Vol. 24, No.2, February 1973 Printed in U.S.A.
FERTILITY AND STERILITY
Copyright © 1973 by The Williams & Wilkins Co.
FOLLICLE RESPONSE TO EXOGENOUS GONADOTROPINS: AN ESTROGEN-MEDIATED PHENOMENON ROBERT L. GOLDENBERG, EDWARD O. REITER,
GRIFF T. ROSS
Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014
Exogenous gonadotropins in doses which produce dramatic weight increases in ovaries of 21-day-old female rats elicit minimal ovarian weight gain when given to rats younger than 8 days of age. 1, 2 The degree of maturation of the follicle attained at the time the immature rat ovary acquires sensitivity to exogenous gonadotropins has been examined previously, but wide disagreement persists in reports relating to this subject.2-4 To document the earliest stage of follicular development in which responsivity to exogenous gonadotropin is seen, and to investigate the reason for the lack of responsivity prior to this time, we have analyzed the age-related changes in ovarian morphology in rats receiving graded doses of pregnant mare's serum gonadotropin (PMSG). We have observed that the initiation of ovarian response to PMSG occurs between 6 and 8 days of age and follows differentiation and epithelioid transformation of the theca interna. MATERIALS AND METHODS
Animals. Immature female SpragueDawley rats, ranging in age from 2-20 days, were received with mothers in litters of 8 from Holtzman Laboratories. All rats were kept at 25° C. with intervals of 14 hr. light and 10 hr. darkness. PU.rina lab chow and tap water were available ad libitum to the mothers. Hormones. Pregnant mare's serum gonadotropin (PMSG) was obtained commercially and was the only source of gonadotropic hormone used in these experiments. 121
The hormone was dissolved in 0.15 M NaCI solution containing 1% bovine serum albumin (1% BSA), and given as a single subcutaneous injection in the back of the neck. Doses ranging from 1-125 I. U. were given to groups of 8 animals on Days 3,5,7, 10, 14, and 18, and the animals were sacrificed 72 hr. later. As a test of the age-related uterine response to estrogens, 100 J-Lg. of diethylstilbestrol (DES) in an aqueous suspension were given daily for 3 days to groups of 8 animals, beginning on Days 3, 5, 7, and 10. This particular dose was chosen because of its known maximal effect on uterine weight in older rats and apparent lack of toxicity in neonatal rats over the time period used. The effect of DES on uterine weight was compared to that following injection of 25 I. U. of PMSG and 1% BSA. Tissue Preparation. At the end of each experiment, animals were sacrificed and the ovaries and uterus were dissected free of extraneous tissue, weighed, and the ovaries fixed in Bouin's solution, serially sectioned at 5 J-L, and stained with hemotoxylin and eosin. To evaluate follicle morphology, random sweeps of slides were made and the first 100 developing follicles sectioned through the ovum were examined. Cross-sectional diameters (including theca interna) of these 100 follicles were measured using an optical micrometer and an average follicular diameter was calculated. In addition, a maximum follicular diameter, defined as the average measurement of the three
GOLDENBERG ET AL.
largest follicles in the midsagittal section of 10 ovaries, was determined. The extent of theca interna development, interstitial cell proliferation, antrum formation, and luteinization was evaluated and graded on a 0-4+ basis, with 0 representing no antra, no theca interna, minimal interstitia, and no corpora lutea, and 4+ representing stimulation of these features comparable to that seen in 18-day-old rats after 25 LV. ofPMSG.
or a single dose of 25 LV. of PMSG (Table 1). The control animals exhibited a progressive age-related increase in each of the tissue indices evaluated except luteinizaAGE RELATED RESPONSE TO GONADOTROPINS
112 108 18 Days
The ovarian weights of animals aged 3-18 days treated with graded doses of PMSG and sacrificed after 3 days are shown in Fig. 1. There was no ovarian weight increase in response to any of the doses of PMSG in animals tested at 3 days of age. At 5 days, treatment with 25 and 125 LV. of PMSG significantly (p < 0.05) increased ovarian weight. In animals treated at 7, 10, 14, and 18 days of age there was a significant (p < 0.02) dose-related increase in ovarian weight, with responses generally maximal at 25 I.V. Thus, ovarian weight increased in response to exogenous gonadotropins by the 5th-8th postnatal days and showed a marked age-related increase. We have compared the average and maximum follicular diameters, and the extent of theca interna development, interstitial cell growth, antrum formation, and corpus luteum formation in ovaries of 3- to 18-day-old rats treated with 1% BSA
E 48 II
36 I I
24 20 /
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----f----... 't = t -----.--'I
5 Days 3 Days
FIG. 1. Ovarian weights ±S.E.M. in rats given a single injection of 0, 1, 5, 25, or 125 LU. of PMSG at the age shown and sacrificed 72 hr. later.
TABLE 1. Follicle Diameters and Ovarian Morphology in Neonatal Rats' Average follicular diameter
Maximum follicular diameter
Days Control 3-6 5-8 7-10 10-13 14-17 18-21
63.4 81.8 91.4 108.3 136.4 172.4
± ± ± ± ± ±
2.0 3.4 3.1 3.8 8_5 10.5
PMSG 65.7 123.4 150.4 179_6 314.0 500.2
± ± ± ± ± ±
2_8 80.4 ± 4_1 117.5 ± 4_7 147.1 ± 5_1 161.9 ± 22.5 269.6 ± 44.3 372.7 ±
ConControl PMSG trol PMSG
0 2.8 82.7 ± 2.8 157.9 ± 6.3 3.7 + 3.8 201.4 ± 8.3 + 5.3 241.7 ± 9.1 + 10.3 443.6 ± 22.3 ++ 7.9 1052.5 ± 48.7 ++
0 0 0 + 0 ++ + 0 +++ + ++ 0 +++ + +++ ++++ ++ ++++ + ++++ ++ ++++ ++ -
+ + ++ +++ ++++
Con- PMSG trol 0 0 0 0 0 0
0 0 0
+ ++ ++++
• First number is age at treatment; second number is age at sacrifice. Mean ± S.E.M. of' follicle diamters (see text). Graded on basis ofOto ++++ (see text). One percent BSA, 0.5 ml. on the day of treatment. PMSG, 25 LU. on the day of treatment.
FOLLICLE GROWTH IN NEONATAL RATS
tion, which is not normally seen in untreated immature animals. Of special interest was the appearance of theca, distinct from surrounding stroma, after 6 days, followed by the beginning of epithelioid transformation of theca interna cells not earlier than 3-4 days later (Fig. 2). PMSG-treated animals generally showed a more advanced state of ovarian development than control animals of the same age. However, animals treated at 3 days and sacrificed on Day 6 were indistinguishable from controls, except for a slight increase in interstitial cell proliferation. By Day 8, rats treated on Day 5 with PMSG had a substantial number of epithelioid theca cells, and in addition had larger follicular diameters, earlier onset of antrum formation, and a greater degree of interstitial cell growth than 8-day-old untreated controls. PMSG treatment of older animals caused even a greater degree of maturation of the parameters evaluated. An epithelioid theca layer can therefore be
found between 5 and 8 days of age, simultaneous with the first significant ovarian weight response to exogenous gonadotropins. Figure 3 presents the uterine weights from animals treated with either 1% BSA saline, 25 I. U. of PMSG, or 0.1 mg. of DES daily and sacrificed 72 hr. later. DES substantially increased uterine weights over control in every age group examined, but PMSG had no effect prior to 6 days of age. DISCUSSION
We have analyzed age-related responses of ovarian weight and morphology to exogenous gonadotropins in immature rats. PMSG, even in massive doses, had no effect on ovarian weight or follicle development before 6 days of age. Prior to this time, the most advanced follicle in both control and treated rats had no more than three layers of granulosa cells, no visible theca interna, and no antra. Two days
FIG. 2. Theca interna development (denoted by arrow) in 6- (A) and 10- (B) day-old untreated rats.
124 IMMATURE RATS
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GOLDENBERG ET AL.
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FIG. 3. Uterine weights ±S.E.M. in rats given either a single injection of 25 I.U. of PMSG and sacrificed 72 hr. later on the day indicated, or DES 100 t'g. q.d. for 3 days,. or 1% BSA q.d. for 3 days, and sacnficed on the day mdicated.
later, at 8 days of age, ovaries from control animals showed a small increase in follicular diameter accompanied by the development of a theca layer distinct from the surrounding stroma. However, ovaries from animals treated with PMSG on Day 5 and examined on Day 8 had a significant increase in ovarian weight, accompanied by an increase in follicular diameter, epithelioid differentiation of the theca interna, antrum formation, and corpus luteum development. The onset of ovarian responsivity to exogenous gonadotropins in the neonatal Holtzman rat, therefore, occurs between the 5th and 8th day of
age, and is coincidental with the epithelioid differentiation of the theca interna .. Previous experiments performed in our laboratory have demonstrated a role for estrogen in stimulating growth of the follicle in both immature and hypophysectomized rats. 5 - 7 Data reported here show that the amount of estrogen secreted in response to PMSG prior to 6 days of age is inadequate to increase uterine weight. The theca interna is believed to be a major site of estrogen synthesis, 8- 11 and in fact, only after the development of an epithelioid theca interna, when the ovary produces sufficient estrogen in response to PMSG to increase uterine weight, does ovarian weight increase in response to PMSG. We suggest, therefore, that the failure of exogenous gonadotropins to stimulate follicle growth prior to 6 days of age may be related to the inability of an undifferentiated theca to secrete adequate quantities of estrogen. Once the theca interna becomes responsive to exogenous gonadotropins and secretes estrogen, granulosa cell proliferation is stimulated, and antrum formation and finally luteinization occur in response to the FSH-like and LH-like activity of PMSG. SUMMARY
Doses of exogenous gonadotropins which markedly increase ovarian weight in 21day-old rats elicit minimal weight gain when given to neonatal animals. We have analyzed the age-related changes in ovarian morphology and found that the initial response, seen at 6-8 days of age, is preceded by differentiation of the theca interna. Coupled with previous observations, these results suggest that the onset of ovarian responsiveness to exogenous gonadotropins is mediated by estrogen produced by the theca interna. Acknowledgments. We wish to express our gratitude to Helen Chambers and Glynss Sherman for assistance in manuscript preparation.
FOLLICLE GROWTH IN NEONATAL RATS
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ment in the ovarian augmentation reaction. Endocrinology. In press. REITER, E. 0., GOLDENBERG, R. L., VAITUKAITIS, J. L., AND Ross, G. T. Evidence for a role of estrogen in the ovarian augmentation reaction. Endocrinology. In press. MOSSMAN, H. W. The thecal gland and its relation to the reproductive cycle. Amer J Anat 61:289, 1937. CORNER, G. W. The sites of formation of estrogenic substances in the animal body. Physiol Rev 18:154, 1938. RYAN, K. J., AND PETRO, Z. Steroid biosynthesis by human ovarian granulosa and theca cells. J Clin Endocr 26:46, 1966. RYAN, K. J., PETRO, Z., AND KAISER, J. Steroid formation by isolated and recombined ovarian granulosa and theca cells. J Clin Endocr 28:355, 1968.