Involvement of lipopolysaccharide in ovarian cystic follicles in dairy cow: Expressions of LPS receptors and steroidogenesis-related genes in follicular cells of cystic follicles
Introduction
High producing dairy cows suffer from a number of periparturient diseases after calving. Of these diseases, cystic ovarian disease (COD) is a factor influencing the reproductive performance of dairy cows (Bartlett et al., 1986) because this disease increases the interval between calving and conception, as well as culling rate (Grohn et al., 1997). The main physiological defect leading to the formation of COD is endocrine imbalance in the hypothalamic-pituitary-ovarian axis (Silvia et al., 2002). COD develops due to a failure of the hypothalamus to trigger the preovulatory surge of luteinizing hormone (LH) in response to estradiol. At the ovarian level, molecular changes in the growing follicle may have a local action and contribute to anovulation and cystic formation (Vanholder et al., 2006). Altered steroid and gonadotropin signaling systems in COD could play a fundamental role in pathogenesis of COD (Ortega et al., 2015). The growth and steroid production of follicles depend on the coordinated interaction between gonadotropins and their receptors. It has been shown that gonadotrophin receptor expression in bovine cystic follicles is altered (Ortega et al., 2015). Moreover, the numbers of follicular stimulating hormone (FSH) and LH receptor in granulosa cells of cystic follicles are decreased when compared to normal follicles (Kawate et al., 1990). Thus, the unresponsiveness of follicular cells to LH surge might be one of the mechanisms of follicular cyst development.
One of the candidate molecules affecting the steroid and gonadotropin signaling systems of cystic follicles is lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. We have previously shown that LPS was detected in bovine dominant follicles and influenced the follicular activity by suppressing transcriptions of steroidogenic enzymes and gonadotrophin receptors such as LH and FSH (Magata et al., 2014a). These results indicate that LPS may reduce the ability of the follicle to respond to gonadotrophins and perturb the stimulation of steroidogenesis. Moreover, LPS inhibits steroid production in granulosa and thecal cells in vitro (Herath et al., 2007; Shimizu et al., 2012; Magata et al., 2014b). Thus, it could be possible that LPS in follicular fluid is associated with the pathogenesis of COD by disrupting the steroidogenic reactions and ovulatory mechanisms. LPS is recognized by its specific receptor complex, toll-like receptor 4 (TLR4), cluster of differentiation 14 (CD14) and myeloid differentiation factor 2 (MD2). Binding of LPS to TLR4 receptor complex results in nuclear translocation of nuclear factor kappa-B components, which leads to the production of pro-inflammatory cytokines and chemokines (Beutler, 2004; Takeuchi and Akira, 2010). In vitro studies showed that bovine granulosa and theca cells express TLR4, CD14, and MD2 transcripts and detect LPS, which leads to the impaired follicular steroidogenesis (Herath et al., 2007; Magata et al., 2014b). Here, we hypothesized that LPS is involved in the pathology of bovine COD, with the local action on the follicular steroidogenesis and gonadotrophin signaling pathways via TLR4 signaling. Thus, the objective of the present study was to examine the molecular characteristics of bovine ovarian cysts in terms of LPS recognition and steroidogenesis.
Section snippets
Sample collection
Ovaries of multiparous Holstein cows were collected at a local slaughterhouse and placed in the ice-cold PBS. Ovaries were washed with 70% ethanol and PBS at the sampling and before aspirating the follicular fluid (FF). FF was aspirated with an 18 G needle connected to a 5-ml disposable syringe to determine the diameter of follicles as described by Murasawa et al. (2005). Follicles were classified into 3 categories based on the calculated follicle diameters, concentrations of E2 and ratio of
Concentrations of steroid hormone and LPS of each follicle
The concentration of E2 and P4 in follicular fluid of POF, EACF and EICF is shown in Table 1. The concentration of E2 in follicular fluid of POF was higher (p < 0.05) than those of EACF and EICF follicles. The concentration of P4 in follicular fluid of EICF was higher (p < 0.05) than those of POF and EACF follicles. The ratio of E/P depended on the endocrine condition of each follicle stage. The concentration of LPS in follicular fluid of POF was lower (p < 0.05) than those of EACF and EICF
Discussion
In the present study, we observed a higher LPS concentration in follicular fluid of E2-active and E2-inactive cystic follicles compared with preovulatory follicles in bovine ovaries. Similarly, Polat et al. (2015) reported that concentration of LPS in follicular fluid of cystic follicles was greater than those of antral follicles. Our previous study demonstrated the high LPS concentration in follicular fluid of large antral follicles from cows with metritis (Magata et al., 2015). Moreover, the
Conclusion
Our present study demonstrated that high LPS was present in follicular fluid of E2-active and E2-inactive cystic follicles, and that LHR expression in granulosa and theca cells is low in cystic follicles regardless of E2 concentration in its follicular fluid. The signal input of LPS might be differ between E2-active and E2-inactive cystic follicle because differential expression of LPS receptors was observed between EAFC and EIFC. These finding revealed the molecular characteristics of bovine
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgement
This study was supported by the Ito foundation, JAPAN.
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