The immunohistochemical analyses of OSE layer using PCNA and Ki67 clearly revealed proliferative activity only in cycling ewes, that too in close proximity to steroidogenic antral follicles and CL, suggesting that ovulation stimulates multiplication of OSE cells. Apparently, this enables the post-ovulatory replenishment of existing cells with new ones. Further, compared to cycling animals, distinct features in follicular development in anoestrous and pregnant ewes were observed.
Pregnancy did not affect early development of follicles, primordial and transitory, but did inhibit the later growth stages (primary, preantral and antral). During pregnancy several factors, notably high steroidal hormones produced in feto-placental unit and CL (Al-Gubory et al. 1994), may retard follicle development, and suppress OSE proliferation. Consequently, large antral follicles were absent in this group. Consistent to our observations, Al-Gubory and Martinet (1986) also reported that the diameter of the largest antral follicles at the onset of pregnancy did not increase more than 1.5 mm, and most of these follicles eventually became atretic.
Presence of large antral follicles in cycling and anoestrous groups can be explained from the reported high level of gonadotropins, especially FSH, in the two groups. Accordingly, the FSH levels in cycling active ewes were found to be similar to that of the anoestrous ewes (Bister and Paquay 1983). FSH is considered to be the most important hormone controlling folliculogenesis, especially at the later stages of follicle growth (Hunter et al. 2004). FSH is also being recognized as a survival factor for antral follicles. Several studies in rodents (Tonetta and diZerega 1989), bovine (Hulshof et al. 1995), sheep (Newton et al. 1999) and human (Roy and Treacy 1993) have revealed to the facts that FSH treatment stimulates differentiation and proliferation of granulosa cells, and reduces the number of in vitro grown atretic follicles.
Another interesting finding was that, relative to cycling, in pregnant and anoestrous ewes negligible or low Ki-67 staining was observed in GC’s in late differentiated follicles. PCNA staining was more intense and followed a similar pattern. Comparing reliability of PCNA and Ki67 as suitable nuclear proliferative markers, it was found that PCNA expression was observed even in the resting primordial follicles with ameiotic oocyte, whereas Ki67 expressed only in the proliferating follicles (at late growth stages and up-regulated in cycling ewes).
It was demonstrated that PCNA immunostaining is better suited for counting ovarian follicles because it strongly labels almost all oocyte nuclei of ovarian follicles at different maturity stages, including the ones in primordial follicles (Muskhelishvili et al. 2005). PCNA expression in primordial follicles’ oocytes can not be recognized as an indicator of cell proliferation because these oocytes are arrested at the first meiosis (Hirshfield 1999).
Possibly, the expression is related to its role in DNA repair process, rather than proliferation per se. Conversely, studies have also indicated that PCNA can be used as a reliable marker for cell proliferation (Tomanek and Chronowska, year?). In this study we have found that Ki67 is a better marker for proliferation studies, and PCNA expression can be confused with DNA repair or other DNA synthetic activities not necessarily associated with cell division. In accordance to our observations, Scholzen and Gerdes (2000) have argued favoring use of Ki67 rather than PCNA as a reliable marker for cell proliferation studies.
Collectively the results indicate that in sheep pregnancy leads to,
- inhibition of proliferative activity in the OSE and
- suppression of follicular growth.
In rodent model, OSE cell differentiation was investigated in presence of exogenously supplemented estrogens and progesterone (Gotfredsonand & Murdoc 2006). The cells varied in types, from simple squamous to stratified. Stratification of epithelial cells increased with estradiol treatment, and was completely suppressed with progesterone. Stratified cells were also found to be highly proliferative and prone to neoplastia. Apparently, progesterone restores the epithelium of postovulatory ovaries to a resting or non-proliferative state.
In our study, though such OSE morphological differentiation was not discernable, estradiol production in cycling antral follicles and CL might have induced proliferative activity in nearby OSE cells. The same explanation also holds true for observed proliferative activity in differentiating follicular GC’s. During pregnancy progesterone seems to have suppressed these events. It has been shown that high levels of progesterone inhibited antral follicles’ growth in cow (Adams et al. 1992).
It is proposed that estrogen-progesterone antagonistic regulation of OSE proliferation near antrum and CL may lead to neoplastic transformations, which may eventually initiate epithelial ovarian cancer, unless for some reasons like apoptosis, such cells are constantly eliminated.
Gonadotropins like LH, FSH and human chorionic gonadotropin (hCG) are the other hormones regulating OSE proliferation and transformation into cancerous cells in mice. Immature CD1 mice were treated with such hormones to stimulate hyper-ovulation (Burdette et al, 2006). The OSE in proximity to antral follicles and CL proliferated significantly in superovulated animals than controls. Proliferation was thought to be an act of healing of surface wounds caused due to ovarian surface rupture after each cycle of ovulation. In course, it was believed that free radicals and other genotoxins, like mitogenic estrogens, generated at ruptured sites induce ovarian neoplastia.
However, it was later observed that proliferation occurs even before ovulatory-caused wounds. This opposed the so called “rupture and repair theory” of proliferation. According to a recent “gonadotropin theory”, the expression of LH and FSH receptors on the surface of OSE cells is a direct response of gonadotropins (Choi et al, 2007). Most likely the hormone-stimulated receptors trigger the localized proliferation events. Besides, these receptors also initiate the oncogenic pathways (Choi et al, 2004). Other than OSE, FSH receptors are also expressed by GC’s, whereas LH receptors by both theca and granulosa.
Using DNA microarrays, it was found that FSH receptors through G-proteins regulate at least 9 genes (Sasson et al, 2003). This process is mediated through cAMP activated protein kinase A through activation of adenylyl cyclase by stimulatory G-proteins. The other gonadotropin-receptor-mediated and proliferation-associated signal proteins are P13K-AKT and EPAC (Wayne et al. 2007). A cascade of gene expression particularly those of proliferation and/or metastasis are triggered through these signals. These include genes for vascular endothelial growth factor (VEGF), metalloproteinase a, tumor necrosis factor-α (for metastasis), and interleukin-1 and 6 (cytokines), epidermal growth factor receptor etc. (for proliferation). Several caspases, mediating apoptosis, are also expressed for routine cell maintenance.
In conclusion, progesterone appears to be a candidate suppressing OSE proliferation and preventing follicular differentiation, thereby not allowing antral follicles to form. Its effect during pregnancy seems to be overriding the positive effect of estrogens on OSE proliferation and follicle development. These hormones regulate OSE proliferative activities through gonadotropins, via stimulating their receptors and signal molecules.
Conditions that suppress ovulation like multiple pregnancies, breastfeeding and use of oral contraceptives suppress follicular differentiation and OSE proliferative activities. This is due to high serum progesterone and low LH, FSH and hCG concentrations. High progesterone may have a direct negative effect on gonadotropin production and thereby it might inhibit events leading to both follicular development and OSE proliferation.
Another likely candidate could be some inhibitory factors of matured follicles, viz. anti mullerian hormone (AMH), which is present in GC’s of all growing follicles up a stage of selection (Durlinger et al. 2002). These factors may prevent early follicle differentiation as already found in case of anoestrous sheep. This is to be noted that AMH is a crucial factor for initial follicle recruitment.