BIOLOGY OF REPRODUCTION 31, 280-286 (1984) Patterns of Follicular Growth During the Four-Day Estrous Cycle of the Rat1 ROY L. BUTCH ER2 and DIANA KIRKPATRICK-KELLER Department of Obstetrics and Gynecology West Virginia University Medical Center Morgantown, West Virginia 26506 -6302 Downloaded from https://academic.oup.com/biolreprod/article/31/2/280/2766498 by guest on 24 September 2021 ABSTRACT Female Sprague-Dawley rats underwent laporatomy during metestrus at 70 to 75 days of age or remained untreated to study the effects of surgical stress on follicular growth. Groups of rats were killed on each day of a 4-day estrous cycle, serial sections of the ovaries were prepared histologically and the number and size of follicles with one or more complete layers of cuboidal granulosa cells were determined. Since no differences due to surgery were found, the data were pooled by day of the estrous cycle (17 or 18 rats/day of cycle) for characterization and comparison of size distribu- tion of follicles on different days of the estrous cycle. Follicles were classified as atretic or healthy and divided into groups by increments of 20 pm of diameter for graphing. Data were analyzed by analysis of variance and least squares means. Significant differences were found in the distribution of both healthy and atretic follicles among days of the estrous cycle. At least 2b follicles/ovary were recruited from <260 pm into >260 pm in diameter between proestrus and estrus, and the follicles for ovulation were selected by diestrus. A greater number of growing follicles of 70 to 100 pm in diameter were present at diestrus. From the disappearance of follicles >260 pm between estrus and proestrus, it appears that arresia is a very rapid process. INTRODUCTION 1978a,b; Hirshfield and DePao!a, 1981; Wels For more than 40 years, attempts to deter- chen and Dul!aart, 1976; Hoak and Schwartz, mine the changes in patterns of follicular growth 1980) have shown recruitment of follicles into during the estrous cycle have been made with a the size group 350 pm in diameter at estrus number of species using counts and measure- and that this recruitment is in response to the ments of follicles in histological sections, radio- increase in plasma follicle-stimulating hormone isotope labeling and mitotic activities. Lane and (FSH) on the afternoon of proestrus and the Davis (1939), using mitotic activity in follicles morning of estrus. The dependence of follicular of rats, determined that large follicles grow at a recruitment on FSH also had been demonstrated faster rate, that follicles of a given size grow at in the hamster (Greenwald and Siegel, 1982). a different rate on different days of the estrous Studies using the mouse have shown an increase cycle, that follicles <250 pm in diameter do in mitotic activity in large follicles, more small not undergo atresia and that more small follicles follicles at diestrus and no atresia in small (30 to bOO pm) are present at diestrus. Mandl follicles (Pederson, 1970a,b). and Zuckerman (1952) reported a homogeneous Unilateral ovariectomy has long been known distribution of follicles <350 pm through- to result in a doubling of the number of ovula- out the estrous cycle, but a different distribution tions in the remaining ovary (Hunter, 1787; with stage of cycle for follicles > 350 pm in Arai, 1920; Mand! and Zuckerman, 1952) and diameter. However, in both of the above studies has been used in studies into the mechanism of the rats that were used had variable lengths of follicular growth and recruitment (Greenwald, estrous cycles, were variable in age and some 1961; Peppler and Greenwald, 1970; Hirshfield, of those classified as estrous had not ovulated. 1982, 1983a). During a study of follicular A number of studies (Hirshfield and Midgley, distribution after unilateral ovariectomy (Zoldak and Bowyer, 1977), it was concluded that surgical stress could alter follicular growth. The present study examined the effect of surgical Accepted May bl, 1984. stress on distribution of follicles. Since no Received December 9, 1983. effect of stress was found, experimental groups ‘This investigation was supported in part by Nih were pooled by day of cycle to provide a larger Research Grant AG-O23bb from the National Institute of Aging. number of observations for characterization of Reprint requests. follicular growth patterns of all follicles in the 280 FOLLICULAR GROWTH IN RATS 281 proliferative pool on each day of the estrous cycle. o., en men en MATERIALS AND METHODS General Female Sprague-Dawley rats at 70 to 75 days of age were housed under controlled conditions of temperature and lighting (lights on 0600-1800 h). Downloaded from https://academic.oup.com/biolreprod/article/31/2/280/2766498 by guest on 24 September 2021 Estrous cycles were monitored by daily vaginal smears . +1 +i +1 and only those rats exhibiting consistent 4-day cycles were used. Surgical procedures using ether anesthesia ‘- ‘.‘I consisted of only flank incisions during the morning of metestrus. A 3 X 4 factorial design with 6 rats per subgroup was used to examine the effects of surgical stress on alterations in distribution of growing follicles. The 3 main groups consisted of: 1) controls killed at 1600 h on each day of the estrous cycle, 2) left flank incisions done between 0830 to 0930 h of metestrus with groups of 6 rats killed at b600 h of each of the next 4 n’ ‘‘ days, 3) identical to Group 2, but groups of rats were + +: +: +1 killed on each day of the third subsequent cycle. The right ovary was recovered, fixed in Bouin’s solution and stored in 70% ethanol. The ovary was subsequently embedded in paraffin, sectioned at bO pm and stained with hematoxylin and eosin. All follicles with ) 1 complete layer of cuboidal granulosa cells surrounding the oocyte were counted, classified as hea4thy or atretic, and 2 perpendicular measurements of the diameter to the basement membrane were made only in the section which contained the nucleolus of the “ T en * en * oocyte. Follicles were considered atretic if two or +1 + +1 +1 v more pyknotic granulosa cells were found in a single section or if the oocyte was degenerating. The ovaries ‘ ‘ were randomized before the follicles were counted and ‘ measured. The person who did the counts and measure- ments was not aware of the treatments or stages of the estrous cycle from which the ovaries were obtained. 2 . Follicles were divided into 4 groups according to dianieter (<70, 70 to 110, 110 to 150 and ‘150 pm), 3 E which gave approximately equal numbers of follicles ,, a a .c per group. Data were analyzed for effects of surgical - stress within day of the estrous cycle by one-way -: analysis of variance (ANOVA). Since no differences N N N Q due to surgical stress were found, groups were pooled within day of the estrous cycle (n=b7 or 18 rats/day) . to examine for changes in follicular distribution. ‘ Data on number of healthy and atretic follicles were analyzed by ANOVA, and when differences were found the least squares means were used to assess differences o_ between days. Since in several studies, differences had . been reported in distribution of large follicles, the “i’ “t en ‘C group 150 pm in diameter was further subdivided into three groups of approximately equal numbers of V W ‘0’0 follicles ( b 50 to 200, 200 to 280, 280 pm in diameter) . ci. E and analyzed by ANOVA and least squares means. Follicular distribution between days of the estrous -5 u cycle was further examined by graphing numbers of follicles within 20-pm size groups. RESULTS 2; No differences were found in follicular “ distribution due to surgical stress when the data were analyzed statistically or plotted graphically. I- 282 BUTCHER AND KIRKPATRICK-KELLER TABLE 2. Number of healthy follicles per ovary in subclasses of the group >150 pm in diameter during the 4-day estrous cycle. Diameter in pm ( ± SEM) 150-200 200-280 >280 Proestrus* 23 ± 2 21 ± 2 15 ± Estrus 26 ± 2 20 ± 2 29 ± Metestrus 25 ± 2 b8 ± b 26 ± Downloaded from https://academic.oup.com/biolreprod/article/31/2/280/2766498 by guest on 24 September 2021 Diestrus 25 ± 2 22 ± I b8 ± N=17 or 18 rats/group. a.baiues in each column without a superscript letter in common are different (P<O.05). Pooling the data by day of the estrous cycle previously reported studies with few rats per provided 17 or 18 rats/day of the cycle (2 rats group. were eliminated from the study due to change Total numbers of healthy and atretic follicles, in the length of cycle). The substantial numbers or their sums, were not different due to the day of rats in the pooled groups allowed a character- of the estrous cycle. However, distribution of ization of follicular distribution within small healthy follicles did differ (P<O.05) in the 70- increments of size, which was not possible in to 110-pm and >150-pm diameter groups 7 #{149} U Proestrus (I) 6 w #{149} S Estrus -J C) tJ---D Metestrus -J 5 -J 0---0 Diestrus 0 n 17 or 18 rats/day >- I 4 I- -J L1J I 3 Lj 0 2 LJ fn z 2 200 240 280 320 360 400 440 480 520 560 600 640 680 DIAMETER (pm) FIG. b. Distribution of number ( ± SEM) of healthy follicles >200 pm in diameter by increments of 20 pm for each day of the 4-day estrous cycle. Each diameter shown includes follicles of that size and the following 19.9 pm eg., 40 pm=40.0 to 59.9 pm).