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.b aiues in each column without a superscript letter in common are different (P 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 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 L J 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). FOLLICULAR GROWTH IN RATS 283 #{149}-. Proestrus among days of the estrous cycle (Table 1). The .-. Estrus differences among days in number of follicles 50 >150 pm were due to differences in number of o---o Metestrus ‘4’ follicles >280 pm (Table 2). Distribution of U, 0--- 0 Diestrus U follicles by 20 pm increments are shown for -J 7 or 8 rats 0 follicles >200 and <200 pm (Figs. 1 and 2, 40 -j respectively). Graphic evidence is shown for -J 0 follicular recruitment between proestrus and Downloaded from https://academic.oup.com/biolreprod/article/31/2/280/2766498 by guest on 24 September 2021 estrus of an additional 21 follicles into the size >- I group >260 pm in diameter. This recruitment is I- 30 -j the difference in the number of nonatretic 4 U follicles >260 pm at estrus minus number of I follicles 260 to 46 1 pm at proestrus. A further 0 20 shift in follicular development occurred by metestrus in the >260-pm size group by a U continued growth and an increase in atresia (Fig. 1). By diestrus, there was a clear separation 2 0 of 8.4 ± 0.5 preovulatory follicles (>400 pm) which continued to grow with 7.2 ± 0,6 non- atretic follicles >480 pm remaining by proestrus. 1 The recruitment of 21 follicles into the size 0 40 80 20 60 200 >260 pm in diameter was not accompanied by a decrease in number of atretic follicles <260 DIAMETER (pm) pm (Fig. 3). An increase (P<0.05) in number of FIG. 2. Distribution of number (X ± SEM) of follicles 70 to b 10 pm in diameter was found at healthy follicles <220 pm in diameter by increments of 20 pm for each day of 4-day estrous cycles. diestrus (Fig. 2; Table 1), and this increase was #{149} U Proestrus 9 #{149} S Estrus C/) o---o Metestrus LU 8 -J o---o Diestrus 0 I n 17 or 18 rats /doy -J -J 7 0 L* 6 0 H LU 5 I- 4 U- 4 0 3 Ui 2 2 120 160 200 240 280 320 360 400 440 480 520 560 DIAMETER (jim) FIG. 3. Distribution of number ( ± SEM) of atretic follicles by increments of 20 pm of diameter for each day of 4-day estrous cycles. 284 BUTCHER AND KIRKPATRICK-KELLER TABLE 3. Number of atretic follicles per ovary by size during the 4-day estrous cycle. Diameter in pm ( ± SEM) Total <150 150-280 ‘280 atretic 63 ± 4 Proestrus 10 ± 2a 41 ± 3 14 ± 1a 38 ± 3 57j Estrus 10 ± 1a 10 ± 1b 3 34 ± 2 Metestrus 14 ± 1b 15 ± 1a 63 ± 4 Downloaded from https://academic.oup.com/biolreprod/article/31/2/280/2766498 by guest on 24 September 2021 36 ± 3 Diestrus 14 ± 1b 14 ± 64 ± 3 N=17 or b8 rats/group. 5 bValues in each column without a superscript letter in common are different (P<0.05). present in each of the 3 original surgical sub- the mechanism of selection within the recruited groups. follicles for ovulation is not known. One pos- Atresia was rarely detected in follicles <100 sible explanation could be that those follicles or >400 pm, with a broad peak in atretic with the greater number of FSH receptors re- follicles between 120 and 260 pm in diameter cruited by the elevation of FSH at proestrus (Fig. 3) which varied with stage of estrous cycle and estrus could survive longer following the (Table 3). The percentage of atretic follicles decrease in FSH at estrus. Techniques such as within a size class increased markedly from 6% that recently reported by Hirshfie!d (1983b), in the 100- to 120-pm to about 50% in the 160- combined with studies of receptors, should help to 280-pm group, and varied from 26 to 48% in the elucidation of the mechanism. atretic among days of the cycle for follicles of There was a significant increase in number of >280 pm in diameter. There was a decrease in follicles 70 to 110 pm in diameter on the day atresia of follicles > 280 pm at estrus, but not of diestrus. This seems to be a real increase in sufficient to account for the number of follicles number of follicles, as it was seen in each of the recruited at estrus. At metestrus, an increase in surgical subgroups before pooling of the data. atresia was seen in the group of follicles recruited Previous reports also have found increased into the size >260 pm in diameter (Fig. 3). numbers of small follicles at diestrus in both rats (Lane and Davis, 1939) and mice (Pederson, DISCUSSION 1970a), but did not characterize their distribu- This study confirms previous reports of tion. Such an increase in number of follicles in recruitment of large follicles between proestrus small size classes, where no atresia is detected, and estrus for the next ovulation (Hirshfie!d would suggest a differential rate of entry of and Midgley, 1978a,b; Welschen and Dullaart, follicles into the proliferative pool with stage of 1976; Hoak and Schwartz, 1980; Hirshfield and the estrous cycle. DePaola, 1981), characterizes the distribution The timing of the increase in number of of all follicles in the growth phase and reports small follicles (diestrus) is out of phase with the differences among days of the estrous cycle for increase in number of large follicles (estrus). number of small preantra! follicles. In addition, One possible explanation for this difference differences in patterns of distribution of atretic could be the ability of large follicles to respond follicles were found among days. The present rapidly to FSH due to the presence of more study, by utilizing a larger number of rats, has FSH receptors in large than in small follicles, or been able to define the size class and number of the FSH could synergize with estrogen. Also, at follicles recruited (21 additional follicles into diestrus, the ovarian production of estrogen has the size class 260 pm in diameter). A further increased, which might locally stimulate mitosis selection and clear separation of the preovula- in small follicles. Specific hormones seem to act tory group of follicles occurred by diestrus. at specific stages of development and could Atresia and a rapid decrease in size occurred in produce differences in both time and rate of the recruited follicles that were not selected for growth of follicles of different sizes (Richards, ovulation. While FSH is known to be responsible 1980; Hirshfie!d, 1983c). Still another possibil- for recruitment (Hirshfield and Midg!ey, 1978b), ity is that the periovulatory surge of FSH could FOLLICULAR GROWTH IN RATS 285 recruit follicles from the nonproliferative pool, becoming atretic vary with day of the cycle. which would not be detectable in the growing From the divergence in pattern of atretic pool until diestrus. Past negative results with follicles at metestrus (Fig. 3), one could con- attempts to increase recruitment of follicles clude that there is an increase in rate of atresia into the proliferative pool with FSH could be at metestrus of the larger follicles present at due to either incorrect timing for counting of estrus in the pool 280 pm in diameter. Hirsh- follicles after treatment, or to relatively small field (1983c) reported atresia by metestrus of the largest follicles present at estrus. An increase increases in total number of follicles combined Downloaded from https://academic.oup.com/biolreprod/article/31/2/280/2766498 by guest on 24 September 2021 with few animals and large variation in total in atresia also was found at metestrus in follicles number of follicles among animals. From <120 pm, but a decreased atresia is seen in the preliminary data (Meredith and Butcher, range 200 to 280 pm in diameter. unpublished) an increased depletion of resting The variable rate of growth and atresia oocytes have been found associated with a among days of the cycle and among sizes of prolonged exposure to elevated FSH. follicles make assessment of follicular growth As with previous studies, atresia was not and atresia even more difficult to study. Hirsh- detected in follicles <80 pm in diameter and field (1983b) recently reported a combined rarely was seen in follicles 120 or >400 pm in technique of radioisotope labeling and mitotic diameter. The percent atresia averaged about arrest to determine rate of follicular growth and 50% for follicles 160 to 280 pm. This agrees time of atresia. She found an increase in rate of with previous findings that once follicles enter follicular growth at estrus and of atresia at the proliferative pool, they do not undergo metestrus (Hirshfield, 1983c). Additional studies atresia prior to antral formation (Lane and by techniques such as those of I-Iirshfield Davis 1939). The first indication of antral (1983b) are needed to determine number and formation, as measured in histological sections size of follicles that become atretic at different in the present study, was intercellular spaces at stages of the estrous cycle. Such techniques 70-80 pm, with a definite antrum present in could help determine if each individual follicle follicles of 120-130 pm in diameter. Failure to <260 pm in diameter grows at the same rate at find atresia in very small follicles (<80 pm) a similar size in development and at different before antral formation begins does not elimi- stages of the estrous cycle. nate the possibility but that rapid atresia has occurred and is not detectable in our histologi- ACKNOWLEDGMENTS cal preparation. However, follicles as large as 150 We wish to thank Dr. William V. Thayne and Mr. pm with mature-sized oocytes rarely are found Steven P. Lerner for statistical analysis of the data. in early stages of atresia, and it is infrequent that follicles <200 pm are in early stages of REFERENCES atresia. It seems unlikely that a significant Arai, A. (1920). On cause of the hypertrophy of the number of follicles of this size with a fully surviving ovary after semi-spaying and the num- grown oocyte could undergo atresia without ber of ova in it. Am. J. Anat, 28:59-79. Greenwald, G. 5. (1961). Quantitative study of several from the 70 ovaries being detected in follicular development in the ovary of the intact the early stage of atresia. or unilaterally ovariectomized hamster. J. Reprod. Large numbers of follicles between 120 and Fertil. 2:351-361. 200 pm in the late stage of atresia are present Greenwald, G. S. and Siegel, H. I. (1982). Is the first and appear to be derived from follicles >200 or second preovulatory surge of FSH responsible for follicular recruitment in the hamster? Proc. pm. As large follicles decrease in size during Soc. Exp. liiol. Med. 170:225-230. atresia, they can no longer be recognized at Hirshfield, A. N. (1982). Follicular recruitment in <80 pm, and the frequency of recognition at long-term hemicastrate rats. Biol. Reprod. <120 pm in diameter is decreased (Fig. 3). The 27:48-53. disappearance in 2 days of most follicles Hirshfield, A. N. (1983a). Compensatory ovarian hypertrophy in the long-term hemicastrate rat: recruited into the size group >260 pm and not size distribution of growing and atretic follicles. selected for ovulation attests to the rapid Biol. Reprod. 28:271-278. decrease itt size of atretic follicles. Changes in Hirshfield, A. N. (1983b). Dynamics of follicular the distribution of atretic follicles with stage of atresia in cyclic rats: A combined continuous- labeling and stathmokinetic study. Prog. 65th the estrous cycle also show either that atresia Ann. Meet. Endocrine Soc., Abstr. #873, p. 299. does not proceed at a constant rate through- Hirshfield, A. N. (1983c). Follicular dynamics during out the cycle or that the number of follicles the postovulatory period in cyclic rats. Biol, 286 BUTCHER AND KIRKPATRICK-KELLER Reprod. 28(Suppl. 1):93. changes in the number of medium and large Hirshfield, A. N. and DePaola, L. V. (1981). Effect of follicles in the adult rat ovary. J. Endocrinol, suppression of the surge of follicle-stimulating 8: 341-346. hormone with porcine follicular fluid on follicular Pederson, T. (1970a). Follicle kinetics in the ovary of development in the rat. J. Endocrinol. 88:67-7 1. the cyclic mouse. Acta Endocrinol. 64:304-323. Hirshfield, A. N. and Midgley, A. R., Jr. (1978a). Pederson, T. (1970b). Follicle growth in the mouse Morphometric analysis of follicular development ovary. In: Oogenesis (J. D. Riggers and A. W. in the rat. Biol. Reprod. 19:597-605. Schuetz, eds.). University Park Press, Baltimore, Hirshfield, A. N. and Midgley, A. R,, Jr. (1978b), The MD, pp. 361-376. Peppler, R. D. and Greenwald, G. S. (1970), Influence role of the FSH in the selection of large ovarian Downloaded from https://academic.oup.com/biolreprod/article/31/2/280/2766498 by guest on 24 September 2021 follicles in the rat. Biol, Reprod. 19:606-611. of unilateral ovariectomy on follicular develop- Hoak, D. C. and Schwartz, N, B. (1980). Blockade of ment in cycling rats. Am. J. Anat. 127:9-14. recruitment of ovarian follicles by suppression of Richards, J. S. (1980). Maturation of ovarian follicles: the secondary surge of follicle-stimulating hor- actions and interactions of pituitary and ovarian mone with porcine follicular fluid, Proc. Nail hormones on follicular cell differentiation. Acad. Sci. 77:4953-4956, Physiol. Rev. 60:51-89. Hunter, J. (1787), An experiment to determine the Welschen, R. and Dullaart, J. (1976). Administration effect of exterpating one ovarium upon the of anti-serum against ovine follicle-stimulating number of young produced. Phil. Trans. 77:23 3- hormone or ovine luteinizing hormone at proes- 239. trus in the rat: effect on follicular development Lane, C. E. and Davis, F. R. (1939). The ovary of the during the oncoming cycle. J. Endocrinol. adult rat. I. Changes in growth of the follicle and 70: 301-306. in volume and mitotic activity of the granulosa Zoldak, J. T. and Bowyer, A. F. (1977). Follicle and theca during the estrous cycle. Anat. Rec. distribution following unilateral ovariectomy of 73:429-442. the rat. Prog. 10th Ann. Meet. Soc. Study Reprod., Mandl, A. M. and Zuckerman, S. (1952). Cyclical Abstr. #75, p. 51.