007). It has been reported that acid phosphatase activity is higher in active and regressing corpora lutea, provides a lurking possibility that during follicular atresia the rise in acid phosphatase activity may not be associated exclusively with regression but also with the formation of interstitial gland tissue. Catalase is generally associated with superoxide dismutase, constituting a reciprocally protective set, while catalase is inhibited by oxyradicals, and SOD is inhibited by H2O2 (Lapluye, 1990). If H2O2 produced by SOD, action on oxyradicals is not removed immediately it will react with super oxide radicals (Haber-Weiss reaction) giving rise to highly reactive hydroxyl radicals (Michiels *et al.,* 1994). However, with the increase in H2O2 concentration, the catalase contribution for its degradation concomitantly increased (Verkek and Jond Kind, 1992). Michiels *et al.,* (1991) reported a 30 percent increase in survival when catalase was injected in combination with SOD, whereas the survival was only 21 percent and 4 percent for SOD and catalase respectively, when independently injected in human fibroblasts. Singh and Pandey (1994) reported an increased catalase activity in the ovary of metaoestrous rats *Pari pasu* with a decline in H2O2 production in the mitochondria and microsomal fraction. In addition to its effects on oxygen free radical metabolism, SOD has been shown to influence cell functions by increasing the levels of the second messenger cGMP (Ignarro *et al*., 1987; Schmidt *et al*., 1993; Burke *et al*., 2005). There are three known forms of SOD with specific subcellular and extracellular distributions (Ravindranath and Fridovich, 1975; Crouch *et al*., 1984; Redmond *et al*., 1984; Tibell *et al*., 1987). The manganese-associated form of SOD is localized in mitochondria of cells, whereas the copper/zinc associated form is found in the cytoplasm. Furthermore, there is an extracellular form of SOD that is secreted from cells. All three forms of SOD are expressed in the ovary (Laloraya *et al*., 1988; Shiotani *et al*., 1991; Sato *et al*., 1992; Hesla *et al*., 1992, Tilly and Tilly, 1995) and the pattern of expression appears to

Apoptosis and Ovarian Follicular Atresia in Mammals 197

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evidence supporting its sufficiency for adult fertility. *Dev. Biol.* 298, 149-154. Burke, C.R., Cardenas, H., Mussard, M.L., Day, M.L. (2005). Histological and steroidogenic

Bussiere, F.I., Zimowska, W., Gueux, E., Rayssiguier, Y., Mazur, A. (2002). Stress protein

Byskov, A.G. (1978). *The Vertebrate Ovary*: Follicular Atresia. In: Jones, R.E. (ed.). Plenum

Chaube, S.K., Prasad, P.V., Thakur, S.C., and Srivastava, T.G. (2005). Hydrogen peroxide

Chaudhary, A., Sharma, R.K., Saini, K. (2004). Antioxidant effect of Mandukparni on

Chen, W., Wang, Z., Zhang, Y. (2003). The effect of zinc on the apoptosis of cultured human

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apoptosis in rat oocytes cultured *in vitro. Apoptosis.* 10, 863-875.

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be related to gonadotropin induced follicular development and luteal steroidogenesis and regression (Laloraya *et al*., 1988; Hesla *et al*., 1992; Sato *et al*., 1992; Tilly and Tilly, 1995; Pangas, 2007). Furthermore, SOD effectively blocks gonadotropin-induced ovulation (Miyazaki *et al*., 1991). The activity of enzyme glutathione peroxidase in follicular fluid and granulosa cells shows a declining trend from healthy to slightly atretic to atretic follicles. It has been reported that glutathione peroxidase (GPx) catalyzes the breakdown of H2O2 with much more affinity than the catalase (Gul *et al.,* 2000). The major protection against both lipid peroxide and H2O2 is reported to be achieved by GPx (Halliwel and Gutteridge, 1985). Land and Verdetti (1989) reported decreased level of GPx with age in kidney and liver in rats, whereas Imre *et al*., (1984) and Hazelton and Lang (1985) reported a decrease in GPx activity with age in liver and kidney and many other tissues of mice. It has been reported that GPx activity remained constant in the caudate putamen and the temporal cortex but decreased in the substantial nigra and the thalamus in rats (Benzi *et al*., 1989).


**4. Conclusions**

The use of microwave plasma treatments has been reviewed both for sintering nanoparticles (referred to as rapid discharge sintering (RDS), as a thermal processing route for crystalline phase changes, as well as for the oxidation of metal surfaces. For the fabrication of porous ceramics, the plasma treatments have the advantage of being rapid and flexible allowing for the tailoring and control of pore structures with processing conditions. Compared to the conventional methods such as air furnace treatment, the plasma treatments were demonstrated to yield improved results in terms of shorter treatment time, lower energy requirement and enhanced performance. The latter was demonstrated in the case of metal oxide layers (NiO and TiO2 ), when evaluated for use in solar energy applications.


**3. Biostimulators to enhance cucumber growth**

#### **3.1 Lactate**

According to previous investigations, lactates (salts of lactic acid) seem to produce bioregulatory effects. The application of lactates was tested as an approach to improve the nutrient balance and plant vitality. Investigations have shown that lactates have more stable bonds with several metal ions than other chelates.

*Growth Promoting and Stabilizing of Cucumber Plants Cultivated in Soilless Cultivation… DOI: http://dx.doi.org/10.5772/intechopen.96536*

Therefore, substances have been used as fertilizers and as bioregulators. Lactates are available under the brand name LACTOFOL® [5, 6]. That suspension fertilizer was designed mainly for foliar application and patented as a plant growth and development regulator. The composition of LACTOFOL O® is shown in **Table 2**.


9. Recommendation

1.Participants have the correct mathematics knowledge and conceptual understanding and transfer it to the physics domain, for example, when

2.Participants reveal the mathematics knowledge but lack the necessary physics knowledge and conceptual understanding, for example, in the kinematics tasks on area and slope, they seem not to know which kinematics relation to use and

3.Participants are unable to blend mathematics and physics knowledge, for example, they do not perceive the kinematic equations as quadratic and represented by a parabola or as a hyperbolic expression and graph.

4.Participants transferred their misconceptions or insufficient knowledge in mathematics to the physics domain. This is evident in the height-slope confusion, area-slope confusion and parabola-hyperbola confusions that occurred in both the mathematics and physics domains. Inaccurate knowledge of the slope as the ratio of change in variables was to a large extent transferred

The results indicated that the majority of participants have an understanding of the physics discipline characteristics with regard to the use of kinematics concepts as variables that can be presented as coordinates on Cartesian planes. In the physics tasks on reading coordinates, they attached symbolic meanings (position and time) to the x and y coordinates. They also recognised correspondences between a linear motion equation and the standard mathematical format for straight lines in a representation task. However, they seem not to have the insight that kinematics relationships can be represented as functions, especially with regard to quadratic (parabolic) and hyperbolic functions. In addition, students failed to attach physical meaning to the area under graphs and slopes of graphs in the kinematics contexts. In order for the participants to solve the physics questions correctly, they did not only have to know the discipline characteristics concerning kinematics graphs but also the discipline characteristics of graphs in mathematics. There are practices that are similar for mathematics and physics, for example, using the Cartesian coordinate system and placing the dependent variable on the vertical axis. Also, concepts such as slope and area are calculated the same in both contexts. Discipline characteristics that differ are, for example, that in mathematics, variables are abstract and have no units, whilst in physics variables, area under graphs and gradients all have physical meanings and units. Another difference is that in mathematics, the horizontal axis has a positive and negative side, whereas in kinematics, the concept time as the independent variable is on the horizontal axis and starts from zero only. The latter difference probably contributed to the significant differences in students' responses on the hyperbolic and parabolic representations. The kinematics graphs only showed the parts of the hyperbola or parabola for which the x-coordinate (time) is positive, which might have prevented students from recognising the

From the results of this study, it is clear that if students know the underlying mathematics, it does not imply that they can use it in another context. There is no automatic transfer from the mathematics domain to the physics domain when using mathematics to solve a physics problem. For a student to be able to solve a certain physics problem, he/she has to know and understand the underlying mathematics

reading coordinates.

Theorizing STEM Education in the 21st Century

what to calculate.

from mathematics to kinematics.

7.3 Discipline characteristics

graph form.

178

In the physics classroom, the students have to be taught how to use their existing mathematics to solve the problems at hand. The instructor has to revise the relevant existing mathematics as well as physics knowledge and draw analogies between aspects such as geometric figures, expressions and graphic representations of functions, etc. Differences in discipline characteristics need to be discussed with the students so that they understand the purpose and applications of graphs in the two contexts.

Further research can be conducted for follow-up years after specific interventions have been done to specifically address the problems identified. This questionnaire can also be used by other lecturers for research purposes or to test their students' abilities and identify areas of concern and come up with intervention strategies thereof.

It is thus recommended that lecturers of undergraduate introductory physics should emphasise the knowledge and skills of algebraic graphs in teaching and learning of kinematics, especially kinematics graphs. This will enable these students to collect data, analyse it, plot graphs and interpret graphs based on this knowledge and relate it to and show physics understanding and knowledge.


**3. Results**

#### *3.1. E*ff*ect of Di*ff*erent Habitat Types and Season on Honey Bees*

Based on the analysis, we found that habitat type and season a ffected the forager abundance and colony weight of bees (*p* < 0.05), although the patterns were di fferent between species and region. In general, habitat types showed to be the most important factor that a ffected both forager abundance and colony weight of bees compared to season and observation time (Table 2). E ffect of habitat type on forager abundance and colony weight of *A. cerana* di ffered between Malang and Bogor. In Malang, the di fference of habitat type significantly influenced the forager abundance and colony weight of *A. cerana,* which were found higher in home gardens and forests compared to the agricultural areas (Figures 2a and 3a). In Bogor, forager abundance of *A. cerana* was not significantly di fferent among habitat type (Figure 2b), while the colony weight of *A. cerana* in forests was significantly higher than in home gardens (Figure 3b). The same pattern was observed with *A. cerana* in Malang for forager abundance of *A. mellifera* in Malang and *T. laeviceps* in Bogor, which were also found higher in home gardens and forests compared to agricultural areas (Figure 2c,d and Figure 3c,d).

**Table 2.** The results of Kruskal–Wallis tests of forager abundance and colony weight of bees in different seasons, habitat types, and observation times.

**Figure 2.** Forager abundance of honey bees and stingless bees in different habitats. (**a**) *A. cerana* in Malang, (**b**) *A. cerana* in Bogor, (**c**) *A. mellifera* in Malang, and (**d**) *T. laeviceps* in Bogor. Boxes with different letters are significantly different at *p* < 0.05 according to Fisher's least-significant difference.

**Figure 3.** Colony weight of honey bees and stingless bees in different habitats. (**a**) *A. cerana* in Malang, (**b**) *A. cerana* in Bogor, (**c**) *A. mellifera* in Malang, and (**d**) *T. laeviceps* in Bogor. Box with different letters are significantly different at *p* < 0.05 according to Fisher's least significant difference.

Based on non-parametric regression, we found that the forager abundance of *A. cerana* in Malang was prone to decrease with increasing observation time (*p* = 0.001) (Table 3). In addition, the colony weights of *A. cerana* and *T. laeviceps* in Bogor as well as *A. mellifera* in Malang tended to increase with increasing observation time (*p* < 0.05).

**Table 3.** Relationship between forager abundance and colony weight of bees and observation time (day) based on non-parametric regression.

#### *3.2. Detection Results of Insecticide Residue in Honey and Body of Honey Bees*

Based on residue analysis, we barely detected insecticide residue (imidacloprid content) both in the honey and the body of bees (Table 4). Two of 18 honey samples (11.11%) contained a small amount of insecticide residue (<5 μg/kg). Insecticide residues were also detected in three bee samples (16.66%). Surprisingly, we did not detect the insecticide residue in the agriculture habitat, yet it was detected in forest and home garden habitats.


**Preface to "Fractional-Order System: Control Theory and Applications"**

Thach Ngoc Dinh would like to dedicate this book to the 70th birthday of his father Dinh Ngoc Thuan and to all his beloved family members for their constant support. Shyam Kamal would like to dedicate this book to his daughter Adhita Kamal and his wife Aprajita Kamal for their constant support. Rajesh Kumar Pandey would like to dedicate this book to his parents and his beloved family members for their constant support.

> **Thach Ngoc Dinh, Shyam Kamal, and Rajesh Kumar Pandey** *Editors*


Thin Films - Growth, Characterization and Electrochemical Applications

*Edited by Fatma Sarf, Emin Yakar and Irmak Karaduman Er*

Published in London, United Kingdom

Thin Films - Growth, Characterization and Electrochemical Applications http://dx.doi.org/10.5772/intechopen.107556 Edited by Fatma Sarf, Emin Yakar and Irmak Karaduman Er

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Arturo Fernández Madrigal, Arelis Ledesma Juárez, Alejandro Altamirano Gutiérrez, Snehal Mandal, Dongmei Dong, Suman Sardar, Emmanuel Ifeanyi Ugwu, Ehsan Rahmani, Daniel Schwarz, Michael Oehme, Erich Kasper, Junjie Yang, Huiwen Deng, Jae-Seong Park, Siming Chen, Mingchu Tang, Huiyun Liu

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**3. Results**

#### *3.1. Species Geographical Distribution*

All locations where the species was observed lie along south-facing, very rocky slopes, at the cliff foot of the mountains Symvolo and Rhodope, at low altitudes (100–150 m asl), approximately 3–15 km from the Aegean Sea, just over the plain (agricultural) area lying between the sea and the mountains. The area of the species appearance is restricted to this altitudinal zone, suggesting that these specific ecological conditions favor its thriving. However, the core population of the species was found more westward with respect to the location indicated in the previous records, at 40◦5816.59 N, 24◦2754.93 E, near the village Chalkero, close to the city of Kavala, approximately 150 km from Thessaloniki (Figure 1), in areas exposed to several anthropogenic actions, such as grazing production, livestock raising, agricultural crops cultivation, presence of vehicles, and generally, in degraded habitats.

**Figure 1.** Map showing the location of the species *Verbascum dingleri* occurrence. Geographical coordinates: 40◦5816.59 N, 24◦2754.93 E.

The climate of this area belongs to the type Csa according to the Koeppen classification system. On the basis of the available data of the nearest meteorological station of Kavala city, the mean annual temperature is 14.6 ◦C, and the mean annual rainfall is 602 mm. The mean monthly temperature during the coldest month (January) is 4.2 ◦C, and that during the warmest month (August) is 26.0 ◦C, while the prevailing wind is from the southeast (SE) [26].

From a geological point of view, the study area is part of the Rhodope massif that consists of metamorphic and plutonic-eruptive rocks. The specific rock types where the species appears are marbles and limestone. The soils are very shallow (depth range 5–19 cm), with an extremely high rock presence (80–85% of the total area) (Figure 2a, Table 1). The soil is alkaline, and the pH value was found to be almost constant (7.8) in all four sampling points, with very slight differences. However, even though the soil is very shallow, it was found to be relatively rich in organic matter (7.18%), with adequate total nitrogen content (0.42%), as well as phosphorous and potassium contents (8.55 ppm

and 336.5 ppm, respectively). Other similar habitats near the species area were investigated during the species flowering period, but we did not locate any additional populations.

**Figure 2.** Photo of the species *V. dingleri:* (**a**) in situ photo near Chalkero village, showing the dominant site characteristics; (**b**) fruits of *V. dingleri*; (**c**) seeds of *V. dingleri* (photos from a stereomicroscope).

**Table 1.** Soil characteristics of the habitat of *V. dingleri*.

The site topography provides a habitat that su ffers from hot and dry weather conditions during the dry summer season. As summer approaches, the sunrays fall almost vertically, causing the soil to dry and exposing the vegetation to high sunlight. This, in turn, results in high summer evaporation rates, causing the drying of *V. dingleri* and other annual plants and eventually their disappearance during the late summer season.

The spatial data analysis showed that the species niche is determined by the afore-mentioned specific site characteristics that favor the species survival and thriving. The species distribution is probably constrained by hard dispersal obstacles or physiological thresholds along environmental gradients rather than by interactions with other species. Thus, the species appears only in dry, rocky, south-faced slopes, with medium inclination, on shallow soils and limestone, under the climate type Csa, and at a short distance from the sea, in the location at 40◦5816.59 N, 24◦2754.93 E.