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The aim of this study was to determine the modulatory effects of peptide 234 (p234) (an antagonist of GPR54 receptors) on kisspeptin and RF9 (an RFamide-related peptide antagonist)-induced changes in reproductive functions and energy balance in female rats. Female Sprague-Dawley rats were weaned on postnatal day (pnd) 21. The animals were intracerebroventricularly cannulated under general anesthesia on pnd 23. Groups of female rats were injected with kisspeptin, RF9, p234, kisspeptin plus p234, or RF9 plus p234, daily. The experiments were ended on the day of first diestrus following pnd 60. Kisspeptin or RF9 alone advanced vaginal opening (VO), which was delayed by administration of kisspeptin antagonist alone. In the rats given kisspeptin plus p234 or RF9 plus p234, VO was not different from control rats. Kisspeptin and RF9 elicited significant elevations in circulating LH levels. Coadministrations of kisspeptin or RF9 with p234 decreased LH levels significantly. The use of p234 alone did not cause any significant change in LH secretion. Kisspeptin decreased both food intake and body weight while RF9 decreased only food intake without affecting body weight. The effects of kisspeptin on energy balance were also reversed by central administration of p234. In conclusion, kisspeptin antagonist, p234, modulates the effects of kisspeptin on reproductive functions and energy balance, whereas RF9 seems to exert only its effects on reproductive functions by means of GPR54 signaling in female rats.

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Non-invasive measurement of cortisol in saliva is of prime importance as it represents a bioavailable neuroendocrine marker for stress. Therefore, in this study, we developed an enzyme immune assay that was suitable for salivary cortisol measurements. For that purpose, rabbit polyclonal antibody was raised against cortisol-3-CMO:BSA conjugate. The test was based on competition of liquid phase cortisol with conjugated cortisol on the solid phase. Primary antibody was used to bind available sites on the conjugate, which was proportional to numbers of cortisol in liquid phase. Biotinylated secondary anti-rabbit antibody was used to detect primary antibodies by addition of streptavidin peroxidase and substrate, respectively. Color formation was stopped and yellow color was read by a plate-reader spectrophotometer. Additionally, validated test was used to met all validation criteria including. Test developed was used to establish cortisol awakening response (CAR) in saliva samples collected in the morning after awakening (0, 15, 30, and 60 min) from women (n = 4) and men (n = 4) at 8 or 4 different days, respectively. Diurnal cortisol levels were assessed (n = 8) at after awaking 60 min at morning, 12:00, 19:00, and 22:00 hr. In conclusion, an enzyme immunoassay test was successfully produced, validated and used for cortisol measurement in saliva samples.

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