Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Ecdysone oxidase and 3-oxoecdysteroid reductases in Manduca sexta midgut: Kinetic parameters

Ecdysone and 20-hydroxyecdysone are converted to their 3-epimers by enzymes in the midgut cytosol of Manduca sexta larvae. A partially purified cytosol preparation has been used to analyze the nature of and the interaction between these enzymes. The cytosol was shown to contain ecdysone oxidase, one or more 3-oxoecdysteroid 3α-reductase(s), and one or more 3-oxoecdysteroid 3β-reductase(s). The reductases reacted at different velocities with NADH and NADPH. With NADH, 3α-reduction was the major reaction; with NADPH, 3β-reduction was the major reaction. The apparent kinetic parameters for the enzymes support the assumed two-step mechanism for the 3-epimerization with a 3-oxoecdysteroid as intermediate.     

Mediation of ecdysone synthesis in Manduca sexta by a hemolymph enzyme

The prothoracic glands of Manduca sexta synthesize dehydroecdysone, which is rapidly converted to ecdysone through the mediation of a hemolymph enzyme, a 3 β-forming-3-ketosteroid reductase. The hemolymph protein fraction (HPF) containing this enzyme was obtained from diapausing and non-diapausing pupae, isolated abdomens, surgically manipulated pupae, etc., and in all cases had the capacity to affect the conversion of dehydroecdysone to ecdysone. The enzyme is heat labile, is inactivated by trypsin, and has a molecular weight of between 20,000 and 30,000. The data indicate that the conversion of dehydroecdysone to ecdysone exhibits linear kinetics and may be dependent on both the enzyme concentration and the concentration of NADPH at the beginning of the reaction but may be limited by the absolute amount of reducing equivalents after 10 min, under the experimental conditions utilized. The capacity of the enzyme to reduce dehydroecdysone was titered in the hemolymph during the last larval instar and during prepupal and pupal life with maximum capacity exhibited at the beginning of the instar, on day 8 of larval life and at day 1 of pupal life. Even at its lowest point at day 5, 1 ml of hemolymph was able to convert 77 pmol (～35 ng) dehydroecdysone to ecdysone in 1 min. These results require a new interpretation of the control of molting in the Lepidoptera.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

Evidence for an endogenous neurocidin in the Manduca sexta ventral nerve cord

Half of the neurons in the abdominal nervous system of the moth Manduca sexta die after adult eclosion. Two physiological signals regulate post-eclosion neuronal death in adult moths. The first is endocrine: a decline in blood ecdysteroids is necessary for the death of neurons in the segmental ganglia. The second signal, which is highly specific for a pair of motoneurons found at the posterior midline in each of the three unfused abdominal ganglia, originates in the nervous system. It is transmitted from the fused pterothoracic ganglion to abdominal ganglion A3 via the intersegmental connectives. To characterize the signal of neural origin, we have developed an in vitro bioassay for neuron-killing factors (“neurocidins”). Aqueous extracts of pterothoracic ganglia were prepared and applied to cultured ventral nerve cords. These extracts exhibited concentration-dependent effectiveness in killing motoneurons. The active component of the extract was heat-stable and protease-sensitive. Size fractionation studies suggested that the active component has a molecular mass between 10 and 30 kD. This is the first report of an endogenous neuron-killing protein from an insect nervous system. © 1995 Wiley-Liss, Inc.     

Inhibitory effects of actinomycin D and cycloheximide on neuronal death in adult Manduca sexta

A decline in circulating 20-hydroxyecdysone permits the emergence of the adult Manduca sexta moth; this endocrine signal also triggers the death of approximately half of the neurons in the unfused abdominal ganglia of the moth central nervous system. This programmed death of neurons was markedly reduced by treatment with either actinomysin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor). Similar results were found after addition of these agents to ventral nerve cord cultures. The effectiveness of these treatments in delaying or blocking neuronal death depended upon their time of administration relative to the normal time of post-emergence death in the particular motoneuron under study: late-dying neurons, for example, could still be saved by these treatments even after early-dying neurons had already initiated degeneration. In both intact moths and cultured ventral nerve cords, the ability of actinomycin D to prevent neuronal death waned at the same time at which replacement of the steroid hormone could no longer block neuronal death. This suggests that the steroid commitment point represents the time at which the genes that mediate cell death are transcribed. Cycloheximide remained effective in delaying or blocking neuronal death until shortly before the onset of degeneration, suggesting that ongoing protein synthesis is essential for the initiation of the degeneration response. 1994 John Wiley & Sons, Inc.     

Alteration of liver microsomal monooxygenases and substrate competition with aniline hydroxylase from rats chronically fed low-fat and high-fat-containing alcohol diets

Male Sprague-Dawley rats fed ethanol (EtOH) 36% of total calories for four weeks in a liquid diet containing either 34% (HF) or 12% (LF) of calories as fat were studied with respect to induction of microsomal monooxygenases (MFO) and substrate competition with EtOH-inducible aniline hydroxylase. The specific activity and turnover of aniline hydroxylase were induced to similar extents by HF-EtOH and LF-EtOH diets. Whereas, both LF-EtOH and HF-EtOH caused a decrease in the turnover of arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and aldrin epoxidase compared to pair-fed (PF) controls, LF-EtOH but not HF-EtOH increased the turnover of ethoxycoumarin and ethoxyresorufin O-deethylase (ECOD and EROD). The increase in ECOD and EROD and the decrease in AHH by EtOH is contrary to the parallel induction of these activities by J-methylcholanthrene (3-MC) and Aroclor 1254 (Aroclor). Benzo(a)pyrene (BaP) stimulated aniline hydroxylase in the HF-EtOH and PF systems, whereas with LF diet, stimulation was seen only in the EtOH group. Ethoxycoumarin (EC) inhibited aniline hydroxylase by microsomes from EtOH- and pyrazole-treated rats, whereas it stimulated aniline hydroxylase by control microsomes, suggesting that the EC effects were associated with EtOH-inducible cytochrome P-450. Ethoxyresorufin (ER) inhibited aniline hydroxylase in EtOH and PF groups, thus the differential effects of EC were not nonspecific O-deethylase effects. The effects of EtOH feeding on ECOD, EROD, and AHH (ie, substrates for 3-MC-inducible cytochrome P-450) displayed a greater differential between the experimental and control group with the LF- than with the HF-containing diet. The findings suggest that the alteration of certain MFO activities by chronic EtOH ingestion can be modified by the content of dietary fat. Moreover, the competition dynamics of MFO substrates toward EtOH-inducible aniline hydroxylase are altered by EtOH feeding and, in turn, modified by dietary fat.     

Manduca sexta lipid transfer particle: Synthesis by fat body and occurrence in hemolymph

Lipid transfer particle (LTP) is present in hemolymph of the tobacco hornworm Manduca sexta. Biosynthesis of LTP, occurrence in hemolymph, and the role of LTP-apoproteins in the lipid transfer reaction were investigated using antibodies specific for LTP or for each of the apoproteins. In vitro protein synthesis followed by immunoprecipitation demonstrated that LTP is synthesized by the fat body and secreted into the medium. In contrast to apolipophorin III, an exchangeable apoprotein of lipophorin (the major lipid transport protein in hemolymph), apoLTP-III could not be detected free in hemolymph. LTP concentrations in the hemolymph were measured by a sandwich ELISA using a mouse monoclonal antibody against apoLTP-III as capturing antibody and rabbit polyclonal antibody against apoLTP-I as detecting antibody. LTP concentration increased during the late fifth instar larval stage, followed by a decrease in the wandering stage. Subsequently, LTP concentrations were strongly increased in hemolymph of adult moths. The role of the three apoproteins of LTP in the lipid transfer reaction was analyzed using apoprotein-specific antibodies. All three, apoLTP-I, -II, and -III, appeared to be important for lipid transfer activity, as shown by inhibition of lipid transfer by antibodies specific for each of the three apoproteins. © 1996 Wiley-Liss, Inc.     

Mechanistic and metabolic studies of sterol 24,25-double bond reduction in Manduca sexta

Larvae of Manduca sexta were used to obtain a cell-free sterol 24,25-reductase. From the midgut of fifth instar larvae fed a mixture of sitosterol and campesterol a microsome-bound 24,25-sterol reductase was prepared that transformed desmosterol (K_m, 3 μM), lanosterol (K_m, 18 μM), and cycloartenol (K_m, 33 μM), to cholesterol, 24,25-dihydrolanosterol, and cycloartanol, respectively. With desmosterol as substrate, the microsome-bound enzyme was found to incorporate tritium into cholesterol from 4S-tritium labelled NADPH. [24-²H]lanosterol was transformed by larvae to [24-²H]24,25-dihydrolanosterol (structure confirmed by mass spectroscopy (MS) and ¹H-nuclear magnetic resonance spectroscopy. A rationally designed inhibitor of 24,25-reductase activity, 24(R,S),25-epimino-lanosterol (IL), was assayed and found to be inhibitory with an I₅₀ of 2 μM. IL was supplemented in the diet of M. sexta with either sitosterol or stigmasterol and found to inhibit development (I₅₀ 60 ppm). The major sterol which accumulated in the IL-treated larvae was desmosterol, confirming the site of inhibition was reduction of the 24,25-bond. IL was converted to [2-³H]IL when fed to the larvae. [2-³H]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques. © 1996 Wiley-Liss, Inc.     

Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M_r = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 ± 20 μg/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH₂-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses. © 1994 Wiley-Liss, Inc.     

Programmed cell death of identified peptidergic neurons involved in ecdysis behavior in the moth,Manduca sexta

The eclosion of the adult Manduca sexta moth is followed by a wave of cell death that eliminates up to 50% of the neurons of the central nervous system within the first few days of imaginal life. While the identity of some of the dying motoneurons has been established, that of most doomed neurons is unknown. Here, we show that the dying cells include peptidergic neurons involved in the control of ecdysis behavior. These cells belong to a small population of 50 neurons that express crustacean cardioactive peptide (CCAP), a potent regulator of the ecdysis motor program, and show increases in cyclic 3′,5′-guanosine monophosphate at each ecdysis. First, we describe new markers for these neurons and show that they are expressed in these CCAP-immunoreactive neurons in a complex temporal pattern during development. We then show that these neurons die within 36 h after adult eclosion, the last performance of ecdysis behavior in the life of the animal, via the active, genetically determined process of programmed cell death. The death of these neurons supports the hypothesis that outmoded or unused neurons are actively eliminated. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 265–280, 1998     

Influence of calcium on Manduca sexta plasmatocyte spreading and network formation

Plasmatocytes are a class of insect hemocytes important in the cellular defense response. In some species, they are phagocytic, protecting the insect from smaller pathogens. In many insects, they work in concert with other hemocytes (particularly other plasmatocytes and granular cells) to form nodules and to encapsulate foreign material. To perform these functions, plasmatocytes attach to, spread on, and surround suitable targets. Because of their importance, because we had previously observed that prolonged incubation of hemocytes in solutions containing the divalent cation chelator ethylenediaminetetraacetic acid (EDTA) inhibited plasmatocyte spreading, and because of the importance of divalent cations in many immune-related functions, we investigated the effect of calcium and magnesium on spreading of plasmatocytes from fifth instar Manduca sexta larvae. On glass slides, plasmatocytes spread more quickly and elongated in Grace's medium containing 5 mM calcium, compared to calcium-free medium. In the presence of calcium, plasmatocyte adhesion, spreading, and network formation were not visibly different in magnesium-free and magnesium-containing Grace's medium. Using immunomicroscopy with a monoclonal antibody specific for plasmatocytes, we measured the length and width of plasmatocytes incubated with several different concentrations of calcium. Plasmatocyte length positively correlated with calcium concentration to 5 mM (maximum concentration tested and approximately the hemolymph concentration). Mean plasmatocyte width was less in 0 and 5 mM calcium than in 0.05 or 0.5 mM calcium. On plastic, hemocytes survived longer than on glass (they survived beyond 24 h) and, in 5 mM calcium, formed an extensive network readily visible by phase-contrast microscopy. This network was never as extensive in the absence of calcium. Network formation in the absence of magnesium, but presence of calcium, resembled network formation in standard Grace's medium.     

Purification and some properties of cytochrome P-450 specific for steroid 17 alpha-hydroxylation and C17-C20 bond cleavage from guinea pig adrenal microsomes

The energy requirements for mitochondrial protein synthesis were investigated in isolated rat liver mitochondria. Controlled changes in coupling efficiency were obtained by titration with FCCP in the presence of various substrates. No relationship was observed between the efficiency of oxidative phosphorylation and the inhibition of protein synthesis. With succinate-ADP as the substrate the ADP:O ratio was decreased by 70–80% with no effect on protein synthesis. In contrast, with acetate-ADP as substrate, a 10–20% reduction in the ADP:O ratio gave complete inhibition of protein synthesis. The data suggest that the rate of ATP production is more important for maintenance of protein synthesis than the efficiency of coupling $\text{per se}$. Thus, certain substrates can support maximal rates of protein synthesis even in relatively poorly coupled mitochondria. Analysis of mitochondrial translation products formed in the presence of increasing FCCP concentrations also showed that decreased efficiency of oxidative phosphorylation had no influence on the nature of the products.     

Utilization of [14C]phenylalanine derived from arylphorin or free amino acid in Manduca sexta pharate adults

The role of arylphorin as a storage protein was studied using 14C-arylphorin. 14C-arylphorin was produced optimally by incubating one-half fat body from Manduca sexta fifth instar larvae at 22 degrees C for 24 h, in 1 ml of medium containing amino acids at 25% of their physiological concentration with [U-14C]-phenylalanine (phe) provided initially without nonlabeled phenylalanine. Nonlabeled phe was provided after 1 h at 16% of its physiological concentration. The specific activity of 14C-arylphorin produced in vitro was 30 times greater than that generated in vivo. Injection of 14C-arylphorin into pharate adults was used to study the distribution of 14C-phe derived from this protein into 14CO2 and tissues for comparison with injection of free 14C-phe during the middle (days 6 to 12 pharate adult) and late (days 12 to 17 pharate adult) stages of adult development. Appearance of 14CO2 from 14C-arylphorin as compared to 14C-phenylalanine showed a slower time course during both the middle and late stages of development, in keeping with the time needed for degradation of the protein. In accord with faster phe turnover near the end of adult development, total 14CO2 production was greater and the retention of 14C in hemolymph and fat body was less compared to the middle stage of development regardless of whether 14C-arylphorin or 14C-phe was injected. In the middle stage of development, the appearance of 14C in the cuticle and head parts was greater, whereas incorporation into abdomen and thorax was less than during the late stage of development. Since the pattern of 14C distribution from 14C-arylphorin and 14C-phe was similar, one major function of arylphorin must be as a storage protein replenishing the supply of free amino acids used for synthesis of adult tissues. These results also suggest a limited contribution of M. sexta arylphorin to formation of the cuticle subsequent to day-6 pharate adult.     

Metabolism of [14C]cholesterol in Manduca sexta pupae: Isolation and identification of sterol sulfates,free ecdysteroids,and ecdysteroid acids

[¹⁴C]Cholesterol was injected into fifth-instar larvae of Manduca sexta, and the metabolites were isolated and identified from 8-day-old male and female pupae. A major portion of the metabolized cholesterol was esterified either with a sulfate group or with fatty acids. The predominant ecdysteroid metabolites were 20-hydroxyecdysone, 20,26-dihydroxyecdysone, 20-hydroxyecdysonoic acid, and 3-epi-20-hydroxyecdysonoic acid. Smaller amounts of ecdysteroids were identified as conjugates of 26-hydroxyecdysone, 3-epi-20-hydroxyecdysone, 20,26-dihydroxyecdysone, and its 3α-epimer. The metabolic profiles were similar for both male and female pupae. The two ecdysteroid acids were identified by nuclear magnetic resonance spectroscopy and chemical ionization mass spectrometry and by mass spectral analyses of their methyl esters. Detection of 3-epi-20-hydroxyecdysonoic acid as a major metabolite is significant, as its occurrence has been scarcely reported. 3-Epiecdysteroid acid formation is discussed as a possible ecdysteroid-inactivating pathway that may be operating specifically in lepidopterous insects or in particular developmental stages such as eggs or pupae.     

Bacteria-induced protein P4 (hemolin) from Manduca sexta: a member of the immunoglobulin superfamily which can inhibit hemocyte aggregation.

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of Mr = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NH2-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and is composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence is 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hemocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects.     

Innate and acquired components of oligophagy in the herbivorous lepidopteran,Manduca sexta

Innate and acquired aspects of oligophagy were investigated in the tobacco hornworm, Manduca sexta L. (Lepidoptera: Sphingidae), a species normally restricted to members of the family Solanaceae. Larvae were reared in the laboratory on solanaceous species tobacco (Nicotiana tabacum L.), strawberry groundcherry (Physalis pruinosa L.), and potato (Solanum tuberosum L.), as well as the non‐solanaceous foods cowpea [Vigna sinensis (L.) (Fabaceae)], rapeseed [Brassica napus L. (Brassicaceae)], and artificial diet. Feeding assays were conducted using the above plants plus corkscrew vine [Vigna caracalla (L.) Verdc. (Fabaceae)], lettuce [Lactuca sativa L. (Asteraceae)], and moist glass‐fiber filter paper. Larval feeding was characterized using two camera‐monitored assays: (1) a quantitative no‐choice disc test that determines consumption over time, delays in initiation of feeding, and % of larvae rejecting the food, and (2) a novel no‐choice emigration test that measures how soon a larva abandons a whole plant. Experimental results from both assays revealed that larvae displayed high levels of acceptance of Solanaceae regardless of whether they had been reared on solanaceous or non‐solanaceous food. We conclude that solanaceous oligophagy in M. sexta is primarily innate and does not require (but may be strengthened by) previous feeding experience on Solanaceae. In contrast, larvae tested on non‐solanaceous plants or moist filter paper showed large variation in both acceptance of foods and emigration times that were strongly dependent on the food on which they had been reared (analogous to the ‘induction of preference’ of earlier literature). Two types of induction were identified: an increase in acceptability of a plant of (1) the same species as the one on which the larva was reared, or (2) a related species. These discoveries both challenge and expand our current understanding of oligophagy in the tobacco hornworm.     

Effects of human choriogonadotropin on mitochondrial and microsomal cytochrome P-450 levels in mouse testes

The mitochondrial and microsomal cytochrome P-450 contents of $\text{C57B1}\text{6}$ mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 ± 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 μg/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N′,N′-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 ± 3.5 and 28.9 ± 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 ± 1.4 and 19.6 ± 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis.