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a Department of Molecular and Integrative Physiology and Ralph Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7401
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In recent years, we have developed a technique to dissociate enzymatically intact preantral follicles from the ovaries of various species and then to determine in vitro steroidogenesis in response to gonadotropins (hamster [9]; human [10]; mouse [11]). The most significant finding is that even preantral follicles of these species respond to gonadotropins. We have now extended these studies to the rat, using as a model prepubertal rats injected with eCG. The objective was to assess the in vitro effects of gonadotropins on steroidogenesis by preantral and antral follicles. As a prelude to the incubation studies, it was thought necessary to determine the temporal changes in FSH and hCG receptors in the ovary, using autoradiography as the endpoint.
Changes in follicular development, serum hormone levels, and other parameters have been studied in the untreated and eCG-treated prepubertal rat. In the untreated rat, serum FSH shows an initial peak between 10 and 20 days and then drops precipitously to baseline values by Day 25. Serum LH sharply increases at Day 15 and then falls and subsequently increases at Day 35 [12]. Another study revealed that at Day 24, large antral follicles constituted 38% of the total follicular population, at a time when serum LH and FSH are at basal levels [13]. It is not until Day 32 that uterine changes indicative of estrogen activity are evident. By Day 21 the granulosa of some antral follicles bind 125I-labeled FSH, whereas it is not until Day 33 that granulosa cells bind 125I-labeled LH [14].
In untreated rats, on Day 25 in small follicles (350–500 µm in diameter), FSH receptor mRNA is localized in granulosa cells whereas LH receptor mRNA is limited to theca and interstitium [15]. After injection of 10 IU eCG on Day 23, by Day 25 ovarian LH receptor mRNA increases nearly 5-fold, whereas FSH receptor mRNA is doubled compared to control values [15]. Ovarian FSH receptor content most rapidly increases between Days 4 and 16 in the untreated prepubertal rat, correlating with maximal serum levels of FSH; the greatest rate of increase of LH receptor occurs between Days 16 and 28, presumably in response to the previous peak of serum FSH [16].
An autoradiographic study [17] considered LH and FSH receptors in large antral follicles throughout the rat estrous cycle and showed that hCG binding to granulosa cells was evident only in preovulatory follicles at proestrus. An earlier study involved topical autoradiography of radioiodinated gonadotropins to serial frozen sections of diestrous rats and revealed localization of 125I-FSH in granulosa of even preantral follicles [18].
With a few exceptions [14, 19], studies employing autoradiography have emphasized the use of the adult rat, focusing on large follicles [17, 18, 20]. To our knowledge, autoradiography has not been used to analyze follicular FSH and LH receptors in the eCG-treated prepubertal rat, especially concentrating on temporal changes in the smallest preantral follicles as well as the interstitium. This was the objective of the present study, which is a prelude to investigating how eCG treatment affects in vitro follicular steroidogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature female Sprague-Dawley rats (23 days of age) were obtained from Sasco Co. (Omaha, NE). The animals were housed under controlled conditions of temperature, humidity, and lighting (14L:10D; lights-on at 0500 h).
Experimental Protocol
At 0900–1000 h on Day 25, a group of 4 animals were killed as a control group; on either Day 23 or 24, two other groups (n = 4 per group) received a single s.c. injection of 5 IU eCG (Sigma Chemical Co., St. Louis, MO) in 0.1 ml of saline. The animals were decapitated on Day 25, i.e., 48 or 24 h post-eCG, respectively. Ovaries were removed and cleaned. One ovary was fixed in Bouin's solution for serial sectioning at 10 µm and staining with hematoxylin and eosin. The other ovary was quickly frozen with freon and stored at -70°C until prepared for topical autoradiography.
Hormone Preparation for Autoradiography
All hormones were provided by the National Hormone and Pituitary Program (NIDDK, NIH, Bethesda, MD). Rat FSH I-8 and hCG CR-27 were used for iodination. These are highly purified reagents specifically prepared for iodination (rat FSH I-8: 4714 IU/mg or 100-strength NIH-FSH-S1; CR-127: 14 900 IU/mg). Bovine FSH B-1 and ovine LH 25 were used as unlabeled hormones to determine nonspecific binding of the tracers.
Topical Autoradiography
The binding procedure was performed according to previously described methods [21] with minor modifications. In brief, 12-µm frozen sections were prepared. The sections were thawed and incubated with labeled FSH (125I-FSH; 10 000 cpm) or hCG (125I-hCG; 10 000 cpm) in 0.01 M PBS, pH 7.0, containing 1% BSA (fraction V; 96–99% albumin; Sigma) alone or with more than 1000-fold excess of unlabeled hormone for 90 min at 37°C. After incubation, the sections were washed with cold PBS. The sections were fixed for 10 min in 3% glutaraldehyde-paraformaldehyde on ice, washed with cold PBS, and dried in air. The sections were then coated with liquid emulsion (NTB-2; Eastman Kodak, Rochester, NY), exposed for 10 days at 4°C, developed in Dektol (Kodak), and stained with hematoxylin. Specific activities were 63 µCi/µg for FSH and 37 µCi/µg for hCG. We calculated that 80 pg and 136 pg of labeled FSH and hCG, respectively, were added to the sections; these amounts were sufficient to saturate the ovarian tissue. The binding characteristics of the tracers used in this study were previously evaluated [22]. Representative sections were photographed with either light- or darkfield optics.
To gain some insight into the rate of growth of the largest and smallest follicles after eCG administration, the hCG autoradiographs were utilized. The first 10 antral follicles encountered were measured at a magnification of x60 in each of 4 ovaries for Day 25 (controls) and 24 and 48 h post-eCG. For the largest section of the follicle, two diameters at right angles to each other were measured and averaged.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Control Ovary on Day 25
In slides stained with hematoxylin and eosin, small preantral follicles with 1 layer of granulosa cells (GCs) showed no trace of theca cells. However, preantral follicles with 2 or more layers of GCs were usually surrounded by spindle-shaped theca cells, 2 layers thick in the smaller follicles and 3–4 layers in large preantral follicles (Fig. 1A). Even in preantral follicles with 3–4 layers of GCs, small isolated pockets of fluid (presumably precursors of the antral cavity) were already noticeable (Fig. 1A). In all preantral follicles, the theca cells were spindle-shaped with an apparent nonsecretory appearance in contrast to the epithelioid theca in antral follicles. However, in large atretic preantral follicles (7–8 GC layers), the theca was hypertrophied and epithelioid in nature. The interstitium consisted of lobulated masses of hypertrophied theca cells surrounding the oocyte; or, ultimately, only the remnants of the zona pellucida, the most resistant portion of the oocyte, were present (Fig. 1A).
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No effort was made to quantitate atresia, but it occurred in both preantral and antral follicles. Large antral follicles were located at the periphery of the ovary, surrounded by 3–4 layers of epithelioid theca cells. Signs of early atresia were present in some of the large antral follicles in the form of crumbling GCs bordering on the antral cavity.
In all cases, excess unlabeled homologous hormone displaced labeled hormone in the autoradiographs. Binding sites for labeled FSH were found on even the smallest preantral follicles with 1–2 layers of GCs, but not in the surrounding interstitial cells (Fig. 1B). The intensity of grains increased according to the stage of follicular development, with a greater reaction in preantral follicles than in large antral follicles (Fig. 1B). However, in some large preantral and small antral follicles, the concentration of FSH receptors was less than in others; and in clearly atretic follicles, the intensity of binding sites in GCs was still further decreased.
On Day 25, hCG receptors in the prepubertal ovary were especially numerous in the interstitium, but in smaller preantral follicles the difference between theca and interstitium was not evident (Fig. 1, C and D); hCG binding was never found on GCs. In large antral follicles, hCG receptors were never found in GCs, but two patterns were observed in theca: in one type, hCG binding sites were found in adjacent interstitial cells but not in the theca, whereas in others, hCG receptors were present in both theca and interstitium.
The largest antral follicles before injection of eCG measured 405 ± 8 µm in diameter (mean ± SEM), and of 40 follicles evaluated, none showed hCG binding to GCs.
Ovary at 24 h After eCG
Striking morphological changes were evident in hematoxylin- and eosin-stained slides: namely, the development of an epithelioid theca around preantral follicles with 2 or more layers of GCs (Fig. 2A). However, follicles with 1 layer of GCs (primary follicles) were unaffected and were still not surrounded by theca cells. Precocious development of an antral cavity was apparent in small follicles with 3 layers of GCs in the form of beginning accumulation of fluid between cells. Mitosis of GCs was greater than that at Day 25. One of the more striking features following eCG treatment was the epithelioid transformation of the edematous interstitium (Fig. 2A).
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Specific binding for FSH was no more intense in the GCs of preantral and small antral follicles than in the controls but was much greater in the largest antral follicles compared to that on the previous day (Fig. 2B).
Specific binding sites for hCG were present in theca and interstitium, but the reaction was not as intense in the latter tissue as in controls (Fig. 2, C and D) . On the small preantral follicles, positive hCG receptors were localized on the theca of follicles with 2–3 layers of GCs but, as anticipated, there was no binding to GCs. In large antral follicles, theca binding was greater than in the controls (Fig. 2, C and D). The largest antral follicles at 24 h post-eCG averaged 459 ± 23 µm; the largest follicle was 637 µm in diameter. Of 40 antral follicles evaluated, only 4 showed hCG receptors in the peripheral GCs, and they were significantly larger than the other follicles (580 ± 21 µm).
Ovary at 48 h After eCG
The proliferative and differentiation changes shown by 24-h exposure to eCG were further accentuated by 48 h, especially in antral follicles. There was still no trace of theca cells surrounding primary follicles (Fig. 3A). In preantral follicles with 4–5 layers of GCs, premature development of a coalesced antral cavity had occurred, and the surrounding theca was divided into an inner epithelial layer and an outer spindle-shaped region. This might represent the differentiation of theca interna and externa. The interstitium was again lush and hypertrophied.
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Specific FSH receptors on preantral and small antral follicles did not differ from the findings at 24 h post-eCG, but the reaction was much more intense in the GCs of large antral follicles (Fig. 3B). In the latter follicles there was continued proliferation of GCs as well as an increased size of the antral cavity.
Specific binding for hCG in the interstitium was definitely less than at 1 day after eCG (Fig. 3, C and D). The size of large antral follicles had further increased to 570 ± 18 µm, with the largest follicle at 1039 µm. More follicles showed hCG in peripheral GCs (Fig. 3, C and D), but this was true of only a minority of follicles. Of 40 antral follicles, only 12 showed hCG receptors in GCs, and the follicles were distinctly larger (614 ± 5 µm) than those in the nonpositive group (496 ± 31 µm).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the current study, primary follicles—with 1 layer of GCs—were unaffected by eCG and did not develop a thecal-interstitial coating. This is interesting because an oocyte-specific growth differentiation factor-9 (GDF-9) first appears in murine primary follicles [29]; and in GDF-9-deficient mice, primordial and primary follicles differentiate, but subsequent folliculogenesis is blocked and the animals are infertile [30]. Moreover, treatment with eCG fails to restore follicle growth beyond the primary stage in the GDF-9-deficient mice [30], a result similar to the unresponsiveness of primary follicles in the present investigation.
On Day 25 in control ovaries, the hematoxylin and eosin slides revealed that spindle-shaped theca cells surrounded preantral follicles with 2 or more layers of GCs, and it was not until the development of antral follicles that the theca converted to an epithelioid variety; it is therefore possible that on Day 25, only the larger follicles are capable of secreting significant amounts of steroids.
The autoradiographic findings were consistent for all 4 animals in each group, i.e., in the Day 25 untreated control group and at 24 and 48 h post-eCG. On Day 25, hCG receptors were restricted to the theca of antral follicles and to the interstitium. The interstitium then consists of scattered patches of various sizes derived from the theca of atretic preantral and antral follicles [31]. The preantral follicles are surrounded by theca-interstitial cells without any distinctive separation of the two cell types, but by 24 h after eCG, the preantral follicles are capped by a wreath of theca cells.
In view of the combined FSH and LH activity of eCG in rodents and other species, its effects on follicular development over the next 48 h are understandable. The first notable action at 24 h post-eCG is the epithelial transformation of the theca of preantral follicles and the interstitium; presumably, this results in increased steroid production. The theca is now discrete around small preantral follicles as evidenced by the distribution of hCG receptors. Preantral follicles—with 2 or more layers of GCs—show increased mitotic activity and beginning formation of an antral cavity in the form of isolated pockets of liquor folliculi. The most significant changes in FSH receptors occur in antral follicles, where there is an increase in the thickness of the granulosa layers; also the intensity of binding is now greater than in preantral follicles. This is especially evident in darkfield photomicrographs. Only a small number of enlarging antral follicles now contain hCG receptors in GCs.
An occasional large antral follicle lacks hCG receptors in the theca, but they are abundant in the adjacent interstitium. These follicles most likely are destined for atresia because of an inadequate source of C-19 steroids. As noted in other studies, antral follicles that are definitely atretic have fewer FSH receptors in the granulosa compartment.
At 48 h post-eCG, the most conspicuous change is further enlargement of some antral follicles, but only 30% have hCG binding in the GCs; these follicles are distinctly larger than smaller, immature ones. The reason that so few antral follicles were positive for hCG receptors in GCs is most likely related to the small dose of eCG administered: 5 IU. In a previous study [32], 25-day-old rats injected with 5 IU eCG and 48 h later with 5 IU hCG were mated. When animals were killed at Day 4 of pregnancy, both ovaries contained 9.35 ± 0.23 corpora lutea (SEM). Thus, the larger the dose of eCG injected, the greater the likelihood of obtaining large antral follicles with hCG receptors developing in GCs.
This study has traced the temporal and spatial changes in FSH and hCG receptors after injection of 5 IU eCG. The following companion paper [33], utilizing this model, considers the functional changes in in vitro steroidogenesis in follicles of various sizes, from preantral follicles with 1 or more layers of GCs to the largest antral follicles, during the 48 h after injection of eCG.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: G. Greenwald, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7401. FAX: 913 588 7430; ggreenwa{at}kumc.edu
Accepted: June 7, 1999.
Received: February 4, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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