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a Gamete Biology Group, Department of Reproductive Biology, German Primate Center, D-37077 Göttingen, Germany
b Reproductive Medicine Group, Department of Obstetrics and Gynecology, University of Göttingen, D-37075 Göttingen, Germany
ABSTRACT
Although it is known that LH receptors are present from the time of thecal differentiation, the role of LH during early follicle development is not yet clear. The effect of LH on preantral follicle development has therefore been investigated in vitro using a culture system that supports the development of intact follicles. We have previously shown that although preantral follicles 150 µm in diameter (2–3 granulosa cell layers) do not require LH to proceed through antral development, smaller follicles (1–2 granulosa cell layers, 85–110 µm in diameter) do not develop beyond the large preantral stage in the presence of only FSH and 5% mouse serum. Follicles of this size were therefore used to determine the effects of LH and serum on their development in vitro. The results showed that although FSH must be continuously present, a low concentration of LH together with a slight increase in serum concentration was necessary, specifically during the primary stage of follicle development (from 85 µm in diameter until the follicles had reached 150 µm in diameter) to induce the capacity for subsequent LH-independent rapid growth and antral development. The in vitro development of maturable oocytes with normal spindle and chromatin morphology was also supported. These results indicate that LH probably induces changes in the early differentiating thecal cells, which are critical for the completion of subsequent follicular and oocyte development.
follicle, follicular development, LH
INTRODUCTION
The glycoprotein hormone, LH, which is secreted by the anterior pituitary gland, together with FSH, is one of the primary regulators of ovarian function. LH may play multiple roles throughout follicular development but most studies have focused on the action in late-stage follicles and during the periovulatory period. In contrast, little information exists on the action of LH in preantral follicle development.
Although a truncated LH receptor (LHR) message can be detected in differentiating rodent gonads even before follicle formation, the first full-length LHR has been detected in rat and mouse ovaries at postnatal Day 5 [1, 2]. This appears to be coincident with the initial morphological differentiation of theca-interstitial cells of the growing primary follicles and their acquisition of the capacity to express basal steroidogenesis [3]. However, the critical stage at which functional responsiveness is acquired has not been investigated. Furthermore, it is not yet clear which follicular stages subsequent to the acquisition of LHRs are LH-dependent and which are not.
The results of Vitt et al. [4] using mouse follicle culture indicate that LH may not be an absolute requirement for preantral follicle development after the 2–3 granulosa cell layer stage, although functional LHRs are present in the theca [5]. This study pointed out that these follicles achieved antral stage in vitro and produced maturable oocytes [6] in an LH-free environment; however, follicles in the 1–2 granulosa cell layer stage grew very little under these conditions. This result suggests a stage-specific difference in requirements for growth, which may involve LH. The objective of this study was therefore to investigate, in vitro, the developmental requirements of preantral follicles smaller than 150 µm and to define the critical stage for the acquisition of functional responsiveness to LH.
MATERIALS AND METHODS
Animals and Tissue Collection
The mice used in this study (C57BI/6J x CBA/J) were bred in the German Primate Center in Göttingen from parents obtained from Harlan Winkelmann, (Borchen, Germany). They were housed in a temperature- and light-controlled room of 12L:12D, and were provided with food and water ad libitum. All experiments were performed according to German animal protection laws. A total of 46 16- to 18-day-old females and 46 20-day-old females were used in this study.
For collection of blood from the heart and subsequent ovary removal, mice were anesthetized with diethyl ether. Ovary donors were 16–18 days old (mean weight 6.36 g ± 0.62 [SD]). Additional serum was also collected from 20-day-old females. For both sources of serum, the samples were pooled at each collection session and stored in aliquots at -80°C until they were used.
Follicle Collection
Preantral follicles were collected and selected using a modification of the procedures and medium supplements described by Vitt et al. [4]. The major differences were, depending on the experiment, that the size of the follicles were 60–160 µm in diameter, and the concentration of insulin was reduced to 3.5 µg/ml in the collection medium.
Follicle Culture
The follicle culture protocol was adapted from Fehrenbach et al. [7] and Vitt et al. [4]. For culture, follicles were transferred from the holding medium into the culture medium, which consisted of 
minimum essential medium (MEM) supplemented with 3.5 µg/ml insulin (I 1882; Sigma, St. Louis, MO), which was a lower amount than had been used in the previous studies; 1 µM L-glutamine (Gibco 15039; Gibco BRL, Karlsruhe, Germany); 10 µg/ml transferrin (Sigma T 5391); 50 µg/ml L-ascorbic acid (A 4544, Sigma); and, depending on the experiment, either 5%, 7.5%, or 10% mouse serum. For the first 2 days of culture, serum from 16- to 18-day-old mice was used; thereafter, serum from 20-day-old mice was used.
Depending on the experiment, culture medium was supplemented with human follicle stimulating hormone (hFSH; Sigma F4021) and human luteinizing hormone (hLH; Sigma 35H0661) in various combinations and concentrations. Although these hormone sources are not absolutely pure, the level of contamination was below the minimum threshold required to influence the development of the follicles. Furthermore, this source of gonadotropins was used to avoid the possible complications caused by the narrowing of isoform range, which could occur with highly purified sources (Vitt et al.[4]). Follicles 60–160 µm in diameter were distributed to the different experimental groups according to the experimental design. Follicles were cultured individually in 96-well Costar ultralow attachment clusters plates (Costar, Bodenheim, Germany) in 40 µl of culture medium as in the previous studies for 5, 6, or 7 days, depending on follicle size at the beginning of the culture. The culture was carried out at 37°C and was gassed with 5% CO2 in air. Each day, the internal and external diameters of the follicles were measured and then transferred into a new well with fresh culture medium.
Histological Investigation of Follicles
At the end of the culture period, follicles were fixed overnight in 3% freshly prepared paraformaldehyde (Merck, Darmstadt, Germany) and 2.5% glutaraldehyde (Sigma) made in 0.1 M phosphate buffer. After fixation, follicles were washed in 0.1 M phosphate buffer for 4 h, which was changed every hour. They were then dehydrated in increasing concentrations of alcohol (30%, 50%, 70%, and 100%). The alcohol was changed every 30 min during the first 2 dehydration steps (2 h); the third step lasted either 4 h at room temperature or overnight in the refrigerator. Finally, the follicles were kept in 100% ethanol for 4 h, which was changed hourly. Afterward, follicles were incubated for 4 h in a 1:1 mixture of 100% ethanol and Technovit 7100 together with hardener I (Heraeus Kulzer GmbH, Germany) according to the product information. This mixture was changed every hour. Incubation was completed by placing the follicles into 100% Technovit together with hardener I for 4 h or overnight. Finally, the follicles were embedded in beem capsules in Technovit 7100 with hardener II for 1 h at room temperature and overnight at 37°C. Serial 0.5-µm sections were cut through the follicles, the sections were placed on glass slides and stained with undiluted Löfflers methylene blue solution (Merck, Germany), and mounted in Neo-Clear (Merck).
In Vitro Maturation of Oocyte-Cumulus Complexes
After the culture was completed, follicles were transferred to Leibovitz L-15 medium supplemented with 0.5 µM L-glutamine, 5 µg/ml transferrin, and 10% fetal bovine serum (FBS; Gibco 16141-079) under silicon oil. They were then opened using 2 Omnican needles. After oocyte-cumulus complexes (OOCs) were removed from the follicle they was washed twice in medium M-199 supplemented with 0.23 mM pyruvate, 50 µg/ml gentamycin, and 10% FBS. For in vitro maturation, 1 IU/ml hFSH (Sigma F4021) and 20 ng/ml epidermal growth factor (EGF; Boehringer Mannheim, Germany, 855731) were added to the medium in which OCCs were cultured for 16 h (Vitt et al. [6]).
Chromatin and Spindle Visualization
Chromatin and spindle configuration were assessed using the fluorescence methods described by Gilchrist et al. [8]. After the cumulus masses were removed with 0.5 mg/ml hyaluronidase, oocytes were fixed for 1 h in prewarmed (37°C) 2% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) with 0.02% Triton X-100 (Serva, Heidelberg, Germany) for 1 h, and stored overnight at 4°C in DPBS with 0.3% (w/v) bovine serum albumin (BSA; Fraction V). For staining, fixed oocytes were incubated in prewarmed 100-µl droplets containing mouse monoclonal anti–tubulin (1:5000; ICN, Meckenheim, Germany) in DPBS with 0.3% (w/v) BSA for 45 min at 37°C. After washing three times (30 min each) in DPBS with 0.3% (w/v) BSA and 1 µl/ml Tween-20 (Merck), oocytes were stained with fluorescein isothiocyanate (FITC) conjugated to goat anti-mouse F(ab') (1:100; ICN) together with 10 µg/ml Hoechst 33258 (Hoechst, Wiesbaden, Germany) in DPBS with 0.3% (w/v) BSA for 45 min at 37°C in the dark. Following another three washes, oocytes were impregnated with Mowiol 4-88 mounting medium (Hoechst) and kept in the dark until they were examined under a Zeiss Axiovert 405M microscope (epifluorescence illumination with Hoechst excitation at 365 nm and FITC at 490 nm). Fluorescence images were recorded on Kodak Ektachrome 400 ASA color-positive film (Eastman Kodak, Rochester, NY).
Experimental Design
Definitions Follicles of 65 to 160 µm were divided into categories according to their size: 65–75 µm (class 1 small), 85–110 µm (class 2 small), 120–140 µm (class 3 small), 150–160 µm (standard). Experiments were carried out with class 2 small follicles (unless otherwise indicated), which represented the minimum size that responded to the investigated conditions. Class 1 small follicles were smaller than the minimum size that could be grown under our culture conditions. A follicle was classified as antral when a confluent antrum could be visualized in the living follicle and the antral threshold was the minimum size at which an antrum was visible. The optimal dose was defined as the dose that produced the maximal growth rate together with healthy follicular appearance as evaluated by morphology.
Experiment 1: Dose Optimization
A pre-experiment was carried out using a three-way factorial design in which the doses of LH, FSH, and serum were varied. Those combinations that produced enhanced growth were then chosen for a more thorough determination of optimal dose combination. The final confirmation of optimal concentrations was carried out as shown in Table 1.
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Experiment 2: Establishment of the Stage Requirements for Each Component
A pattern of culture combinations is shown in Table 2. These were tested to determine the effect of each individual component and to establish whether the requirements for the first 24 h differed from the subsequent culture days.
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Statistical Analysis
Only follicles that grew and remained intact for the entire culture period were considered for analysis (>95%). One-way ANOVA was used for all statistical comparisons. P values of < 0.05 were considered significant. Follicle size was graphed as mean and standard deviation.
RESULTS
Under Standard Follicle Culture Conditions (5% FSH, Mouse Serum, and No LH), Only Follicles of at Least 150 µm in Diameter Had the Capacity to Grow Through the Antral Stage
This follicle stage has 2 to 3 layers of granulosa cells and a multilayered theca consisting of flattened cells. They grew rapidly (around 50 µm/day to a final size of 396 ± 4.7 µm after 5 days; Fig. 1). In contrast, under these conditions, follicles that were smaller than 150 µm (class 3 and class 2 small) grew slowly (around 20 µm/day) and did not reach antral size. Figure 2A illustrates a standard starting size of a follicle 160 µm in diameter; Figure 2B shows a Class 2 small follicle of 85 µm in diameter.
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An Increased Concentration of Serum Is Synergistic with LH and FSH in Supporting the Growth of Small Follicles
Whereas in the culture for follicles that had a starting size greater than 150 µm, 5% mouse serum has been shown to be optimal, the serum requirements for smaller follicles are unknown. Therefore, class 2 small follicles (mean diameter 95 ± 2.5 µm) have been cultured in different concentrations of serum to determine the optimal concentration, and the results were confirmed on the larger class 3 small follicles. The effect of 5% (standard concentration), 7.5%, and 10% mouse serum was examined in a prestudy under a range of hormonal concentrations. The comparative effects of serum concentration in the presence of the optimal hormonal concentrations are shown in Figure 3A (10 mIU/ml LH and 400 mIU/ml FSH).
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Even in the presence of optimal concentrations of both FSH and LH, the standard concentration of serum supported rapid growth (~50 µm/day) only for the first 24 h. Thereafter, the growth rate reduced to an average of 23 µm/day, which led to a final mean follicle size of only 281 ± 20.7 µm (n = 50). That standard conditions (FSH with 5% serum but no LH) did not produce rapid growth on the first day (Fig. 1) indicated that LH played a role in this initial growth induction. However, the increase of serum to 7.5% (FSH, no LH) induced first-day rapid growth, whereas 7.5% serum without gonadotropins did not (Fig. 3b). This indicated that FSH in synergy with the increased serum also played a role that could not be detected at the lower serum concentration.
These findings considered together indicate that all three factors were playing complementary roles in this initial development stage. This is substantiated by the fact that the presence of all three factors (i.e., 7.5% serum together with optimal FSH and LH) appeared to produce a potentiating effect over any two-factor combinations. The initial rapid growth was maintained over a longer time; an additional 2 days (3 days total). This resulted in a high proportion (85%) of follicles forming antra and a Day 7 mean diameter of 369 ± 6 µm (n = 41) was achieved. In contrast, with a further increase in serum to 10% (in the presence of both FSH and LH), initial rapid growth was sustained for only 2 days, resulting in a smaller mean final diameter (323 ± 14.6 µm; n = 51) and a major reduction in the proportion of follicles (36%) attaining antral stage. This growth and developmental inhibition indicated a serum overdose.
An Optimal Concentration of LH Is Necessary for FSH and Higher Serum Concentrations to Induce Small Follicle Rapid Growth
Class 2 small follicles were cultured under a range of LH concentrations together with 400 mIU/ml FSH and 7.5% serum to determine a dose-response based on follicle growth rate (Fig. 3B). The mean follicle growth increased significantly from 0 to 2.5 mIU/ml LH, but the follicles remained below the antral threshold. The growth increased progressively and significantly until the dose of 10 mIU/ml was reached and the antral threshold size was surpassed (mean end size, 371 ± 5.8 µm; n = 26). Furthermore, 10 mIU/ml LH produced a significantly higher rate of growth over the first 24 h (53 µm/day) compared with 2.5 mIU LH (34 µm/day). In contrast, although the same end size was reached, the highest dose tested (20 mIU/ml; n = 25) significantly depressed follicle growth for the first 6 days below that produced by 10 mIU/ml (316 ± 8.1 µm vs. 349 ± 7.2 µm), which is indicative of an overdose.
Although the zero LH control (with optimal FSH and 7.5% serum) also produced rapid growth for the first 24 h, this growth was not sustained (final mean follicle size was only 275 ± 20 µm; n = 33) and there was an associated degeneration of the oocyte and surrounding cumulus cells by the end of culture (Fig. 4B), indicating the essential role played by LH during this early stage. That the degeneration as well as the initial growth was triggered by FSH was shown by the absence of apparent degeneration and slow growth when the follicles were grown only with 7.5% serum.
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FSH Is Required for LH and Higher Sera to Stimulate Small Follicle Growth Through the Antral Stage
Furthermore, the effect produced by LH in combination with higher sera is dependent on the presence of FSH (Fig. 3C). Without FSH, LH produced a very specific effect during the first 24 h by inducing very rapid growth (60 µm/day) but, thereafter, growth was extremely slow (12 µm/day; mean final size 242 ± 7.4 µm; n = 38). The resulting follicles were nonantral (Fig. 4A) but, in contrast to FSH without LH (Fig. 4B), contained healthy-appearing oocytes.
The presence of FSH together with optimal LH and serum produced a dramatic dose-related improvement in growth rate. One hundred mIU/ml FSH (n = 29) produced a significant increase in growth above zero although follicles did not pass the antral threshold. Two hundred mIU/ml (n = 31) produced a further, significant increase in mean end size, which allowed the follicles to pass the antral threshold size (average growth rate 36 µm/day, mean size 351 ± 4 µm). A further significant increase in mean end size was observed with 400 mIU/ml (369 ± 6.6 µm; n = 30) and best overall growth rate was achieved with this dose. This was most apparent over the first 3 days in which the growth rate was 54 µm/day for the first 24 h, with a subsequent growth rate of 36 µm/day. The next higher dose, 800 mIU/ml, produced an equivalent end size but signs of overdosing were indicated by suppression of growth for the first 2 days and poorer end follicle and oocyte quality.
Morphologically, the class 2 small follicles grown for 7 days under optimal conditions (Fig. 4C) were antral and similar in appearance to those of a standard start size (>150 µm diameter at the start of culture) after 5 days of culture under standard conditions.
The Minimum Start Size That Was Responsive to the Optimized Conditions for Small Follicle Growth Was 85 µm
Three size categories of follicles were tested under optimal conditions (Fig. 5); class 1 small (65–75 µm), class 2 small (85–110 µm), and class 3 small (120–140 µm). The smallest class 1 size did not reach antral size under these conditions (mean final size 237 µm ± 11.07 µm; n = 31) and was therefore considered to be below the minimum threshold size for our test conditions. Class 2 follicles had a mean final diameter of 371 ± 4.9 µm; (n = 29) after 7 days of culture, whereas class 3 follicles reached an endpoint diameter of 387 ± 6.47 µm (n = 35). For both class 2 and 3 follicles, more than 85% were antral at the end of the culture under the optimized conditions, and both were significantly larger than under standard conditions (Fig. 1) in which they did not reach antral size.
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Optimized Conditions Were Required Only During the First 24 h
Under optimal conditions, over the first 24 h of culture, the class 2 small follicles reached diameters of 150 µm or larger, which is equivalent to the minimum threshold size for growth under standard conditions. The question was, therefore, whether these in vitro-developed follicles had reached a development stage that was equivalent to their in vivo counterparts.
In order to evaluate the requirements of class 2 small follicles for LH and serum after the first 24 h in culture, the follicles were thereafter transferred from optimal to standard conditions (FSH and 5% serum) for the rest of the culture (two-phase treatment). The follicles grown under two-phase conditions (n = 52) reached a final size of 380 ± 5.3 µm (Fig. 6). This was not significantly different from the end size reached by those that had been grown under optimal conditions for the entire time (n = 54; 370 ± 5.4 µm; Fig. 4, D and C, respectively). Further, the equivalence of the antral formation (97.8% and 96.9%, respectively) as well as subsequent oocyte maturation rates (67.68% and 71.17%, respectively) strongly indicated that after 24 h in culture, the class 2 small follicles had reached not only a size equivalence with the standard-sized follicles, but a functional equivalence as well. Figure 7A shows a typical example of an in vitro-matured oocyte from a follicle that was grown with the two-phase treatment, Figure 7B shows the normal chromatin and spindle arrangements.
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Finally, in order to confirm whether FSH was specifically required over these first 24 h for the complete response, a parallel two-phase experiment was performed in which the mixture for the first 24 h did not contain FSH (n = 42). After 1 day, the follicles without FSH had reached the same size range as those that had the complete mixture, and were transferred to standard conditions containing FSH and no LH. The mean rate of subsequent growth was slower than when FSH was also present and, as a result, the mean end size was significantly smaller than when FSH was present during the first 24 h (331 ± 10 µm vs. 380 ± 5.3 µm; Fig. 6). Further, only 55% of small preantral follicles grew to the antral stage and excessive theca cell proliferation was obvious. These results confirm that all three components of the mixture are required to induce the full developmental competence in follicles of 85 µm diameter.
DISCUSSION
Intact preantral mouse follicles with a minimum size of 150 µm and between 2 to 3 granulosa cell layers [7, 9, 10] can be routinely cultured to the preovulatory stage in a virtually LH-free mixture containing FSH and 5% serum [4] in 3–5 days, depending on follicle size at the beginning of culture. In contrast, preantral follicles from 140 µm down to 85 µm in diameter (between 1 to 2 layers of granulosa cells) were shown in this study to additionally require LH together with an increase in serum to support rapid growth and antral development. Furthermore, after follicle development under these conditions, the oocytes were shown to have the ability to mature in vitro and to have normal spindle and chromatin configurations. This advance in technique has considerable implications for humans and other species.
This result is consistent with the literature that identified the first appearance of LH receptors [1, 2] at around this stage of follicle development. The differentiating thecal cells acquire LH responsiveness and the capacity for basal androgen production coincident with the initial appearance of the typical morphological characteristics of thecal cells [11, 12]. This flattening and proliferation begins after the initiation of follicle development, around the time the granulosa cells have become cuboidal and begun to proliferate [13].
Although the in vivo results of Flaws et al. [14] suggest that LH may be important in small preantral follicle growth recruitment, never before has it been shown that LH has an obligatory and stage-specific role in primary follicle development. It is also important to point out that, although individually, LH and each of the two other factors we studied could initiate rapid preantral growth, the normal pattern of subsequent development occurred only when LH was present in optimal concentrations together with the optimal combination of other factors. The LH dose was found to be critical in its effect on the small preantral follicle, with a very narrow optimal dose threshold. The dose sensitivity suggested that LH level may be an important synchronizing factor in inducing appropriately timed, healthy primary follicle growth.
LH also appeared to act in some way as a survival factor. For follicles that were cultured for the entire 7 days with LH (but no FSH) together with higher serum, the health and growth of the oocytes were maintained despite slow growth after the first day and lack of antral formation. Cortvrindt et al [15] suggest that LH exerts its general "survival" effect on the oocytes of attached follicular cell complexes through thecal cell induction of granulosa cell proliferation. However, we found no apparent difference in number of granulosa cell layers (unpublished data) between follicles grown with LH only vs. FSH only. Without the protection of LH, the follicles exposed to FSH with high serum had a poorly structured appearance, degenerating cumulus cells, and a degenerated oocyte, indicating that, although FSH is necessary in combination with other factors, it does not promote small follicle survival and may, in fact, have a negative effect.
Homologous serum has previously been shown to be essential for the maintenance of normal intact follicle organization [9]. In this study, we have shown for the first time that follicles in primary stage development have a higher requirement for serum factors than those in later development, and that this was important in potentiating rapid growth from 85 µm to the 150 µm threshold and greater. In addition to the continuous presence of FSH, the responsiveness to LH during this early phase depended on a slightly increased serum concentration (above that required for follicle growth after they reach 150 µm). This indicates that certain growth factors that are present in serum may play a more critical role for 85-µm follicles than at later stages, perhaps acting both on the thecal and the granulosa cells.
The optimal follicle response was obtained with an FSH dose of 400 mIU/ml (at an FSH:LH ratio of 40:1 in terms of IUs), although by the time the dose of 200 mIU/ml was achieved, the follicles were already over the antral threshold by the end of culture, indicating a wider dose tolerance than for LH. It is possible that FSH also plays a role in promoting thecal differentiation through its action on the granulosa cells. Magarelli et al. [16] showed that FSH induces, in a dose dependent manner, the production of not-yet-characterized thecal differentiating peptide(s) by granulosa cells, which supplemented the effect of LH on thecal cells.
The stage specificity of the effects of LH and increased serum concentration were shown by the removal of LH after the first 24 h and a return to the standard serum concentration (two-phase treatment). This return to standard conditions did not produce a reduction in follicle growth or oocyte capacity for maturation, indicating that the follicles had reached a developmental stage that was equivalent to their in vivo counterparts. That FSH was essential to the effect, specifically during this first 24 h, was shown by its removal from the mixture over this time. This produced a much poorer subsequent development under standard conditions than when FSH had also been present during the first 24 h. Recent studies with FSH-receptor knock-out mice [17], which are unresponsive to their own FSH but have an elevated level of circulating LH, are suggestive that a similar effect is obtained in vivo. This is a further indication that FSH may be inducing granulosa cell-thecal differentiating factors (as suggested by Zachow and Magoffin [18]), which are specifically required during early preantral development.
In conclusion, this study has shown that specific combinations of factors at appropriate doses and times are required to induce the correct balance of stage-specific differentiation. In order to reach the LH-independent 2–3 granulosa cell-layer stage (150 µm), primary follicles of 1–2 granulosa cell-layer stage (85 µm in diameter) require a threshold concentration of LH as well as more serum than is necessary later. Both LH and serum are critical for developmental progression through this specific preantral stage; with the acquisition of competence for further development, FSH differed in that its presence was obligatory throughout all stages. All three factors are probably involved in the completion of thecal cell differentiation, which is essential for the further progression. Follicles smaller than 85 µm in diameter do not respond to this treatment, thereby defining a specific lower threshold in growth requirements. These results strongly indicate the existence of specific threshold stages of follicle development, which can be characterized by their differing growth requirements.
ACKNOWLEDGMENTS
The authors thank J.K. Hodges for his consistent support of this project.
FOOTNOTES
First decision: 24 August 1999.
1 J.W. was supported by research grant GB 9601/2 from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie. 
2 Correspondence: Penelope L. Nayudu, Department of Reproductive Biology, German Primate Center, Kellnerweg 4, D-37077 Göttingen, Germany. FAX: 49 551 3851288; pnayudu{at}gwdg.de
Accepted: February 22, 2000.
Received: July 26, 1999.
REFERENCES
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