BGB324 Previous studies have shown the harmful effects of
Previous studies have shown the harmful effects of NEFA on ovarian functionality, such as inhibition of bovine granulosa cell survival and proliferation, steroidogenesis, and follicular development and alteration in oocyte development [36,45,46]. Therefore, the higher levels of NEFA found in the follicular fluid of cysts could be altering the normal functionality of the cells, and could thus be affecting steroidogenesis. It has been reported that increased NEFA concentrations may have a putative direct toxic effect at the ovarian level . The granulosa cell layer is a non-vascular compartment surrounded by follicular fluid and the basal membrane, whereas the theca interna cell layer is rich in blood capillaries. In agreement with that found by Leroy et al. , in the present study, NEFA concentrations were lower in the follicular fluid of dominant follicles than in blood serum, which indicates that granulosa BGB324 might be exposed to lower NEFA concentrations than theca cells. However, NEFA metabolism is different in both compartments, with granulosa cells having a higher sensitivity to NEFA than theca cells . In addition, in cows with COD, we detected similar NEFA concentrations in plasma and follicular fluid of cysts. NEFA, especially saturated long-chain fatty acids, seem to affect the reproductive physiology adversely. When granulosa and theca cells are incubated with high NEFA concentrations, their viability and steroidogenic capacity are hampered [4,45]. In this sense, we have previously detected altered expression of steroidogenic enzymes  and steroid hormone concentrations [22,23] in serum and follicular fluid of cows with COD, which could be related to altered NEFA concentrations, as reported in the present study. The lower progesterone concentration detected in cows with COD is in agreement with the in vitro study reported by Vanholder et al.  and could negatively modulate the ovarian functionality. On the other hand, we herein corroborated that cows with COD had lower plasma insulin levels than controls, and, as we have previously shown, this was accompanied by lower protein expression of some components of the insulin signaling cascade . Many authors have demonstrated that the actions of insulin in the ovary, including steroidogenesis, growth of granulosa and theca cells [48,49], follicular maturation [50,51] and normal postpartum ovarian function , occur through the increased steroidogenic capacity of the dominant follicle . Furthermore, it has been demonstrated that alterations in insulin concentrations can affect the development and maturation of follicles and the response to stimulation by LH, which can lead to anovulation and cyst formation . Additionally, in vitro studies with bovine theca cells have shown that adiponectin decreases the androgen and progesterone production induced by insulin . Even more, different mRNA levels of adiponectin and adiponectin receptors have been detected in granulosa and theca cells of dairy cows at different follicular stages, suggesting that adiponectin may exert an important physiological role . In granulosa cells, the growth of bovine healthy follicles was correlated with an increase in the gene expression of adiponectin and its receptors, whereas in theca cells, adiponectin expression decreased across follicular development . Moreover, adiponectin has been reported to have suppressive effects on progesterone and androstenedione production and LH receptor expression in theca cells of bovine large follicles, probably regulated by LH and IGF-1 , which could be related to the lower concentrations of progesterone detected in cystic follicles [22,23]. Also, adiponectin increases insulin sensitivity and consequently insulin would be negatively regulating the mRNA levels of AdipoR1 and AdipoR2 via activation of PI3K [19,58]. Although several studies have reported lower concentrations of adiponectin in women with polycystic ovary syndrome (PCOS), mainly associated with obesity [19,57,59,60], here we detected that cows with COD had higher concentrations of adiponectin than control cows. In addition, in a retrospective study carried out by our group, we observed that the body condition at the voluntary waiting period did not influence the incidence of cysts . In cattle, the body condition is related to fertility, mainly in the peripartum period, when overfeeding cows lipomobilize their reserves, increasing the levels of NEFA and related to a negative energy balance [62,63]. Furthermore, Ohtani et al.  measured serum adiponectin in cows at different stages of lactation, and observed an increase in postpartum lactation, probably associated with the energy deficit occurring during this period. In several species, the action of adiponectin has been studied in tissues such as liver and skeletal muscle, and it is known that this adipocytokine is involved in homeostasis and glucose and lipid metabolism, and participates in the control of reproductive functions [55,, , ]. In the present study, we found an increase in AdipoR2 and AMPK expression in theca and granulosa cells, respectively. Probably, the increased binding of adiponectin to its receptor favors glucose metabolism through AMPK . Furthermore, PPAR-stimulated adiponectin expression would increase fatty acid oxidation and energy consumption [19,68], which could be related to the higher protein expression of CPT1 and ACOX1 in cows with COD detected in our study. Adiponectin, AMPK and PPARs may be key signals regulating the amount of energy required for the growth of follicles, oocytes, and embryos . Therefore, the activation of these sensors could be related to the low intraovarian concentrations of glucose and triacylglycerol herein observed, as it has been widely studied in skeletal muscle and liver [19,68].