Ovary is highly dynamic organ with constantly changing environment.The ovary is an organ that is constantly subjected to change. Periodic and regular formation and development of fFollicles contribute to production of a large number of somatic and gametic cellsstart to grow at all times and as they develop they produce large numbers of cells throughout lifespan. One of the ovarian dynamisms is degeneration and clearance ofHowever, most of the growing follicles degenerate at some stage of their development and have to be cleared from the ovary. Accordingly, There are considerable numbers quantities of cells that are produced arend disposed of within short time intervals.
Considering the small size of the ovary and with limited accommodation it is imminent that tThe emergence and removal of follicles must be well balanced, as the ovary could not maintain its limited size. RegularThe constant production and eliminationremoval of follicles lead to causes pre-determined movement of cells and cell complexes within the ovary, such as: small follicles grow into large ones, displacing already existing large follicles, andothers; corpora lutea form and moves soon displaced from the ovarian wall periphery towards the cortex,entre from where they disintegrateappear. There exists difference among species so far as follicular cycle is concerned speed with which follicles develop varies considerably in different species but a dynamic flow is always maintained.
The Oorderlyed event of follicular growth and differentiation depends on functional interrelationships between the hypothalamus, the pituitary and the ovary, particularly at the level of pituitary hormones, whose. The ovarian steroids are the messengers between the ovary and the hypothalamic-pituitary unit. The requirements varies for pituitary hormones change as the as follicle develops. During the follicular phase of a sexually activemature female, a functionalthe interactions between the hypothalamus, pituitary and the developing follicle is may be sustained until ovulation is over, because there is no interference in the feedback relationships between them.
By contrast, during luteal phase or pregnancy, when there isare high levels of progesterone, the positive influence ofsensitivity of the hypothalamus and pituitary overto the requirements of the developing follicle is collapsed, andimpaired. Under these conditions follicular growth is unlikely to be sustained through its ultimatefinal developmental stages.
Follicles atin different stages of development have been described during pregnancy in many species; humanwomen (Govan 1968),; hamsters (Greenwald et al. 1967) and; mice (Greenwald and Choudary 1969).
In our study, wWe have investigated the follicle dynamics in pregnant ewe, and our results were consistent with a general notion,with previous studies. Apparently, pregnancy didoes not affect follicle formationgrowth initiation, or initial development, thoughbut it suppressed the later stages of down regulated follicular development and differentiation. Not just the development was affected, theThis study has shown that the proliferative activity of granulosa cells of growing follicles also decreased during pregnancy decreased compared relative to the non-pregnant animals. An appreciable difference in the This may be explained by the alteration of circulating gonadotropins and steroidal hormones may attribute to follicle suppression and which occur during pregnancy leading to an alteration in steroidogenesis in granulosa cells.
Pituitary gonadotropins, FSH and LH levels are low during pregnancy (Taya and Greenwald 1981b; Taya and Greenwald 1981a). FSH has been reported to acceleratetivate granulosa cell proliferation and differentiation in cow (Hulshof et al. 1994),; sheep (Newton et al. 1999) and human (Roy and Treacy 1993).
In agreement with our results, in golden hamster (Greenwald et al. 1967) and cow (Guilbault et al. 1986) duringAt the late stage of pregnancy, a pool of transitory follicles washas been observed, whereas the and large healthy follicles were missing as they were degeneratedabsent during this stage of pregnancy. In agreement with our results, it was reported that all large follicles were degenerate during late stage of pregnancy in golden hamster (Greenwald et al. 1967) and in cow (Guilbault et al. 1986).. High levels of progesterone mightcould be involved in attenuation the inhibition of follicular development that occurs during pregnancy. Progesterone inhibits gonadotropin induced follicular growth in hypophysectomised rats (Fukuda et al. 1980), and also as well as inhibitsing the the induction of aromatase activity in rat granulosa cells responsible for steroid production (Fortune and Vincent 1983).
Loss of follicular estrogenic activity could be a reason responsible forof the down-regulation suppression of follicular development. Besides that, it has been suggested that the conceptus exerts follicle suppressive effects in pregnant ewes (Al Gubory and Abdennebi 1996), , especially the and it could be that the factors inhibiting antral follicle’s developmentular growth during pregnancy.. Moreover, Another study revealed that their may be residual local inhibitory effects of the CL of pregnancy on on folliculogenesis in pregnant cows may reduce the number of antral follicles (Bellin et al. 1984).
Apart from the anti-Not only the proliferative actionvity of pregnancy on granulosa cells of growing follicles was affected by pregnancy, another influence happens to be on compartment of the ovary showed a consistent response under the influence of pregnancy, the ovarian surface epithelium (OSE).. As previously described, The OSE is the layer that covers the ovary, and; it is separated from the ovarian stroma by a basement membrane and the tunica albugenia (TA).
Johnson and his colleagues, in their paper in Nature (Johnson et al. (2004) provided evidence for the existence of proliferative large ovoid germ cells in the surface epithelial cell layer of embryonic, juvenile and young adult mice that give rise to oocytes and follicle production in the the postnatal period of development of mice. They (Johnson et al. 2004) provide results from a systematically executed set of experiments that strongly indicate that ovaries of juvenile and young adult mice contain large ovoid cells, resembling germ cells of fetal mouse ovaries, in the surface epithelial cell layer. Another study by Bukovsky et al (Bukovsky et al. 2004) revealed prevalence ofported a dynamic population of differentiating and regressing ovarian follicles in adult women. This study was based on athe hypothesis that mesenchymal cells in the TAtunica albuginea are bipotent progenitors for both granulosa cells and germ cells.
It wasThey demonstrated that cytokeratin positive mesenchymal cells of TAtunica albuginea differentiate into surface epithelium, by a mesenchymal- to- epithelial cell transition, and that. They reported that germ cells derived from surface epithelial cells that overlayingcover the TA tunica albuginea, wereenter and are transported by a vascular network to beand incorporated into the epithelial nests associated withwith the blood vessels (Bukovsky et al. 2004). These studies contradicted the validity of an established doctrine that Results from previouse study showed no evidence that stem cells of bone marrow cells pass on to the ovary through peripheral vascular system and contribute to the formation of mature, and ovulated oocytes.
But (Eggan et al. (2006). They concluded that, the cells that travellinged to the ovary through bloodstreamthe bloodstream exhibited properties characteristic of committed blood leukocytes rather than and the inability of these cells to form ovulated oocytes contributing to the demonstrates that any such cells that might exist do not contribute to oocyte pools destined for available for fertilization (Eggan et al. 2006).
All theseBoth studies, Johnson et al. (Johnson et al. 2004) and Bukovsky et al. (Bukovsky et al. 2004) studies providedrepresent evidence that OSE layer may have a role in follicular renewal from germline stem cells in the postnatal mammalian ovary. However, these findings remain controversial due to a residual concerns over the physiological relevance of putative marrow-derived or circulating germ cell precursors and their functional capacity to enter into the pool of mature, ovulated oocytes (Telfer et al. 2005).
Away from involving the OSE in the mentioned challenging findings, eEpithelial ovarian cancer (EOC) is the primary ovarian malignancy affecting women. Although tumors originate from almost every cell type present in the ovary, approximately, 90% of the cancers originating in this organ are referred to as common epithelial tumors. As early as 1872, Sir Spencer Wells suggested that these tumors originated from surface epitheliumthe OSE. A lot of eEfforts have been made over the years to understand the biology of the surface epithelium with ain the hope that thissuch information mayight explain the overwhelming tendency of these relatively inconspicuous rare ovarian cells to undergo malignant transformation.
The etiology of epithelial ovarian cancer is poorly understood, partly one of the most important reasons for this is due to the lack of suitable animal models. Most established risk factors for the disease relate to reproductive events. Malignant transformation of normal ovarian epithelial cell is caused by genetic alterations that disrupt regulation of proliferation, programmed cell death and senescence. The rate of proliferation is a major determinant of the number of cells in population and.
To prevent excessive proliferation, DNA synthesis and cell division are normallyordinarily restrained. There is a general agreement that on the protective effects of parity and oral contraceptive use (Whittemore et al. 1992; 1987; Negri et al. 1991); the risk of ovarian cancer decreases with increasing parity and longer duration of oral contraceptive use (Whittemore et al. 1992; 1987; Negri et al. 1991). Further,It has infertile been reported that women not going for alternate methods of pregnancy who have never been pregnant because of infertility develop epithelial ovarian cancer at 40% higher rate than normal women who chose to remain non-have never attempted to become pregnant (Rodriguez et al. 1998).
Using immunohistochemistry method andfor different cell proliferation markers (PCNA, Ki67 and BrdU), we have demonstrated that results of this thesis indicated that pregnancy alleviated inhibited the proliferative activity of OSE cells during early and the early and late stages of pregnancy (sheep and marmoset monkeys). OSE cells of non- pregnant animals showed a variation in the labelling indexes depending on the underlying ovarian compartments. We proposeattributed that the suppression affect of OSE cells’ proliferative activity due to pregnancy is a consequence of on OSE cells proliferative activity to the prolong exposure of these cells to high level of progesterone, specifically if it is accompanied with low level of gonadotropin. Over the first month of pregnancy, maternal LH and FSH decline strongly with the increase in trophoblast hCG (Jaffe 1991).
hCG also stimulates the CL to continue producing progesterone and not to regress (Yen 1994). An direct inhibitory effect of progesterone on OSE cells’ proliferation washas been demonstrated in cell cultures of OSE from bovine ovaries (Chapter 6). Consistent with our finding, it was indicated that progesterone treatment inhibitsed the proliferation of OSE cells obtained from pre- and postmenopausal women pre and post menopausal (Ivarsson et al. 2001). Supplementing Administration of progesterone to sheep OSE cultures resulted in a decrease in both basal and estradiol-17β-stimulated proliferation rates (Murdoch 2002).
A popular In literature, the most popular explanation for the onco-protective responseeffect of pregnancy is that it leads to a temporary cessation of ovulation (Fathalla 1971). This would reduce constant ovarian epithelial disruption and repair that may induce ovarian neoplasia, possibly through the accumulation of mutations in tumor suppressor genes such as pP53 (Schildkraut et al. 1997).
The results with heifers described in chapter 6 showeddemonstrate that progesterone inducesd apoptosis in cultured bovine OSE. Moreover, and the in vivo observations revealed showed that pregnancy led to high induced apoptotic indexsis in OSE cells and inin cells lining ofthe inclusion cysts. In contrast, comparing to the non-pregnant animals which did not produceshow any evidence supportingof apoptosis in both OSE and inclusion cysts. Apparently, This finding support the hypothesis that elevated progesterone during pregnancy plays a role in regulating programmed cell death inof the OSE. Consistent with our finding, Rodriguez et al. and colleagues (1998) demonstratedindicated that, in vivo administration of progesterone to monkey’s OSE cells induced apoptosis (Rodriguez et al. 1998).
The protective role of progesterone during pregnancy could be the result of a clearing process or exfoliation of the ovary (particularly the OSE layer and inclusion cysts) from any DNA damage that could be in the cells of these structures and might lead to malignancy through apoptosis. Apoptosis may be a key factor to avert suchserve an important role in preventing malignant transformation, by way of specifically elimination ofng the cells that have undergone mutations.
Progesterone may also up-regulate the tumor suppressor gene Pp53, which is a critical regulator of cell cycle arrest and apoptosis in response to DNA damage. Mutations of the Pp53 tumor suppressor gene is the most frequent genetic lesionevent described in human cancers (Berchuck et al. 1994), and it is known to play a pivotalcentral role in regulation of both proliferation and apoptosis (Braithwaite et al. 1987; Rotter et al. 1983). In normal cells, P53 protein protein exerts its tumor suppressor activity by binding to transcriptional regulatory elements of the genes that act to arrest cells atin G1 phase. Additionally, P53 is thought to play a role in preventing cancer by stimulating apoptosis of cells that have undergone excessive genetic damage (Kuerbitz et al. 1992). In this regard, P53P53 has been described as the “guardian of the genome” since it delays entry of cells into S phase until the genome has been cleansed of mutations. If DNA repair is inadequate, P53 may initiate apoptosis, thereby eliminating the cells alteredwith genetic damaged genotype.
In this study, it was shown that P53 was expressed more within OSE cells over the CL, with less expression over the large antral follicles which might be preovulatory follicle (Cchapter 7). In contrast, P53 protein was completely absent within the OSE cells in all the areas away from ovulation events. Moreover, tThe in vitro results demonstratereported in Chapter 7 shows that progesterone administration significantly increasedup-regulates P53 expression in bovine OSE cells.
Expression of P53 within OSE cells over the CL may contribute to the re-epithelizsation process. P53 might either induces cell cycle arrest cell cycle, enabling to allow the DNA repair before replication, or it might induces apoptosis if DNA damage is not repairableirreparable. Therefore, therefore it could be up regulated in OSE cells prior to ovulation to induce apoptosis within the OSE cells close to follicle rupture.
besidesAs well as the cessation of ovulation, pregnancy is also associated with elevated progesterone and this may have a protective role by inducing since it also affects P53 expression. A report by Schildkraut (1997) revealed that exposure to a high calculated lifetime number of ovulatory cycles was associated with increased risk of P53 over-expressing ovarian cancer (Schildkraut et al. 1997). – (sentence not clear either it is P53 overexpression or cancer overexpression)
HighAn increased risk of ovarian cancer is invariably associated with elevated gonadotropin secretion particularly at postmenopausal stage leading to increased estrogenic stimulation (Cramer and Welch 1983).
In marmoset monkeys, treated with GnRH antagonist, OSE proliferation was found to be diminishedinhibited (chapter 5). Thisese finding is consistent with the theory that pregnancy protection role is a combination of low gonadotropin and high progesterone for a certain period.
Histological studies indicatesuggest that the growth and function of OSE cells is regulated by paracrine and/or endocrine pathways and many investigations have been begun to clarify which hormones and growth factors might be involved. The OSEovarian surface epithelium is an avascular tissue, thereforesuggesting a predominant influence oflargely paracrine rather than endocrine factors is expectedinfluence of hormonal factors (Clement 1987). Both epidemiological and experimental investigationsobservations have implicated sex steroids in the pathogenesis and growth regulation of ovarian cancer. Most of the ovarian steroidogenesis occurs in the granulose and theca cells of developing and mature follicles under the control of the gonadotropins, FSH and LH in the granulose and theca cells of developing and mature follicles (Clement 1987).
Estrogen biosynthesis peaks sharply in the granulosa cells prior to ovulation (Carr 1993)., Aas follicular growth distends the surface of the ovary, the epithelial cells multiply and get flattened in shape (Gillett et al. 1991) until ovulation,, when the epithelial cell’s proteases dissolve the follicle apex and rupture it (Kruk et al. 1994). It was suggested that the epithelium during this stage is exposed to more paracrine influences of the granulosa and theca cells (Hafez et al. 1980), or this is mediated through diffusion of follicular fluid (Carcangiu and Chambers 1992), as follicular fluid before ovulation this fluid may contain high concentration of estradiol (Clement 1987).
Results from chapter 4 showed that in vitro administration of estrogen and progesterone did not stimulate OSE proliferation, suggesting neither of the steroids affect cultured ovine OSE cells, whereas. Whereas, EGF and IGF-1 significantly inducedd proliferation in cultured ovine OSE. Moreover, observed The stimulatory effect that occurred after of the follicular fluid treatment may be related to another ovarian factors other thanrather than steroids, such as EGF and IGF-1.
Immunohistochemistry data (PCNA, Ki67 and BrdU) indicated that, the proliferative activity of OSE cells is related to the ovulation and post- ovulation repair process. Although immunohistochemistry results in chapters 3 and 5 showed a variation in the percentage of immunureactivity within OSE cells, the over all proliferative indexes were very low compareding to the other ovarian compartments such as granulosa cells. It seems that during ovarian cycles, the epithelium proliferates at times when mitogenic influences are relatively greater, and increased mitotic activity is likely to enhance the risk of mutations occurring. This genetic aberration could then accumulate with additionally epithelial cell divisions in further cycles during re-epithelizsation.
In this study, some results were ironically different from what was expected, and further study is desirous to unravel the facts. Firstly, there was marker-based difference in recognition of the proliferative activity among different cell types, i.e. epithelial cells, granulosa cells and oocytes (chapters 3 and 5). In cycling ewes, a baseline PCNA staining in granulosa cells of all follicular stages was consistently observed. In contrast, Ki67 marker was restricted to only the late follicles. Our explanation is that DNA damage and repair activity takes place even in non-dividing primordial follicles, possibly due to pre-granulosa cell formation and accommodation of oocytes.
Correspondingly, PCNA staining was discernable in oocytes, suggesting that high turnover of DNA in these cells might create an environment for the surrounding cells to undergo DNA modulation. Thus, despite Ki67 negativity we can not entirely rule out that the primordial-transitory follicles are non-proliferative. In marmoset monkeys, low but significant BrdU staining of “resting” OSE over stroma was seen, which indicates that these cells are also dividing albeit slowly. We hypothesize that this discrete proliferation accounts for the formation of the germ cells. Secondly, during early-pregnancy the OSE proliferation was reasonably high in marmoset especially over CL (chapter 5), whereas progesterone concentration must have been high enough to suppress this proliferation.
It appears that pro-proliferative factors originating from CL might supersede the progesterone’s action, and this effect may extend to early pregnancy. The nature of this factor is not known. Thirdly, in heifers during mid-pregnancy number of inclusion cysts increased and epithelial cells of these cysts and OSE exhibited substantial apoptotic index revealing reasonable cell death (chapter 7). Elevated P53 expression in these cells corroborated to the fact that apoptotic activity was indeed high. In normal course, formation of inclusion cyst and epithelial apoptosis is confined to ovulation process. It is reasonable to assume that at mid-pregnancy, due to some unknown factors, there is a tendency of neoplastic transformation of OSE cells and high apoptosis stimulated by progesterone is a mechanism to avert this.
This work embodiedtook in this thesis is a comparative approach using three animal models systems, different timings through the ovarian cycle and different cell proliferation markers to elucidate. tThe possible mechanisms by which the pregnancy affects the OSE are not fully cleared. It wasThis study concluded that the possible protective role of pregnancy onin the progressionetiology of ovarian cancer iscould be at least in part related to the an inhibition of OSEovarian surface epithelium cell proliferation and accelerated cell death regulation in this layer. WWe provide an evidence to support anfor the involvement of progesterone in apoptosis induction and cell growth inhibition in OSE cells.
Additionally, data from pP53 tumor suppressor gene indicated that, this gene whose expression is governed by progesterone and/or some other hitherto unknown factors, might play an important role in the onco-protection mechanisms,. either under the control of progesterone or under unknown factors that induce This feature the expression of P53 and therefore saves the genome from any progressive accumulation ofin mutations. We suggest that any defect in the P53 pathway may lead to malignancy either by mutation in pP53 gene, and/or by down-regulation of a cascade of reactions responsible for cell cycle attenuation and apoptosis. that gene when it is necessary to be up-regulated.
More investigations should be done on the Eexpression of pP53 during pregnancy has to be dealt with care as using advance techniques since this protein hasve a short-half life, and thisat may give provide a false negative labelling and erroneous results during data analysis.
One of the objectives was In our experiments we used different animals as a model systemto select , trying by that to choose a suitable animal model that has a similar ovarian physiology to that of such as the human ovary. We studied tThe proliferation activity of the OSE in ewes (with monoovular cycle), animals; cows (with low ovulation rate) and in marmoset monkeys (high ovulation rate) was investigated. Interestingly, Oovine ovaries provided us with good information onabout what is happenings in the OSE during the anoestrus period, or this period is a resting period from ovulation., Oour results indicaterevealed that the OSE cells were in a quiescence state and this could be an explaination why ovarian cancer does not occur in these animals.
Cows turned out to be good models but there were experimental are a limitations; the animals brought from local slaughterhouse were young and it was good model for study but most of the materials which we have got are from young adult animals, it could be too early to detect any defect or malformation within OSE cells or inclusion cyst.
Moreover, because the animal we are using are from the local slaughter, so we do not know the age or the number of parity for these animals was not known. If older animals were brought it would have been possible to , and maybe if these animals live longer they could be the best model for analyze the etiology of ovarian cancer in the quiescence statestudying the biology of ovarian surface epithelium and the etiology of ovarian cancer.
Overall, the picture from tThe in vitro supplementation of hormones/growth factors on study on OSE cells including treatment administration, induction of apoptosis induction or stimulation of proliferation stimulation did not entirely match with the corresponding analysis on OSE layer in the are different than in vivo models.study, For e.g. progesterone had practically no influence over ewe’s epithelial cultured cell proliferation, but high progesterone level during pregnancy remarkably suppressed the OSE cell proliferation within ovary. Apoptosis induction by progesterone was clearly discernable in heifer OSE cultured cells but was not observed in late pregnancy, at which stage progesterone level should have been high.
It is likely that the absence of the TAtunica albuginea (TA) layer in the in vitro study may alter the physiological response of OSE cells to different treatments. Cultured OSE are grown in a different environment and are exposure directly to the effectors in the the mediuma containing the treatments. , whereas inside the ovary The TA may selectively hinder exposure of plays an important role in protecting the OSE cellslayer to the effectors produced within from the stroma or follicles, or are circulated from the vascular system.l components and its effects.
The exact effect of such effectors And that situation can be better judged notice within the in the epithelium of inclusion cysts which are hen they invaded in the ovarian ovarian stroma, and are more exposed to the compounds.l, We do find new epithelial characteristics in most of these structures gained new characteristics that is different from there OSE layer, such as, they tend to change the shape and undergo malignant growthreal phenotype. So Hence, besides searching for the challenge in finding a suitable animal model for studying OSE physiology, we need to fabricate uniqueprovide an advance OSE culture system that mimics the conditions prevailing with a similar order as it is in vivo.
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