MicroRNA miR-152 can support ovarian granulosa cell functions and modify apigenin actions.

The study aimed to evaluate the involvement of apigenin, microRNA (miR)-152, and their interrelationships in the control of basic ovarian granulosa cell functions. The effects of apigenin (0, 10, and 100 µg/mL), miR-152 analogues or miR-152 inhibitor, and their combinations with apigenin on porcine granulosa cells were examined. Expression levels of miR-152, viability, proliferation, apoptosis, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 release were analyzed. Apigenin increased the expression of miR-152, cell proliferation, and estradiol release and reduced apoptosis, progesterone, and IGF-I output. MicroRNA-152 analogues promoted cell viability and proliferation, as well as the release of progesterone, IGF-I, oxytocin, and prostaglandin E2; however, it inhibited apoptosis and estradiol output. miR-152 inhibitor had the opposite effect. Moreover, miR-152 analogues suppressed the effect of apigenin on cell apoptosis and estradiol release. These observations 1) confirm the involvement of apigenin in the control of basic ovarian cell functions; 2) are the first demonstration of importance of miR-152 in the control of these functions; 3) show the ability of apigenin to promote miR-152 expression and the ability of miR-152 to modify apigenin effects on ovarian cells.

Rooibos (Aspalathus linearis) and its constituent quercetin can suppress ovarian cell functions and their response to FSH.

Rooibos (Aspalathus linearis Brum. f) can directly influence female reproduction, but whether rooibos can influence the response of ovarian cells to FSH and whether the rooibos effects are due to the presence of quercetin remain unknown. We compared the influence of rooibos extract and quercetin (both at 10 µg/ml-1) on porcine ovarian granulosa cells cultured with and without FSH (0, 1, 10 or 100 ng/ml-1). The expression of intracellular proliferation (PCNA, cyclin B1) and apoptosis (bax, caspase 3) markers in the cells was detected by immunocytochemistry. The release of progesterone (P), testosterone (T) and estradiol (E) were evaluated with ELISAs. Administration of both rooibos and quercetin reduced the accumulation of proliferation markers and promoted the accumulation of apoptosis markers and the release of T and E. Rooibos stimulated, but quercetin inhibited, P output. Administration of FSH increased the accumulation of proliferation markers, decreased the accumulation of apoptosis markers, promoted the release of P and T, and had a biphasic effect on E output. The addition of both rooibos and quercetin mitigated or prevented the main effects of FSH. The present observations suggest a direct influence of both rooibos and quercetin on basic ovarian functions - proliferation, apoptosis, steroidogenesis and response to FSH. The similarity in the major effects of rooibos and its constituent quercetin indicates that quercetin could be the molecule responsible for the main rooibos effects on the ovary. The potential anti-reproductive effects of rooibos and rooibos constituent quercetin, should be taken into account in animal and human nutrition.

Interrelationships between kisspeptin and FSH in control of porcine ovarian cell functions.

The existing knowledge of the direct action of kisspeptin on the ovary needs to be expanded. In our study, the direct effects of kisspeptin on basic ovarian cell functions and their response to FSH were examined. We studied the effect of kisspeptin alone (0, 1, 10, and 100 ng/mL) and of kisspeptin (1, 10, and 100 ng/mL) in combination with FSH (10 ng/mL) on cultured porcine granulosa cells. Markers of viability, proliferation (accumulation of proliferating cell nuclear antigen [PCNA] and cyclin B1), and apoptosis (accumulation of bax and caspase 3), as well as the release of steroid hormones and IGF-I were analyzed using the trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. Addition of kisspeptin at lower doses (1 and 10 ng/mL) increased cell viability, the accumulation of PCNA and cyclin B1, decreased the accumulation of bax and caspase 3, and promoted release of progesterone, estradiol, and IGF-I, but not testosterone. A high dose (100 ng/mL) of kisspeptin had the opposite, inhibitory effect. The addition of FSH increased cell viability, proliferation, decreased apoptosis, and promoted progesterone, testosterone, estradiol, and IGF-I release. Kisspeptin at lower doses supported the stimulatory action of FSH on viability, PCNA and cyclin B1 accumulation, and release of progesterone and estradiol, promoted its inhibitory action on bax and caspase 3 accumulation, but did not modify its action on testosterone and IGF-I release. On the contrary, kisspeptin at a high dose inhibited and even reversed the FSH effect. FSH mimicked and promoted both the stimulatory and inhibitory action of kisspeptin on all examined ovarian functions besides IGF-I release. These observations show that kisspeptin can directly regulate basal ovarian cell functions. Furthermore, they demonstrate the functional interrelationships between kisspeptin and FSH in direct regulation of ovarian functions.

The inhibitory influence of toluene on mare ovarian granulosa cells can be prevented by fennel.

The influence of environmental contaminant toluene and of plant fennel (Foeniculum vulgare Mill.) on reproduction are reported, but the mechanisms of their action and the protective effect of fennel on contaminant influence remain to be elucidated. In this study, we hypothesized that toluene and fennel directly affects basic ovarian cell functions, and that fennel can be used as an appropriate natural protective agent against the potential adverse effects of toluene. This study aimed to examine the action of toluene (20 µg/mL) and fennel extract (0, 1, 10, 100 µg/mL), and assess their combination on viability, proliferation, apoptosis, and hormone release by cultured healthy mare ovarian granulosa cells. Viability, proliferation (percentage of PCNA-positive cells), apoptosis and release of progesterone, oxytocin and prostaglandin F were evaluated by using Trypan blue exclusion tests, immunocytochemistry and enzyme immunoassays, respectively. Toluene, when given alone, inhibited viability, proliferation, apoptosis, progesterone, prostaglandin F and IGF-I. However, it did not affect oxytocin release. Moreover, Fennel, when given alone, inhibited viability, progesterone, and prostaglandin F release, as well as stimulating proliferation and oxytocin release. In addition, Fennel did not affect apoptosis. When given in combination with toluene, fennel was able to suppress, and even invert, the effects of toluene on viability, proliferation, apoptosis, prostaglandin F, and IGF-I. However, it did not alter its effect on progesterone release. Moreover, fennel induced the inhibitory effect of toluene on oxytocin output. The findings of our study suggest direct adverse effects of toluene on the basic ovarian functions of mares. Lastly, we also observed the direct influence of fennel on these functions, as well as its ability to be a natural protector against the action of toluene on the ovarian functions of mares.