Reproductive Biology Analysis

Using monoclonal cytokeratin antibody, which signifies the development of cytoskeleton in growing epithelial cells and hence determines the proliferation ability, we detected in estrous sheep a high degree of multiplication in both OSE layer within the ovary and in cultured OSE cells isolated from this layer. Keratin markers distinguish OSE cells from the underlying stroma and other ovarian cells. For cell culture, M199/MCDB 105 medium and antibiotic combinations were used, which is an appropriate medium routinely used for sheep OSE cells (Gubbay et al. 2006). There are, however, discrepancies so far as the selection of medium for given mammalian OSE cell culture is concerned (Auersperg et al. 2001).

Rodent OSE cells can propagate in FBS-supplemented Waymouth medium 752/1 whereas human cells quite like ovine cells can grow in FBS-containing M199/MCDB105 medium. This is because external calcium regulates OSE cell growth in culture medium through surface calcium-sensory receptors. While human OSE propagates at > 0.8 mM Ca2+ concentration, which is the constituent of M199/MCDB105, for growing rat cells lower concentrations, found in Waymouth medium, are suitable.

Hence, the nutrient requirement of ovine OSE cells is similar to the of human cells. So far as proliferation-associated marker selection is concerned, another complexity with mammalian OSE cells is that in several growth passages epithelial cobblestone compact cells tend to transform into mesenchymal phenotype (fibroblast-like shape) (Auersperg et al. 2001). Consequently expression of several markers, including keratins, also changes. It appears that the ovine keratin marker is rather stable.

The addition of FCS in the serum-free medium enhanced synthesis of cytokeratin proteins compared to controls. Apparently, FCS contains some undefined compound(s) that stimulates proliferation. Serum supplementation provides essential nutrients (amino acids, vitamins, lipids) and adherence material for the growing animal cells (Master, 2000). Because serum-free medium can support growth, ewe’s OSE cells are probably capable of synthesizing these components. Reports also suggest that some undefined serum factors may influence epithelial-mesenchymal cell transformation (Auersperg et al. 2001). There are two possibilities how serum might influence OSE proliferation. Follicular fluid is partially an exudate of serum and therefore it is expected to contain at least some of the mitogenic factors found in serum (Murray et al. 1998).

Fetuin, a glycoprotein component of FCS that contributes to attachment of the cultured cells, may cause adherence of the epithelial cells in a way that happens in re-epithelialisation of OSE layer. It has been found that Fetuin-A, a Ca2+-dependent adhesion factor in FCS induces cell-to-cell adhesion leading to tumorigenesis (Kundranda et al. 2005). Initial adherence may influence synthesis of cellular Ca2+-dependent adhesion factors like N- and E-cadherins, and these in turn may influence proliferative activity.

In this study, follicular fluids from medium and large follicles and extracts of CL induced proliferative activity over controls. These extracts/fluids contain a number of anterior pituitary glycopeptide hormones, FSH and LH/hGC, steroidal hormones, estrogen and progesterone and several localized growth factors like IGF and HGF. Besides, receptors of all these follicular modulating hormones are also present on the surface of OSE cells.

While most of the steroidal hormones regulate follicular cycle and maintain luteal cycle, their role in OSE lining is not clearly elucidated. Estrogens are implicated in OSE neoplastic transformation. In order to examine whether steroidal hormones affect OSE proliferation, the follicular fluids/CL extracts were processed by passing through charcoal to remove steroidal components. Interestingly, even such de-steroidal fluids also accelerated OSE proliferation over serum-free controls. Apparently, steroids have no direct effect on in vitro OSE cell multiplication. In order to further verify the ineffectiveness of steroidal hormones, pure estrogen (estradiol) and progesterone at graded concentrations were added to the cultured OSE cells.

Corroborating to our assumptions, both hormones did not make any significant difference in proliferative activity of OSE cells over controls. So far the results suggest that factors other than steroids may be responsible for in vitro proliferation of OSE. The likely candidates could be gonadotropins and the local growth factors produced in follicles, which trigger sequence of reactions leading to proliferative response. FSH in particular has implicated in increasing in vivo OSE proliferation (Choi, Wong, Huang & Leung, 2007).

However, in vitro results were conflicting, and in various species stimulatory, inhibitory as well as non-responsive influences of these hormones were noticed. One standing explanation for such variation was differential expression of surface gonadotropin receptors and follow-up downstream signaling molecules affecting proliferation. It has been reported that hGC and estradiol may regulate OSE proliferation through IGF and HGF.

Further, receptors of FSH and LH mediate up-regulation of IGF-1 and HGF. These growth factors utilize multiple signal pathways to suppress OSE apoptosis, and thus may lead to mitogenic effect resulting in OSE malignancy. It was a matter of interest to examine whether such localized growth factors have a direct influence in multiplication of ewe OSE cells. We indeed observed that relative to controls IGF-1 stimulated over 2-fold the proliferative response.

It can be presumed that in cycling ewes’ estrogen response on OSE proliferation could be in part mediated by an increase in the transcription of IGF-1 and HGF. Most likely FSH and LH also mediate, through their receptor expression, an independent transcriptional activation of such growth factors, and thus the ultimate manifestation of estradiol and gonadotropins is the same. One of the underlying signal transduction operating transcriptional control is the cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of transcriptional factors (Gubbay et al. 2006).

One such factor is cAMP response element binding protein (CREB) and activating transcriptional factor-1 (ATF-1). In sheep model, CREB/ATF-1 was accounted for survival of OSE through stimulation of proliferative activity and prevention of apoptosis. OSE cells are exposed to gonadotropin inflammatory response during ovulation and genotoxic interleukins-1, -6 etc. may induce oncogenic response. Elimination of such cells at rupture site by apoptosis during post-ovulatory epithelialisation is presumably an oncoprotective mechanism. In the surrounding regions gonadotropins stimulate an entirely opposite response. This is by evading apoptosis and up-regulating CREB/ATF-1 expression.

Thus collateral OSE cells take over the rupture site and contribute to healing process. Additionally, in human, gonadotropin cAMP-PKA signaling pathway was found to down-regulate N-cadherin protein (Pon, Auersperg & Wong, 2005). N-cadherin, a Ca2+-dependent cell-to-cell adhesion protein, was found to be controlling survival capabilities of OSE. In the absence of adhesion due to N-cadherin down-regulation, the cells were non-aggregated and had tendency was to undergo apoptosis. This feature is observed on the post-ovulatory rupture site. Thus, follicular hormones appear to have both antagonistic effects on OSE cells at rupture site and at the surrounding region.

Although the expression of gonadotropin receptors in the OSE has been reported in several species (Murdoch et al. 1999; Parrott et al. 2001; Syed et al. 2001), the distribution of receptor expression across the ovarian surface has not previously been considered. We found that the expression of LH and FSH receptors over large follicles (5 mm or larger) were 2- and 4-fold higher than those over stroma and CL respectively, suggesting that OSE proliferation over growing follicles results, at least in part, is from a local increase in the sensitivity of OSE cells to circulating gonadotropins.

It has been reported that treatment of anoestrous sheep with estradiol increased LH receptor expression in the OSE (Murdoch et al. 1999), and in granulosa cells both estradiol and IGF-1 up-regulate the expression of gonadotropin receptors (Knecht et al. 1984; Hirakawa et al. 1999). Therefore an increase in gonadotropin receptor expression in OSE overlying large follicles may be mediated by paracrine follicular influences.

In conclusion, OSE proliferation in cycling sheep is associated with underlying developing follicles and CL, mediated through, at least in part, up-regulation of gonadotropin receptors and facilitated by the action of mitogenic glycopeptides and growth factors but not the steroids.

References

Auersperg, N., Wong, A.S.T., Choi, K.C., KANG, S.K. & Leung, P.C.K. (2001). Ovarian Surface Epithelium: Biology, Endocrinology, and Pathology. Endocrine Reviews, 22(2), 255-288.

Choi, J-H., Wong, A.S.T., Huang, H-F. & Leung, P.C.K. (2007). Gonadotropins and Ovarian Cancer. Endocrine Reviews, 28(4), 440–461.

Gubbay, O., Rae, M.T., McNeilly, A.S., Donadeu, F.X., Zeleznik, A.J. & Hillier, H.G. (2006). cAMP response element-binding (CREB) signaling and ovarian surface Epithelial cell survival. Journal of Endocrinology, 191, 275–285.

Kundranda, M.N., Henderson, M., Carter, K.J., Gorden, L., Binhazim, A., Ray, S., Baptiste, T., Shokrani, M., Leite-Browning, M.L., Jahnen-Dechent, W., Matrisian, L.M. & Ochieng, J. (2005). The Serum Glycoprotein Fetuin-A Promotes Lewis Lung Carcinoma Tumorigenesis via Adhesive-Dependent and Adhesive-Independent Mechanisms. Cancer Research, 65(2), 499-506.

Master, J.R.W. (2000). Animal Cell Culture (Third Edition). New York: Oxford University Press.

Pon, Y.L., Auersperg, N. & Wong, A.S.T.(2005). Gonadotropins Regulate N-cadherin- mediated Human Ovarian Surface Epithelial Cell Survival at both Post-translational and Transcriptional levels through a Cyclic AMP/Protein Kinase A pathway. Journal of Biological Chemistry, 280(15), 15438–15448.