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Figure 1 | Reproductive Biology and Endocrinology

Figure 1

From: NTPDases in the neuroendocrine hypothalamus: Possible energy regulators of the positive gonadotrophin feedback

Figure 1

Experimental design for testing the proposed hypothesis. Animals will be ovariectomized (ovx) 7 days prior to further experimentation. Seven days after ovariectomy, animals will receive intracerebral (i.c.) stereotaxic injections into the arcuate nucleus as follows: Group 1 will receive 17beta-estradiol (E2) 20 minutes after initial injection of saline; Group 2 will receive E2 20 minutes after initial i.c. injection of suramin (NTPDase-inhibitor); Group 3 will receive two consecutive injections of physiological saline (sham control) with a timing presented above. An additional group of animals (Group 4) not subjected to i.c. injections will be investigated. Animals will be sacrificed 2-6-10-18 and 26 hours after the i.c. injections. Blood samples will be drawn from each individual and plasma LH will be determined. Brains will be quickly removed and the medial part of the hypothalami will be isolated. Tissue samples from left and right sides will be separately handled. Tissue samples will be homogenized and protein concentrations quickly determined. Based on sample protein concentrations, aliquotes will be prepared from tissue homogenates for further determination of NTPDase3-levels by Western blot analysis. Mitochondrial oxygen consumption (mitochondrial respiration rates, especially ADP-dependent state 3 respiration and total mitochondrial respiratory capacity) will be determined from synaptosomal preparations of tissue aliquotes without further inhibition (suramin) of the sample (i.e., the left-sided medial hypothalamus), or with further suramin-pretreatment of the contralateral (i.e., the right-sided) sample.

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