Some biochemical studies related to steroidogenesis in the porcine corpus luteum
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A brief summary of the work described in this thesis is given below:- 1. The cholesterol side-chain cleavage reaction was investigated in preparations of porcine luteal mitochondria. The enzyme system was found to be similar to that studied in other steroidogenic tissues in that it was located in the mitochondrial subcellular fraction, was associated with cytochrome P450 and required NADPH as an electron donor. 2. A method for determining the oxygen content of incubation media was described] it was based on the stoichiometric oxidation of added NADH, catalysed by phenazine methosulphate. Using values for oxygen concentration obtained by this method, some respiratory characteristics of luteal mitochondrial preparations were investigated. Respiratory control and ADP:0 ratios of these preparations were found to be significantly lower than those measured for porcine liver mitochondrial preparations. 3. The efficiency of several tricarboxylic acid cycle intermediates as electron donors for cholesterol side-chain cleavage activity was investigated: citrate, isocitrate, succinate, fumarate and malate supported greater activity than NADPH. 4. Studies with respiratory inhibitors and uncoupling agents indicated that NAD+- linked substrates could donate electrons to the NADPH - cytochrome P450 reductase by an energy-independent pyridine nucleotide transhydrogenase. In contrast, succinate supported the reaction via an energy-dependent electron transfer pathway. Experimental evidence was presented which indicated that this latter route might not involve reduction of NADH. 5. In view of the natural abundance of cholesterolfatty acid esters in luteal tissues, it was thought relevant to investigate the utilization of such com¬ pounds by porcine luteal mitochondrial preparations. [4-14^C] cholesteryl oleate was shown to be hydrolysed, and the cholesterol thus liberated underwent side-chain cleavage. The capability of the fatty acid moiety to act as an electron donor for this reaction was also demonstrated. Palmitylcarnitine was shown to support high levels of cholesterol side-chain cleavage activity, via an energy dependent electron transfer process. 6. A schematic hypothesis of electron transfer pathways in porcine luteal mitochondria was presented. Its main features embodied electron transfer connections between the mitochondrial "respiratory" and "steroido¬ genic" chains at two different levels: (a) a reversible, non-energy dependent pyridine nucleotide transhydrogenase, and (b) an energy-dependent electron transfer route from reduced flavoprotein to the NADPHcytochrome P450 reductase. 7- As a speculation it was suggested that LH might stimulate cholesterol side-chain cleavage activity, and hence steroidogenesis, by promoting electron transfer from respiratory substrates to the NADPHcytochrome P450 reductase via these connections, at the expense of mitochondrial respiratory electron flow.