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Ovarian changes in Sprague-Dawley rats produced by nocturnal exposure to low intensity light Eric Beys, Thomas Hodge & Gerhard J. Nohynek Drug Safety Department, Seine, Cedex, France
Rhone-Poulenc Rorer S.A., B.P. 14, 13, quai Jules Guesde, 94403 Vitry-sur-
Summary This study was performed to investigate the potential effects of nocturnal, low-intensity light upon ovarian morphology of female Sprague-Dawley rats and to investigate the cause for ovarian changes which had been observed in an earlier study following transient exposure of control female Sprague-Dawley rats to indirect light of minimal intensity during the nocturnal 12-h dark cycle. Twenty female Sprague-Dawley CD rats (initial age: 5 weeks) were kept for 8 weeks under our standard laboratory conditions including a daily 12-h light cycle (light intensity: approximately 200 lux) followed by a 12-h dark cycle with exposure to an indirect light source of low intensity (approximately 30 lux). Ten female control rats of comparable age from a concurrent toxicology study housed in an adjacent animal room under our standard 12 h light/dark cycle served as controls. At the end of the study the rats were sacrificed, necropsied and the ovaries were evaluated histopathologically. In 5 of the 20 animals we found ovarian atrophy consisting of decreased number and size of corpora lutea and increased number of tertiary follicles and/or follicular cysts. Most corpora lutea present in these ovaries were old, indicating the absence of recent ovulations. In contrast, the incidence of ovarian changes in the control group was 0/10. In conclusion, nocturnal exposure of female Sprague-Dawley rats to light of minimal intensity produced a substantial incidence of ovarian changes and suggests that the incidence of ovarian atrophy observed in a previous study may have been due to transient exposure to indirect nocturnal light of minimal intensity. Keywords
Ovarian atrophYjlow intensity lightj Sprague-Dawleyrat
During the histopathological evaluation of a recent study in Sprague-Dawley CD rats we observed an elevated incidence of ovarian atrophy (6/1Sl in control females (RPRunpublished report, 1993). However, in control females of comparable age from concurrent toxicology studies performed in our laboratory we observed no ovarian changes of comparable incidence or severity. The ovarian changes in this study may have been due to exposure of the animals to minimal illumination (approximately 30 lux) by an indirect light source during their daily 12-h dark cycle. The nocturnal exposure to light occurred Accepted 16 September 1994
during the initial 7S-day period of the study (initial animal age: 5 weeks). Through the subsequent period of 126 days, however, the animals were kept under standard 12/12-h light/dark conditions. Although it has been reported that continuous 24-h exposure to light of daylight intensity may be associated with ovarian changes in female rats (Singh 1969, Campbell &. Schwartz 19801,little is known about the effects of light of low intensity on ovarian morphology and its reversibility. Therefore, we performed the present study to investigate whether nocturnal exposure of female rats to an Laboratory Animals (1995) 29. 335-338
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indirect light SOllce of low intensity may be responsible for ovarian changes detected in the earlier study. MATERIALS
AND METHODS
Twenty female Sprague-Dawley Crl:CDTM(SD)BRrats were supplied by Charles River France, 59, rue de la Paix, 76410 Saint-Aubin-Ies-Elbeuf, France. At the initiation of the study, the animals were approximately 5 weeks of age and weighed between 120 and 220 g. In addition, 5 male animals were housed in the same animal room to provide the necessary environmental condition for normal oestrus function. Housing conditions were as follows: temperature 22±2°Cj humidity 40 to 70%; 15 to 20 air changes per hour; animals housed in
intensity at the level of the animal cages of approximately 200 lux during the light cycle and approximately 30 lux during the dark cycle (light intensity determined manually using a digital Lux Tester (Sper Scientific, Prolabo, rue Pelee, 75011 Paris, France)). An outline of the animal room and the illumination is shown in Fig 1. The animals were kept for 8 weeks in this animal room under identical conditions as the rats of the earlier study which had resulted in the high incidence of ovarian changes in control animals. The animals were observed daily for mortality and clinical signs. At the end of the study all female rats were anaesthetized with pentobarbital and killed by exsanguination via the abdominal aorta. Partial necropsies were performed and the ovaries from all females were collected and preserved in
individual stainless steel cages with free
Baker's solution lformol-calciumj. Sections
access to food (DAR A04, DAR, 7, me Gallieni, Villemoisson, 91360 Epinay sur Orge, France) and tap water. A 12-h light/ dark cycle was maintained in the animal room itself while the illumination in the ante-room was maintained continuously (Fig 11resulting in a light
of all ovaries were processed, sectioned at 5/Lm, stained with haematoxylin, eosin and saffron and examined microscopically. Ten female rats of the same strain and comparable age housed under standard 12/12-h light/dark conditions from a concurrent toxicology study performed in an adjacent room served as controls. RESULTS
Animal
Cage .
In-life observations No indication for adverse effects were observed during the duration of the study. Behaviour, food consumption and body weight gain were typical for rats of this strain and age.
ttery
Room
Anteroom
D
Door Fig 1 Outline of the animal room. 0 = location of the light sources in the anteroom providing 24-h continuous illumination resulting in a light exposure to approximately 30 lux of the animals during the 12-h dark cycle.
Pathological findings Ovarian atrophy was observed in 5 of the 20 (25%) female rats (Table 11exposed to the daily 12-h nocturnal illumination by indirect, low-intensity light but was not seen in the concurrent controls which remained unexposed during their 12-h dark cycle. Ovarian atrophy as defined in this study, consisted of decreased size and number of corpora lutea and increased number of tertiary and/or cystic follicles. Corpora lutea were either small and
Ovarian changes in Sprague-Dawley
rats
337
Table 1 Incidence and severity of ovarian changes in Sprague-Dawley rats. Group A: 8 weeks of continuous nocturnal exposure to a low intensity (30 lux) light source. Group B: concurrent control group. (Group C: data from an earlier uncontrolled study are included for comparison: animals of that study had been exposed for 7S days to low intensity light (30 lux) during their 12 hour dark cycle, followed by 126 days with 12h/12h light/dark cycle.) Group A
B
c
No examined • No normal b
20 15
10 10
15 9
Decreased/small corpora lutea: -minimal -mild -moderate Total
1 2 2 5
3 2 6
Increased tertiary follicles/follicular cysts: -minimal -mild -moderate Total
1 2 2 5
1 3 2 6
1
a Number of animals examined b C
Number of animals with normal ovaries Not detected
undergoing regression, or were absent. Most follicles were tertiary follicles and many displayed degenerative changes in the granulosa cells and ova. The control females had numerous, large corpora lutea and had follicles in all stages of development (Fig 2). The ovaries from light-exposed rats were small with the above atrophic changes ranging in severity from minimal to moderate (Fig 3). One animal had minimal lesions, 2 animals had mild lesions and 2 animals had lesions graded as moderate. Most of the corpora lutea which were present in the 5 affected animals were small and were regressing, indicating absence of recent ovulations. DISCUSSION/CONCLUSION Ovarian atrophy as defined in the present study and the earlier investigation is
Fig 2 Ovary (partial view) from a control rat. Note prominent corpora lutea (long arrows). Primary, secondary and tertiary follicles are present (short arrows). Magnification: 9.9X.
known to occur spontaneously in laboratory rats, particularly in senescent animals (Greaves & Faccini 1984). However, given the age of the animals in our study, the observed incidence was far in excess of values reported in the literature (Peluso & Gordon 1992, Alison & Morgan 1990) and of the incidence observed in our laboratory with rats of comparable age « 1%). It has been reported that continuous exposure of female rats to artificial light corresponding to daylight intensity (300 to 600 lux) may disrupt the oestrous cycle (Singh 1969, Campbell & Schwartz 1980) by lowering peak levels of LH, FSH and progesterone ITakeo 1984) whereas diurnal exposure to ISO lux combined with nocturnal exposure of female rats to very dim light (0.2 lux) was reported to produce no effect upon normal
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In conclusion, even minimal light exposure during the nocturnal period should be avoided in toxicology studies in rats. References Alison RH, Morgan KT 119901Ovary. In:Pathology of the Fischer Rat. (Boorman GA, Eustis SL, Elwell MR, Montgomery CA, MacKenzie WF, eds·1 San Diego: Academic Press, Ch. 26, p431 Campbell CS, Schwartz NB (1980) The impact of constant light on the estrus cycle of the rat. Endocrinology 106, 1230-8 Greaves P, Faccini JM (1984) Ovary and oviducts. In: Rat Histopathology-A Glossary for Use in Toxicity and Carcinogenicity Studies. (Greaves P, Faccini JM, eds.l Amsterdam, New York: Elsevier Science Publishers, pp 175-6 McCormack CE, Sridaran R (1978) Timing of ovulation in rats during exposure to continuous light: evidence for a circadian rhythm of luteinizing hormone secretion. Tournal of Endocrinology 76, 135-44 Peluso Gordon LR (1992) Nonneoplastic and neoplastic changes in the ovary. In: pathology of the Ageing Rat, Vol I. [Mohr U, Oungworth OL, Capen CC eds.) Washington DC: ILSI Press, pp 351-64 Rh6ne-Poulenc Rorer (1993) RPR/RD/CRVA/ SM91-711 (unpublished report) Singh KB (1969) Induction of polycystic ovarian disease in rats by continuous light. I. The reproductive cycle, organ weights and histology of the ovaries. American Tournal of Obstetrics and Gynecology 103, 1078-83 Takeo Y (1984) Influence of continuous illumination on estrus cycle of rats: time course of changes in levels of gonadotropins and ovarian steroids until occurrence of persistent estrus. Neuroendocrinology 39, 97-104
n,
Fig 3 Ovary from a rat exposed to continuous low-level light during the 12-h dark period. The ovary is small and corpora lutea are absent. Tertiary and cystic follicles (arrows) are predominant. Magnification: 9.9X.
cyclic ovulation (McCormack & Sridaran 1978). Therefore, the presence of ovarian changes in our study indicates that a light intensity of 30 lux during the dark cycle is in excess of the threshold light intensity producing ovarian changes.
