© 1963 Nature Publishing Group 1310 NATURE June 29, 1963 voL. 190 possible to elaborate such a conditioned reflex 1-3 h later (Fig. IB). The signal stimuli evoked 50-60 per cent positive responses, that is, the same as in I-day-old guinea pigs•. After tying the cord animals also showed increased motor activ;ity and a strong reaction to indifferent external stimuli. Independent rhythmic r espiratory movements appear only in frotuses shortly before term. Only animals aged 60-66 days survived. The inability of frotuses with an intact placental circu- lation to form temporary connexions is interesting in view of the fact that guinea pigs are born with a nearly com- pl etely differentiated cortex both morphologically 3 and enzymatically' and bioelectrically•. Probably the start of 1·espiration is particularly important. I thank Dr. J. Sedlacek for his help in this work. VL. HLAVAOKOVA * De partment of Physiology, Charles University, Prague. • Pre sent address: Institut e of Physiology, Acndemy of Scicuce, Sal- 111ovs k6. 1, Prague 2. ' SedlM\ek, J., Phys.fol. BohernfJslol! ., 11, 313 (1962). 'Scdla1:ek, J., and Hlav6.~kova, VI., Act. Nerv. Sup., 4, 2 (1962). 'Peters, V. Il., and Flexncr, L. B., Amer. J. Anat., 86, 133 (1060). ' Kreps, E. M., Zurn. vyss. ner·v. dej., 2, 46 (1952). 'Flcxncr, L.B., Tyler, D. B., and Gallant, L. J., J. Neurophysiol., 13, 427 (1950). Hormonal Dependence of Oxidative Enzymes in the Testis of the Rat PITUITARY and chorionic gonadotrophins accelerate the synthesis of testosterone from acetate in dog testes in vivo 1 and in rabbit testes in vitro•. It is known that reduced triphosphopyridine nucleotide (TPNH) is required for steroid synthesis 3 , and t, here is some evidence indicating that gonadotrophic hormones influence dchydrogenase activit,y in target glands. Thus, Samuels• showed st , imu- lation of 3-B-ol-dehydrogonase by chorionio gonadotrophin in rat testis and Niemi• examined histochcmically hor- monal influences on d-1-~-hydroxybutyric and hydroxy- steroid dehydrogenascs. In previous work 6 , one of us found that addition of ~-hydroxybutyrate brought about a marked increase in oxygen consumption in testis homogenates of post- puberal but not of pre-puberal rats. These changes coincide with the onset of t estosterone synthesis. In a series of experiments designed to elucidate the effect of gonadotrophins at the cellular level, we have examined various metabolic steps endeavouring to find t, he pathways concerned in the production and supply of energy for steroid synthesis. The e xperiments reported here concern the effect of chorionic gonadotrophin on isocitric, glucose-6-phosphatc and lactic dehydrogenascs Immature A x C male rat ,s inbred weighing 23-25 g at the onset of the experiment wern injected with chorionic gonadotrophin for 5 days, after which the rats were d ecapitated. Effectiveness of treatment was checked by the weight of the testes and seminal vesicles (Table I). Tho testes were dissected free of their capsule, 100 mg of Table 1. EFFECT OF ClONADOTROPHIU HORMONE ON ORGAN WEIGH1' AND ON SPECIFIC ACTIVITY OF DEHYDROGENASE Total Glncosc-6- Tsocitrlc Lactic No. of dose Testis Seminal pho•phate de- dehydro- dehydro- :Lnimals G dnys weight vesicle hydrogenasc genase genase (I.U.) weight acltlvity activity activity 12 GOO 79 715 141 67 0 12 100 43 570 123 61 0 6 1 38 480 132 44 s• 6 2·5 80 295 85 31 14* • Not st:1tistically significant. All figures are expressed as percentage increase over those of untreated controls or the same age. Weights of organs were computed relntive to JOO g body-weight. Aconit11se activity was not modified. tissue were homogenized in 0·25 M sucrose in a motor- driven Potter-Elvchjem homogenizer at 2,000 r.p.m. for 90 soc in ice, and then centrifuged for 30 min at 14,000g under refrigeration. The oxidative enzymes examined here were located mainly in the post-mitochondrial fraction remaining in the supernatant . Isocitric dehydrogenase was detArmined by the method of Ochoa', glucose-6-phosphatc dehydrogenase by the m ethod of Kornberg and Horecker•, aconitase by Anfinsen's• and lactic dehydrogenase by Neilands's 10 procedure. Proteins were estimated by the biuret method as d escribed by Layne 11 • Chorionic gonadotrophin caused a significant increase in the specific activity of testis iAocitric and glucose-6- phosphatc dehydrogcnases (Table 1). Lactic dehydro- genase activity was not increased. All figures were well beyond statistical error at a probability-level of 0·001 by Student's t test, except th e lowest figure for isocitric dehydrogenase which was in the limit between P = 0·05 and O·l. Doses as small as 0·5 u a day were effective; smaller doses have not been tried. The effect appears to be organ- specific since it could not be obtained in liver homo- genates. Our results also showed that in rat testis these enzymes arc TPN dependent. No reduction of DPN could be obtained with testis homogenates using isocitratc and glucose-6-phosphat ,e as substrates. These findings provide evidence that enzymes belonging to the respiratory system and to the hexose monophos- phate shunt are stimulated in tho t estis by chorionic gonadotrophin; but we cannot say at the moment whether the specific action of the hormone is mediated by these systems. The gonadotrophic stimulation of dehydrogenases may be indirect, since Field 12 found no stimulation in vitro of glucose oxidation by luteinizing hormone or human chorionic gonadotrophin. If the hexose monophosphato shunt is involved in steroid synthesis in the testis it may be stimulated by a different mechanism from that postulated by Haynes 13 in adrenal cortex, since cyclic adenosinc monophosphate, which mediates the action of ACTH on phosphorylase in the adrenal cortex, does not stimulate the conversion of 14 O-acetate to 14 O-testostorono in vitro, according to Brinck - J ohrnmn and Eik-Noss 1 . Further experiments are in progress to elucidate some of the points raised hero and to investigate other systems in rat testis which may be influenced by gonadotrophic hormones. One of us (N. A. S.) received financial support from the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina. Institute of Collular Biology, Univorsidad Nacional de C6rdoba, Argentina. N. A. SCHOR J. CARA A. PERE7. 'Mason. N. R., and Samuels, L. T., Endocrinol., 68, 890 (1961). Brinck-Johnsen, T., and Eik-Ness, K. , ibid., 61, 676 (1957). 'Hnll, P. F., and Eik-Ness, K., Biochim. Biophys. Acta, 63, 411 (l962). 'J,ynn, jun., S. W., and Browi:i, R.H. , J. Biol. Chem., 232, 1015 (1058). Pinc us, G., Proc. Fourth Intern. O,mo. Bior:hem., 4, 61 (1958). • Samuels, L. T., and Hcmreich, M. L., Endocrinol., 58, 435 (1956). 'Niemi, M., and Ikonen, l\f. I. , Endocrinol ., 70, 167 (1962). • Schor, N. A., Rev. Soc. argellt. Bfol. (in the press). 1 Ochoa, S., Methods in Enzymology, edit. by Colowick, S. P., and Kaplan, N. 0 .. 1, 609 (Academic Press, New York, 1057). • Kornbug, A., nnd Horccker, n. L., l'n Methods in Enzymology, 1, 323 (Academic Press, New York , 1057). • Anfinsen, C. A., in Methods in Enzymology, 1, 695 (Aca.demic Press , New York, 11J57). "Nelland.s, J.B., in Methods in Enzymology, 1, 449 (Academic Press, New York, 1957). 11 Layne, E., in Methods in Enzymology, 450 (Academic Pres&, New York, 1957). 12 Field , J. n., Pastan, I., Herring, B. , nnrl Johnson, P., Endocrinol., 67, 801 (1960). "Haynes, R. C., .J. Biol. Chem. 233, 1220 (1958).