Effects of clomiphene citrate on the expression of kisspeptin dynorphin A and neurokinin B in female Sprague-Dawley rats

Abstract

Clomiphene citrate (CC) is the leading treatment for women with anovulatory infertility. The precise mechanism of action of the drug on the hypothalamic-pituitary-gonadal (HPG) axis has yet to be determined. Neurons expressing kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn), collectively called KNDy neurons, in the arcuate nucleus (ARC) of the hypothalamus have been shown to play an integral role in the estradiol (E2) feedback pathways of the reproductive system in mammals. KNDy neurons are found in the ARC and the anteroventral periventricular nucleus (AVPV) in humans but have been predominantly reported to not express NKB and Dyn in rodents. The axons of these neurons project to the medial eminence (ME) in the region where the gonadotropin-releasing hormone (GnRH) terminals and fibres are located. It was hypothesised that CC upregulates the gene expression of kisspeptin and neurokinin B while down-regulating the gene expression of dynorphin A which results in a leutenizing hormone surge and an increase in oestradiol which causes ovulation. This was a randomized experiment which included 18 female Sprague-Dawley rats in which the aim was to analyse the expression of kisspeptin, NKB and Dyn in the ARC and the AVPV as well as blood plasma levels of oestradiol and leutinizing hormone (LH) in female rats after CC administration. Six of the rats constituted the control group that received a vehicle solution. The second group of 6 rats received the intervention in the form of CC and the third group of six rats received CC as well as p234-penetratin, a kisspeptin antagonist (KpA). The mRNA expression of the KNDy genes were analysed using real-time quantitative polymerase chain reaction (qPCR) and the plasma levels of E2 and LH were analysed by enzyme-linked immunosorbant assays (ELISA). ELISA results show that the E2 concentration in the group that received CC plus KpA was found to be marginally lower than that of the control group but there was no significant difference between the E2 concentrations of the control group and the group that received only CC. The LH concentration in the group that received CC plus KpA was significantly higher than both other groups but again, there was no significant difference between the LH concentration control group and the group that only received CC. The qPCR showed that in the AVPV, the kisspeptin expression of the CC group and the CC plus KpA groups are marginally higher than that of the control group. Conversely, the Dyn expression of the CC group and the CC plus KpA groups are marginally lower than that of the control group in the AVPV. There were no significant differences in NKB expression across the three groups. In the ARC, there were no significant differences in kisspeptin or Dyn expression across the groups. The NKB expression of the CC group was marginally lower than that of the control and there was no significant difference between the CC plus KpA group and the control group. In summary, CC appears to have a marginal effect on the kisspeptin and Dyn mRNA via the positive feedback systems in the rat AVPV as well as a significant decrease of NKB mRNA via the negative feedback systems in the ARC. To increase the validity of similar future studies, higher sample sizes, different drug administration doses, possibly more precise surgical techniques and more accurate age determination methods or ovariectomised rats could be used.