Changes in the superoxide radical and superoxide dismutase levels in the uterus of Rattus norvegicus during the estrous cycle and a possible role for superoxide radical in uterine oedema and cell proliferation at proestrus M. LALORAYA, G. P. KUMAR, AND M. M. LALORAYA~ School of Life Sciences, Devi Ahilya Vishwavidyalaya, Vigyan Bhawan, Khandwa Road, Indore - 452 001, Madhya Pradesh, India Received April 25, 1990 LALORAYA, M., KUMAR, G. P., and LALORAYA, M. M. 1991. Changes in the superoxide radical and superoxide dismutase levels in the uterus of Rattus norvegicus during the estrous cycle and a possible role for superoxide radical in uterine oedema and cell proliferation at proestrus. Biochem. Cell Biol. 69: 313-316. Superoxide anion radical, a radical whose toxicity is well documented, is present in the uterus of albino rat. The cyclic changes that it undergoes during the reproductive cycle are also illustrated. An inverse correlation is seen between the levels of superoxide radical and the enzyme dismutating it, i.e., superoxide dismutase (EC 1.15.1.I), which is also reported to be present in the uterus of albino rat and is shown to exhibit cyclic changes during its oestrous cycle. The high levels of superoxide radical at pro-oestrus enable us to hypothesize that superoxide may be involved in regulating the oedema and cell proliferation of the uterus during proestrus. Key words: superoxide anion radical, superoxide dismutase, uterus, oestrous cycle, uterine oedema, cell proliferation. LALORAYA, M., KUMAR. G. P., et LALORAYA, M. M. 1991. Changes in the superoxide radical and superoxide dismutase levels in the uterus of Rattus norvegicus during the estrous cycle and a possible role for superoxide radical in uterine oedema and cell proliferation at proestrus. Biochem. Cell Biol. 69 : 313-316. Le radical anionique superoxyde, un radical dont la toxicitk est bien connue, est prksent dans l'uttrus du rat albinos. Les changements cycliques qu'il subit durant le cycle de la reproduction sont kgalement illustrks. I1 existe une corrklation inverse entre les taux du radical superoxyde et l'enzyme qui en catalyse la dismutation, la superoxyde dismutase (EC 1.15.1.I), qui est Cgalement prksente dans l'utbus du rat albinos et qui manifeste des changements cycliques durant le cycle oestrien. Les t a w klevks du radical superoxyde au stade pro-oestrien nous permettent de supposer que le superoxyde puisse jouer un r81e dans la rkgulation de l'oedtme et de la proliferation cellulaire de l'utbus durant le pro-oestrus. Mots clbs : radical anionique superoxyde, superoxyde dismutase, utkrus, cycle oestrien, oedkme utkrin, prolifkration cellulaire. [Traduit par la rkdaction] Introduction The reported toxicity of superoxide anion radical (Fridovich 1981) has evoked considerable debate, since if it is truly toxic, all aerobes should be endowed with superox- ide dismutase activity. However, recent work has shown the complete lack of superoxide dismutase in strictly aerobic oxygen-requiring and -consuming microorganisms, viz., pathogenic strains of Neisseria gonorrhoeae, its related strains, and Lactobacillus plantarum (Fee 1981). The absence of superoxide dismutase in these organisms should be explained if superoxide is toxic. We have recently demonstrated the presence of superox- ide anion radical and superoxide dismutase in the ovary of Rattus norvegicus (the Norway rat) and have illustrated the cyclic changes undergone by them during the oestrous cycle. A useful role for this superoxide anion radical has been sug- gested by us; i.e., in conjunction with superoxide dismutase, this radical seems to regulate luted steroidogenesis (Laloraya et al. 1988). In this paper, we present the changes in uterine superox- ide anion radical and superoxide dismutase during the oestrous cycle of Rattus norvegicus and implicate a func- tional significance for the presence of superoxide anion radical in the uterus of albino rats. ABBREVIATIONS: EPR, electron paramagnetic resonance. '~uthor to whom all correspondence should be addressed. 2~resent address: Ravi Shankar Vishwavidyalaya, Raipur 492 002, Madhya Pradesh, India. Printed in Canada / Imprime au Canada Materials and methods Trizma base, Trizma HCl, Triton X-100, diethyldithiowbamic acid (sodium salt), and Hepes were purchased from Sigma Chemical Company Inc., St. Louis, MO, U.S.A. N-t-Butyl-a-phenyl nitrone was obtained from Aldrich, Milwaukee, WI. EDTA was from BDH, India, while pyrogallol was from Loba Chemie, India. Tris- HCl buffer (50 mM, pH 8.2) was prepared as described elsewhere (Laloraya et al. 1988). Female albino rats (Wistar strain) were bred in our institute col- ony and housed in rooms of controlled temperature (27 f 1°C) and light (14 h light : 10 h dark; the lights came on at 0600 and went off at 1900). They were provided with food and water ad libitum. Experiments were conducted on mature female rats of 4-6 months age. The reproductive cycle was identified by examination of vaginal smears (Stockard and Papanicolaou 1917). Only those females that showed a regular 4- or 5-day oestrous cycle were used for the above study. The rats were sacrificed by cervical dislocation at each stage of the reproductive cycle, namely dioestrus I, dioestrus 11, proestrus, and oestrus. The uteri were dissected and freed of adhering fat, blood was removed by washing several times with chilled physio- logical saline (0.154 M NaCl), and they were subsequently used for analysis. The tissues were weighed before processing for superoxide anion radical quantitation or superoxide dismutase assay. In all the experiments, three replicates were taken for each analysis. The superoxide radical was trapped as a N-t-butyl-a-phenyl nitrone adduct as described in our previous paper (Laloraya et al. 1988). EPR spectra of the N-t-butyl-a-phenyl nitrone - superoxide radical adduct were recorded on a Varian E-104 EPR spectrometer equipped with TM 110 cavity. Instrument settings employed were as follows: scan range, 100 Gs (1 Gs 0.1 mT); field set, 3237 Gs; Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Beijing University of Aviation and Aerospace on 06/06/13 For personal use only.