THE INVESTIGATION reported in this article was devoted to an analysis of

..., Polyovular Follicles in the Immature Hamster Ovary II. The Effects of Gonadotropic Hormones on Polyovular Follicles CHARLES W. BODEMER, ph.d., AND STELLA WARNICK, B.A... THE INVESTIGATION reported in this article was devoted to an analysis of the influence of gonadotropic honnones on polyovular follicles in the immature hamster ovary. Many uncertainties exist regarding the mode of origin and developmental significance of polyovular follicles, particularly in the ovaries of fetuses and young mammals. The fate and potentialities of polyovular follicles have not been adequately documented. Thus, some authors consider them as "abnonnal" structures, destined to atresia/' 8 whereas others think of them as capable of ovulation and as a mechanism for multiovular twinning.s. 4, 9 An earlier paper described polyovular follicles in the ovaries of intact immature hamsters.1 From this study emerged the observation that a certain percentage of polyovular follicles develop antra and appear to progress through a nonnal course of maturation. Further study suggested that polyovular follicles may be capable of nonnal development and ovulation. The general lack of information regarding the potentialities of these follicles and the debate attendant upon the question of their ovulation warrants exploration of the behavior of multiple follicles under conditions favoring precocious growth and ovulation. It is known that follicular growth and ovulation may be induced from 6 to 10 days prior to the first spontaneous ovulation in the hamster by injections of gonadotropic honnones. 2 The response of the ovaries From the Department of Anatomy, University of Washington, Seattle, Wash. The study reported here was supported by funds from Research Grants 5820 and 4241, U. S. Public Health Service, Department of Health, Education, and Welfare. 353

... 354 BODEMER & WARNICK FERTILITY & STERILITY to these hormones offers a means for ascertaining the growth and ovulatory capabilities of polyovular follicles. MATERIALS AND METHODS Sixty-two ovaries removed from 31 immature golden hamsters ranging in postpartum age from 21 to 33 days were studied. The animals were obtained from several colonies in order to minimize possible effects of strain differences. Only hamsters for which there were accurate birth records were utilized. While in our laboratory, the hamsters were fed Purina Fox Chow* with a greens supplement at least once a week; water was supplied ad libitum. In order to study the effects of gonadotropic hormones, female hamsters were injected at ages of 21, 22, 24, 25, 26, 27, 29, 31, and 33 days. Single subcutaneous injections of pregnant mare's serum t (PMS) at concentrations of 10 or 20 I. U. were followed 54 hours later by a single subcutaneous injection of chorionic gonadotropint (ICSH). All injections were made into the nape of the neck with a No. 27 hypodermic needle, and care was exercised to prevent loss of material. Within 18 hours after the last injection, the animal was killed by decapitation, and the ovaries removed, trimmed free of tissue, and immersed in Bouin's fluid. The ovaries were embedded in paraffin and sectioned longitudinally at 7 fl. The mounted sections were stained with hematoxylin and eosin or according to Bodian's protargol method. Direct counts were made of the uniovular and polyovular follicles at a magnification of 44 times. Records were kept of the total number of ovarian follicles, antral follicles of either type, and corpora lutea. Any ovum surrounded by at least one layer of cuboidal epithelial cells was counted; any follicle possessing a clearly defined antral cavity regardless of its size, was designated as an antral follicle. OBSERVATIONS Antral Follicles in the Untreated Animal No antral uniovular follicles were observed in the ovary of the intact immature hamster before Postpartum Day 24. Between Days 24 and 33, however, the proportion of antral uniovular follicles to the total number of follicles in the ovary increased from 0.05 to 5.3 per cent (Fig. 1). The first * Ralston Purina Company, Seattle, Wash. t Equinex, Ayerst Laboratories, New York. t "A.P.L.," Ayerst Laboratories, New York.

VOL. 12, No.4, 1961 POLYOVULAR FOLLICLES II 355 spontaneous ovulation in the hamsters of our colony occurred between Day 33 and Day 36 postpartum; the age-related increase in incidence of antral uniovular follicles apparently reflects progressive prepubertal maturation of the ovary. The number of polyovular follicles that develop antra in the intact immature hamster ovary is small by comparison with the number of uniovular 12 II).l!! IO -S! 8.!:! 6 13 4 2 14 21 25 26 27 29 31 33 Age -days t 12... IO 8 C) C) 6...... li4 2-0-----:... ---------0--------- 21 22 24 25 26 27 29 31 33 Age-days Key: triangles, solid line: untreated animal; open circles, dotted line: 10 LV. of PMS, 20 LV. of ICSH; closed circles, solid line: 20 LV. of PMS, 20 LV. of ICSH. Fig. 1. Percentage of antral follicles relative to total number of ovarian follicles. Note that in this and Fig. 2 the normal represents counts made on the day specified; in the case of animals treated with gonadotropins, the response is aligned with the day on which the first injection was given.

... BODE MER & WARNICK FERTILITY & STERILITY follicles. No antral polyovular follicles were observed before Day 24. On this day, 0.03 per cent of the total number of ovarian follicles was comprised of antral polyovular follicles. During succeeding days, the relative proportion of these follicles did not vary greatly, remaining consistently below 0.5 per cent. Antral polyovular follicles were absent on Day 33 (Fig. 1). The proportion of antral polyovular follicles relative to the total number of follicles does not, then, increase gradually with progressing age as does the proportion of uniovular follicles. When, however, the number of antral polyovular follicles relative to the total number of polyovular follicles only is considered, the incidence of antral polyovular follicles does conform to the general trend established by uniovular follicles (Fig. 2). It should be emphasized, however, that regardless of how they are estimated, the number of polyovular follicles with antra in the normal intact hamster is never large. ". Antral Follicles in Treated Animals Single consecutive injections of PMS and ICSH significantly affected the incidence of antral uniovular follicles in the immature hamster ovary. Antral uniovular follicles were observed in ovaries removed from animals injected as early as Day 22 with either 10 or 20 LV. of PMS in combination with a subsequent injection of 20 LV. of ICSH. The number and relative proportion of antral uniovular follicles increased gradually according to increasing postpartum age. Thus, in animals injected with 10 LV. of PMS on Day 24, 0.92 per cent of the total number of ovarian follicles were antral uniovular "', follicles, and in those injected on Day 31, 4.64 per cent of the total number of follicles were antral uniovular follicles. In addition to the effects of postpartum age, there is some correlation between the PMS dosage and the percentage of antral uniovular follicles. The ovaries of animals receiving 20 LV. of PMS on Days 24 and 31 contained antral uniovular follicles amounting to 3.31 per cent and 6.6 per cent, respectively, of the total follicle population (Fig. 1). It may be noted that from Day 22 onward, an increasing percentage of injected animals ovulate; all treated animals 27 days old or older ovulate. 2 The percentages of antral uniovular follicles recorded here, then, are somewhat lower than they would be in the absence of ovulation. Hamsters injected with 20 LV. of PMS on Day 31 ovulate an average of 21 ova; including these with the average number of counted follicles raises the incidence of antral uniovular follicles relative to the total number of ovarian follicles from 6.6 to 10.6 per cent. The increasing effectiveness of the higher dosages of gonadotropins in the induction of ovulation in the older prepubertal animals may explain the reduction in relative numbers of antral uniovular follicles after Day 29 (Fig. 1 and 2).

VOL. 12, No.4, 1961 POLYOVULAR FOLLICLES II 357 [ncorporating, as in the preceding example, the average number of ova ovulated with the number of antral uniovular follicles relative to the total number of follicles, the relative numbers of antral follicles are as follows: 29 days, 16 per cent; 31 days, lo.6 per cent; 33 days, 12 per cent. It is clear that polyovular follicles, like uniovular follicles, respond to the influences of injected gonadotropins. The size of the polyovular-follicle population appeared to remain unaltered following single consecutive injec-.... 30.<'> 25 lo. 20 S!. 15... 10 5 0. ---, --'[:-:-:-=-==-=-:; 0'" -0----" " 21 22 24 25 26 27 Age-days 29 31 33,".. 35 30 15..-:: 25 1; 20 I:) 15 15 5 21 22 24 25 26 27 Age-days 29 31 33 Key: triangles, solid line: untreated animal; open circles, dotted line: 10 I.U. of PMS, 20 I.U. of ICSH; closed circles, solid line: 20 I.U. of PMS, 20 I.U. of ICSH. Fig. 2. Percentage of antral follicles relative to other follicles of same type.

358 BODEMER & WARNICK FERTILITY & STERILITY tions of PMS and ICSH. The number of antral polyovular follicles, however, increased significantly in hamsters injected with gonadotropic hormones. Polyovular follicles with antra do not appear consistently in animals injected before Day 24, although they may occasionally occur earlier (Fig. 1). There is a relation between age and the incidence of antral polyovular follicles. The percentage of the total number of follicles formed by antral polyovular follicles increased from 0.09 per cent (in animals given lo 1 U. of PMS) or 0.42 per cent (those given 20 I. U. of PMS) in animals injected on Day 24 and afterward. These follicles reached their maximal number between Days 27 and 29, when they formed 1.39 per cent (10 1 U. of PMS) or 2.6 per cent (20 1 U. of PMS) of the total follicle population. Their incidence decreased thereafter, and in hamsters injected on Day 33, only 0.29 per cent (lo lu. of PMS) or 0.23 per cent (20 lu. of PMS) were antral polyovular follicles. Although antral polyovular follicles represent a small fraction of the total number of ovarian follicles, it is of interest that a rather large proportion of these follicles develop antra following administration of gonadotropic hormones. As compared with the uniovular follicles, a much larger percentage of polyovular follicles develop antra under the influence of the gonadotropins. Almost 40 per cent of the polyovular follicles developed antra in hamsters receiving 20 1 U. of PMS on Day 29. The number of antral polyovular follicles was correlated with the dosage of PMS administered (Fig. 2). Polyovular follicles, then, although substantially fewer in number than the uniovular follicles, display a comparable reaction to the influence of gonadotropic hormones. The commonest antral polyovular follicles were those containing two ova. These are most commonly found in the early prepubertal stages. On Day 27 and later, antral follicles containing ova were observed. Biovular follicles are, however, the predominant polyovular type in the hamster.1 Corpora Lutea and Antral Follicles In the untreated intact immature hamsters studied, no corpora lutea were observed in ovaries removed from animals ranging in age from 22 to 33 days. In animals that had received injections of gonadotropic hormones, however, corpora lutea were found in all groups, beginning on Day 22. The average number of corpora lutea per ovary increased with increasing postpartum age and with increased PMS dosage (Fig. 3). Figure 4 illustrates the relation between the number of corpora lutea and the total number of antral follicles according to dosage of administered PMS and postpartum age. It may be seen that at a dose of 10 I.U. of PMS (upper graph), the number of antral follicles increased gradually, reaching its peak of about 6 per cent on

VOL. 12, No.4, 1961 POLYOVULAR FOLLICLES II S59 Days 27-29 and thereafter declining to approximately 3 per cent on Day 33. The number of corpora lutea, on the other hand, remained relatively constant from Day 24 to Day 29, then increased abruptly to about 5 per cent on Day 33., 24 22 20 18 t:j 16.!!? 14 12 e. 10 8 6 4 2 21 22 0.. - 2-----0---------... ----- o 0_----0-""------- 24 25 26 27 29 Age - days...... /0 ",...,..0"".--' Key: open circles, dotted line: 10 I.U. of PMS, 20 I.U. of ICSH; closed circles, solid line: 20 I.U. of PMS, 20 I.U. of ICSH. Fig. 3. Number of corpora lutea per ovary at various dosages of PMS. No corpora lutea were observed in the untreated animal. 31 33 The effects of 20 J.U. of PMS are illustrated in the lower graph of Fig. 4. The percentage of antral follicles is higher throughout, although the general pattern of gradual increase and decline remains clear. By contrast with the relatively constant number of corpora lutea in animals receiving 10 J.U. of PMS, corpora lutea are more numerous between Days 24 and 29 in animals given 20 J.U. of PMS. These data indicate that both postpartum age and dosage of administered PMS significantly influence the number of antral follicles formed and the number of ovulations or corpora lutea that may be found. These observations reinforce the earlier statement that the apparent reason for the decline in antral follicles during the later prepubertal period is the increasing number of ovulations. DISCUSSION Polyovular follicles have been identified in the ovaries of many mammals. 7 There is general agreement among the various investigators who have stud-

360 BODEMER & WARNICK FERTILITY & STERILITY ied multiple follicles that they most commonly occur in fetuses and young animals. 4-6, 10 There is, by contrast, no consensus regarding their origin, significance, or potentialities. Hartman states, with particular reference to the opossum, that the high incidence of polyovular follicles in this marsupial may be correlated with its fecundity, although he hesitates to designate it as a direct causal relation. Arguing that "there are good reasons for ascribing 14 'Yo- 12% e- o 10%... 8%.l!!. 6%... 4% 2% 11 - --cr'.,,'"./ /....,,<>"...-- 14% <::i :!! 12% IO% a "b 8% II).l!! 6%... 1) 4% 2% 21 22 21 22 24 25 26 27 29 Age -days o,,-..-" ---0-----;,- 31 o --- -..--..-- -- -'" o I 24 25 26 27 29 31 Age-days ---- Fig. 4. Total number of antral follicles (solid line) and corpora lutea (dotted line) relative to the total number of ovarian follicles. Above: 10 LV. of PMS; below: 20 LV. of PMS. No corpora lutea were observed in normal animals. 33 I 33 <.

VOL. 12, No.4, 1961 POLYOVULAH FOLLICLES 11 361 not more, but rather less, viability to the atypical follicles as compared with normal structures," Hartman considers it certain that most polyovular follicles degenerate. Lane reached a similar conclusion from his study of the immature rat. Engle concluded that in the mouse follicles containing ova of unequal size, the small ovum degenerates and only one viable ovum results. Dawson stated that polyovular follicles with ova of equal size are most likely to survive but did not mention their ability to ovulate. The possibility that polyovular follicles may ovulate has been suggested, albeit not demonstrated, by various authors. O'Donoghue recorded the ovulation of a ripe biovular follicle in the opossum. Lillie compared the number of ova recovered from the genital tract in cattle with the number of corpora lute a and in 126 recorded counts noted only 1 instance in which twin calves were found in the uterus with only 1 corpus luteum in the ovary. Corner thought that in the pig the rare cases of embryos in excess of the corpora lute a might be explained by polyovular follicles; he did not omit single-egg twinning as a source of the discrepancy. Davis and Hall found that in 21 per cent of the wild Norway rats they studied, there were 1-10 embryos in excess of the corpora lutea. Dissection of embryos from their membranes in all cases excluded for these authors the possibility of single-ovum twinning. They considered it "a more likely explanation... [that] some follicles release more than one viable ovum." Lane noted that the incidence of polyovular follicles in the immature rat was not materially altered by injections of various hormone preparations (Amniotin, F.S.H., L.H.). This brief statement is, to our knowledge, the only comment in the literature regarding the effect of hormones upon polyovular follicles. The investigation described in this article reveals that polyovular follicles in the ovary of the immature hamster are quite responsive to the effects of gonadotropic hormones injected in dosages sufficient to induce ovulation. It may be noted that our quantitative data substantiate Lane's remark that the incidence of multiple follicles is essentially unaffected by these hormones. The phenomenon of immediate interest in our study, however, is the effect of gonadotropins upon the incidence of antral polyovular follicles. The maximal concentration of antral polyovular follicles in the untreated hamster during the prepubertal period is 0.5 per cent of the total number of ovarian follicles and 4.7 per cent of the total number of polyovular follicles; in gonadotropin-treated animals, the maximal density of antral polyovular follicles may amount to 2.8 per cent of the total number of follicles and 37.8 per cent of the total number of polyovular follicles. The size of the polyovular-follicle population may, indeed, remain unaffected by gonadotropic hormones; the follicles comprising that population, however, are dramatically reactive to the influences of these hormones.

362 BODEMER & WARNICK FERTILITY & STERILITY Under the same experimental conditions, the polyovular follicles behave in a manner thoroughly comparable with the preponderant uniovular follicles. The percentage of both antral uniovular and antral polyovular follicles increases with increasing postpartum age and varies directly with the dosage of administered PMS. Antral follicles of both types are present in the greatest relative density in animals injected with PMS on Days 27-29. The relative number of antral follicles declines between Days 29 and 33. The reason for the decreased number of antral uniovular follicles probably is related to the increased number of ovulations induced by the gonadotropins as the animals approach puberty (Days 33-36). This may not explain the decline in the antral-polyovular-follicle population. In our initial study, it was demonstrated that the percentage of polyovular follicles increased from about 7 per cent on Day 21 to approximately 15 per cent on Day 27 and thereafter declined to approximately 6 per cent on Day 33. The percentage of antral polyovular follicles in the untreated hamster generally revealed a comparable pattern; however, antral polyovular follicles were absent from the ovary on Day 33. Approximately 12 per cent of the polyovular follicles in animals receiving 20 I.U. of PMS on Day 33 developed antra; these follicles, however, accounted for a mere 0.23 per cent of the total number of ovarian follicles. The decreased incidence of antral polyovular follicles in animals receiving gonadotropins after Day 29 may well be correlated with the overall reduction of the polyovular-follicle population during this period. The number of multiple follicles that develop antra, however, suggests that ovulation may not be completely unrelated to the decline in their incidence. We have no direct evidence regarding actual ovulation of polyovular follicles in immature hamsters. Our results indicate that these follicles are not "abnormal" by virtue of refractoriness to gonadotropic hormones. Polyovular follicles seem to differ little from typical ovarian follicles in their response to hormones that elicit follicular growth and maturation. The physiologic substrate upon which the gonadotropins exert their influence, then, appears to be comparable in follicles of both types. Follicular growth and formation of a large antrum does not necessarily lead to ovulation of a follicle, and complete follicular development with ovulation does not mean that any or all of the ova ovulate and are viable. It is quite conceivable that supernumerary ova embedded in the walls of a multiple follicle may, even if the follicle ruptures, never emerge from the collapsed follicle. Similarly, ova surmounting separate cumuli oophorus may be of unequal viability and/or may degenerate in the oviduct. It should also be remarked that the follicular response to administered gonadotropins may not necessarily occur under the influence of these hormones at the titer characteristic of the prepubertal period, although the presence of antral (,,

VOL. 12, No.4, 1961 POLYOVULAR FOLLICLES II 363 polyovular follicles in the untreated hamster does not lend support to this argument. There are, then, good reasons for withholding a definite conclusion concerning the ovulation capacity of polyovular follicles. The evidence can be interpreted only to indicate that there is no basis for considering polyovular follicles in the immature hamster ovary as physiologically inert oddities. The response of these follicles to gonadotropins suggests rather that they are morphologic, not physiologic, variants. Although their relative scarcity would appear to preclude polyovular follicles as a major source of viable ova in the adult, it is conceivable that they may sometimes ovulate. This possibility warrants further experimentation..> SUMMARY Intact immature hamsters ranging in postpartum age from 21 to 33 days were given single consecutive injections of PMS and ICSH in dosages and manner sufficient to induce ovulation. The effects of this regimen on the large population of polyovular follicles in the ovary of the immatur hamster was studied. The number of polyovular follicles was not significantly altered by administration of gonadotropic hormones. The number of polyovular follicles that develop antra was, however, profoundly influenced by these hormones. The number of antral polyovular follicles varied directly with postpartum age and dosage of administered PMS. The comparable response of polyovular and uniovular follicles to the gonadotropic hormones suggests that polyovular follicles represent morphologic, not physiologic, variants, and that they may be capable of ovulation. University of Washington, Seattle 5, Wash. REFERENCES 1. BODEMER, C. W., and WARNICK, S. Polyovular follicles in the immature hamster ovary. 1. Polyovular follicles in the normal intact animal. Fertil. & Steril. 12:159, 1961. 2. BODEMER, C. W., RUMERY, R. E., and BLANDAU, R. J. Studies on induced ovulations in the intact immature hamster. Fertil. & Steril. 10:350, 1959. 3. CORNER, C. W. The problem of embryonic pathology in mammals, with observations upon intra-uterine mortality in the pig. Am. J. Anat. 31 :523, 1923. 4. DAVIS, D. E., and HALL, O. Polyovuly and anovular follicles in the wild Norway rat. Anat. Rec. 107:187, 1950. 5. DAWSON, A. B. Histogenetic interrelationship of oocytes and follicle cell. Anat. Rec. 110: 181, 1951. 6. ENGLE, E. T. Polyovular follicles and polynuclear ova in the mouse. Antflt. Rec. 35:341, 1927. 7. HARTMAN, C. C. Polynuclear ova and polyovular follicles in the opossum and

364 BODEMER & WARNICK FERTILITY & STERIL1TY other mammals with special reference to the problem of fecundity. Am.]. Anat. 37:1, 1926. 8. LANE, C. E. Aberrant ovarian follicles in the immature rat. Anat. Rec. 71 :243, 1938. 9. LILLIE, F. R. Supplementary notes on twins in cattle. Biol. Bull. 44:47, 1923. 10. NUNES, P. J. Les follicules poly-ovulaires chez la lapine. Compo Rend. Soc. Biol. 120:242, 1926. 11. O'DONOGHUE, C. H. The corpus luteum in the non-pregnant Dasyurus and polyovular follicles in DasyuTUs. Anat. Anz. 41 :353, 1912. Annual Convention of the Pacific Coast Fertility Society The Tenth Annual Convention of the Pacific Coast Fertility Society will be held at the EI Mirador Hotel, Palm Springs, Calif., Nov. 9-12, 1961. Inquiries should be addressed to the Secretary, Gregory Smith, M.D., 909 Hyde Street, San Francisco, Calif.