| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ovary |
a Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
developmental biology, follicle, follicle-stimulating hormone, follicular development, ovary
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
It is possible that, in addition to this indirect regulation of follicular dominance, there are other direct interfollicular interactions playing a role in regulating follicular dominance [8]. Dominant follicles might produce factors that act directly on subordinate follicles from the growing cohort to impair their development (such direct regulatory processes functioning alongside the systemic regulation described above). Direct interactions might also occur between follicles at earlier stages of development, thus regulating which follicles first become dominant or subordinate. Hypotheses regarding direct follicular interactions have been suggested now for many years, but have proven difficult to investigate in vivo [1]. Despite such difficulties, several extracts of follicular fluid have been proposed to play a role in follicle-follicle interactions [9–14], but their in vivo roles have yet to be established.
More recently, culture techniques have been used to investigate the effects of interactions between follicles in a more direct manner. Such in vitro work allows follicle-follicle interactions to be examined in the absence of the possible intervention of endocrine, or indeed any extrafollicular, action. Results indicate that interactions are dependent on the stage of follicle development and possibly on culture conditions. Mizunuma et al. [15] have shown that large, antral mouse follicles secrete a factor, most likely activin, that inhibits the growth of small primary follicles in culture. Conversely, Zhao et al. [16] found that the in vitro growth of preantral rat follicles was stimulated by coculture with other preantral follicles. Spears et al. [17] showed that when two equivalent late preantral mouse follicles were cultured in contact through the antral stage, one follicle invariably became "dominant" over the other, with the growth of the "subordinate" follicle becoming severely impaired over the culture period. This effect appeared to be due to a contact-mediated process, rather than to secreted factors, as no effect was found when cocultured follicles were placed apart. It was possible, however, that inhibitory and/or stimulatory secretory factors were present in these cultures but were undetected by the specific experimental paradigm used. For example, such secretory factors may have become too dilute in the culture medium, or their actions overridden by other factors (such as high levels of FSH or secretory factors stimulating growth). The experiments here were designed 1) to investigate whether secretory factors are produced, 2) to determine how they might influence the growth and development of other like-sized follicles, 3) to examine how secretion/action of such factors interacts with the FSH environment, and 4) to begin to establish the nature of such factors.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Twenty-one- to 25-day C57Bl/6 x CBA/Ca F1 female mice were housed in an environmentally controlled room on a 14L:10D photoperiod. Animals were provided with food and water ad libitum and kept in accordance with U.K. legal requirements.
Follicle Isolation and Culture
Mice were killed by cervical dislocation and their ovaries removed to watch glasses containing Leibovitz L-15 medium (Gibco-BRL, Renfrew, U.K.) supplemented with 3 mg/ml BSA (Fraction V, Sigma, Poole, U.K.). Individual late preantral follicles (180 ± 10 µm in diameter) were manually dissected using fine needles and placed in the wells of microtiter plates (Iwaki, Osaka, Japan) containing 
-Minimal Essential Medium (Gibco-BRL) overlaid with silicone fluid (Merck, Poole, U.K.) to prevent evaporation. Six to 12 mice were used in each run of each experiment, and all follicles from each animal were randomly allocated across all treatment groups. Culture media was supplemented with recombinant human FSH (rhFSH, Serono-Ares, Geneva, Switzerland) at the concentration indicated in the individual experiments, 5% mouse serum and 50 µg L ascorbic acid sodium salt per milliliter (Sigma, Poole, U.K.). Recombinant human FSH was supplied by Serono-Ares as a bulk preparation for most experiments but as the commercial preparation Gonal-F in experiment 2a and experiment 3b. Follicles were moved to new wells of media daily, except in experiment 1 in which 50% of media was exchanged with fresh media daily. Follicles were cultured in 30 µl medium overlaid with 75 µl silicon fluid in round-bottomed wells, except in experiment 1 in which 150 µl medium was overlaid with 75 µl silicon fluid in flat-bottomed wells (to accommodate the presence of the polycarbonate membranes in the wells). This culture paradigm supports the development of follicles in a manner that closely resembles development in vivo and allows the oocytes obtained at the end of the culture period to undergo fertilization and subsequent embryo development [18].
Production of Conditioned Medium for Experiments 2–4
Conditioned medium was obtained by setting up a culture of follicles (termed the "conditioning culture") one day prior to setting up the culture of experimental follicles. Levels of rhFSH present in the conditioning follicle medium were in each case identical to the medium in which the experiment follicles were later cultured (see individual experiments for details).
Experiment 1: Is Physical Contact Between Follicles Necessary for Follicle-Follicle Interactions?
Follicles were cocultured across polycarbonate membranes as follows:
Membrane preparation Polycarbonate membranes (Isopore, Millipore, U.K.) were used in this experiment. Pores measuring 0.1 µm in diameter do not allow the growth of cellular processes across the membrane, whereas membranes with 10-µm-diameter pores do permit this cellular growth, thus allowing the establishment of direct contact across the membrane [19, 20]. Membranes, which are supplied coated with a wetting agent, polyvinylpyrrolidone (PVP), were boiled in deionized water for at least 1 h to remove the PVP coating, as the PVP was found to encourage adhesion of cultured follicles resulting in follicle rupture. The undersides of membranes were examined by scanning electron microscopy following the culture of preantral follicles for 48 h on these membranes, showing clear evidence of cellular protrusions on the undersides of the 10-µm-diameter pore membranes only (results not shown). Prior to use in the culture experiments, treated membranes were wrapped individually in aluminium foil, autoclaved, and allowed to dry. Under sterile conditions, 0.1- and 10-µm pore-size membranes were cut into rectangles approximately 2 x 4 mm and folded. Two membrane pieces of the same type were attached as in Figure 1 using a surgical adhesive (Histoacryl Blue, B. Braun Medical Ltd., Sheffield, Coventry, U.K.). This allowed one follicle to be placed on either side of the vertical membrane. The two-piece assembly of the membrane was then secured into flat-bottomed 96-well plates (Dow Corning, Coventry, U.K.) using a silicon-based sealant (RTV 32, Dow Corning). The surgical adhesive and silicon-based sealant were both tested previously to ensure that they presented no toxicity problems for follicle development. When a row of wells had been filled with either 0.1- or 10-µm-pore membrane inserts, the tray was left for 24 h at 37°C to allow the sealant to cure. Sterility was ensured by UV irradiation of trays for 15 min immediately prior to the addition of culture medium.
|
Follicle culture Late preantral follicles were cultured in media as described above in a constant level of 1 IU rhFSH per milliliter throughout. The medium was equilibrated for 1 h in a humidified incubator at 37°C with 5% CO2 before follicles were added. Like-sized follicles were paired and placed directly across from each other on either side of the upright membrane (Fig. 1). Paired follicles cultured on polycarbonate membranes in separate wells served as controls. After 24 h, cocultured pairs were examined by an unbiased observer who assessed if they were still suitably juxtapositioned, and repositioned follicles if they were not. Measurements of growth were recorded daily. At this time, an unbiased observer assessed if the cocultured pairs of follicles were still positioned directly across from each other. If a pair was no longer considered to be in position on any day of the experiment after the initial 24 h, it was excluded from the experiment. Fifty percent of the medium was extracted daily using an angled gel-loading pipette tip and replaced with the same volume of previously equilibrated medium. The culture continued for 4 days, at which time all follicles were transferred to a watch glass for a more accurate final measurement and assessment of size.
This first experimental paradigm, coculturing across polycarbonate membranes, indicated that follicles do secrete factors that affect the growth and development of like-sized follicles. However, due to the necessity of excluding pairs of follicles that may not have remained directly across from each other throughout the culture period, the experimental paradigm was changed in subsequent experiments to confirm the results found here using a different experimental set up and then to further investigate the effect in a system that did not exclude the results of any follicles moving during the culture period.
Experiment 2: Is the Production/Action of Secretory Factors Affected by the Level of FSH?
Follicles were cultured with either 1 IU rhFSH per milliliter throughout the culture (experiment 2a: high FSH) or 1 IU rhFSH per milliliter for the first 2 days of culture and then lowered to 0.1 IU rhFSH per milliliter for Days 3 and 4 of culture (experiment 2b: lowered FSH). In each case, experimental follicles were placed in wells containing media that had supported the culture of a conditioning follicle for the last 24 h (conditioned media), but with 10 µl of conditioned media replaced with 10 µl of fresh media. Conditioning follicles were cultured in the same levels of FSH as the experimental follicles (1 IU rhFSH per milliliter throughout in experiment 2a and 1 IU rhFSH per milliliter for the first 2 days of culture, lowered to 0.1 IU rhFSH per milliliter for Days 3 and 4 of culture in experiment 2b). Because it has previously been shown that 10 µl media was sufficient to support one follicle for 1 day in culture [21], the final 30 µl of media should contain sufficient support for the experimental follicle for 1 day. Follicles dissected from the same ovaries but cultured in fresh, unconditioned media throughout served as controls. All follicles were measured daily.
Experiment 3a: What Is the Nature of the Inhibitory Factor?
Follicles were cultured as in experiment 2b (1 IU rhFSH per milliliter for the first 2 days of culture, lowered to 0.1 IU rhFSH per milliliter for Days 3 and 4 of culture). Control follicles were cultured in 30 µl fresh medium throughout. For experimental follicles, media was collected daily from the conditioning culture and pooled. Twenty microliters of conditioned medium was then mixed with 10 µl of fresh medium, and experimental follicles were placed in this conditioned media/fresh media mixture. There were two experimental treatments: in one experimental group conditioned medium was left untreated, whereas in the other group the conditioned medium was heat-inactivated (by heating to 56°C for 30 min) before mixing with fresh medium. All follicles were measured daily.
Experiment 3b: Is the Inhibitory Factor Activin?
In this experiment, the rhFSH used was supplied by Serono as the commercial preparation Gonal-F, and it was found that lower concentrations could be successfully used to support follicle growth. Thus follicles were cultured in 0.5 IU rhFSH per milliliter for the first 2 days of culture and lowered to 0.05 IU rhFSH per milliliter for Days 3 and 4 of culture. Control follicles were cultured in 30 µl fresh medium throughout. Two groups of experimental follicles were cultured in conditioned medium, which was collected daily and pooled as in experiment 3a. In one group, conditioned medium was left untreated. Ten nanograms recombinant follistatin was added daily to each milliliter of conditioning medium in the other group to negate the action of activin. Mizumuma et al. [15] used 2 ng/ml follistatin to successfully abolish the effect of activin in other coculture experiments. Smitz et al. [22] have shown that maximum production of activin by cultured mouse follicles was 2–5 ng/ml of culture medium. Follicles were measured daily.
Statistics
To determine if there was an effect of coculture on growth in experiment 1, a chi-square test was performed comparing each experimental group with controls. Student t-test was then used to establish if there was a bimodal population present by comparing the difference in size between cocultured pairs and control follicles and to compare the final size of dominant and subordinate follicles with singly cultured control follicles. Finally, a chi-square test was used to assess if there was any difference between the two experimental groups. In experiments 2–4, final follicle size was examined. Probability values (P values) were determined by analysis of variance (ANOVA), and where appropriate paired comparisons were made using Student t-test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
There was a strong tendency for follicles to migrate during the culture period. As a result, many pairs of cocultured follicles did not remain directly across the polycarbonate membrane from each other for the entire culture period. Only those pairs that an independent assessor was certain of being directly across from each other throughout the experiment were included in the analysis. Figure 2A shows a frequency histogram of the final diameter of the nonmoving follicles cultured across the membranes with 10 µm diameter pores (n = 16 pairs), and Figure 2B shows that for the 0.1-µm-diameter pores (n = 12 pairs), both compared with distribution of control follicles, paired for subsequent analysis but cultured singly in individual wells (n = 20 pairs). The data from both experiments are illustrated as a box-and-whisker plot in Figure 2C.
|
In both cases, culture of follicles across the membranes resulted in a bimodal distribution of final follicle size, whereas that of the control follicles showed a normal distribution. Size of the follicles at the end of the culture was divided into bins so that approximately equal numbers of control follicles fell into each bin (<285, 286–310, 311–335, 336–355, and >356 µm).
For the 10-µm-diameter pore membranes, chi-square test returned a highly significant difference (P < 0.001) between the distribution of control and experimental follicles across the size bins. This shows that coculture does have an effect on growth, but it cannot confirm that a bimodal population is present in the experimental group. To ascertain if there were distinct dominant and subordinate populations in the experimental groups (i.e., if there was a bimodal distribution), Student t-test was performed comparing the differences in size between dominant and subordinate follicles within each pair in the experimental group with the difference in size between paired, singly cultured controls. This returned a highly significant difference between the two groups (P < 0.001) showing a bimodal distribution. When dominant and subordinate follicle final size was compared with that obtained by control follicles using the Student t-test, the subordinate follicles showed no significant difference (subordinate: 333.1 µm, SEM ± 7.4; control: 326.4 µm, SEM ± 0.2), but the dominant follicles were significantly larger (dominant: 396 µm, SEM ± 4.3, P < 0.001).
For the 0.1-µm-diameter pore membranes, chi-square test returned a significant result (P < 0.01) when cocultured follicle growth was compared to single controls. When difference in size between paired control and experimental follicles was compared, the Student t-test returned a highly significant difference between the populations (P < 0.001), again illustrating that there is a bimodal distribution. Comparing dominant and subordinate follicle growth to controls (again using the Student t-test), the subordinate follicles showed no significant difference (subordinate: 324.2 µm, SEM ± 9.4; control: 326.4 µm, SEM ± 0.2); however, the dominant follicles were significantly larger (dominant: 370 µm, SEM ± 9.6, P < 0.001).
A chi-square test for contingency tables was performed to establish if there was an overall statistical difference between the two experimental groups. This test failed to find any significant difference between the two groups.
Experiment 2: Is the Production/Action of Secretory Factors Affected by the Level of FSH?
When follicles were placed in high levels of FSH throughout the culture (high FSH culture), there was no difference between the size of control and experimental follicles (cultured in conditioned media) on the final day of culture (Day 4; Fig. 3). Culture in conditioned medium did in fact produce a stimulatory effect earlier on, with the growth of experimental follicles significantly greater than that of control follicles on Days 1, 2, and 3 of culture (experiment 2a; P < 0.05, P < 0.005, and P < 0.01, respectively). In contrast, when the level of FSH was lowered during the final 2 days of culture (lowered FSH culture), culture in conditioned medium resulted in a significant inhibition of follicle growth (experiment 2b, P < 0.01; see Fig. 4).
|
|
Experiment 3a: What Is the Nature of the Inhibitory Factor?
At the end of the culture period, there was no difference between the size of control follicles and that of follicles cultured in conditioned medium that had been heat-inactivated. Both of these groups of follicles had grown to a significantly greater size than the follicles cultured in untreated (non-heat-inactivated) conditioned medium (P < 0.01 for both; Fig. 5).
|
Experiment 3b: Is the Inhibitory Factor Activin?
As in experiments 2 and 3a, when follicles were cultured in conditioned medium with FSH levels lowered during the culture period, follicles grew less well than control follicles and were significantly smaller than control follicles at the end of the culture period. Addition of follistatin to the conditioned medium did not alter its effect. There was no difference between the final size of the two different groups of follicles cultured in conditioned medium (those cultured with or without follistatin added). The follicles treated with conditioned medium plus follistatin were significantly smaller than the control follicles at the end of culture (Fig. 6; P < 0.001).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previous work, mainly in the cow and sheep, has shown that follicles secrete factors that can inhibit various aspects of development in other follicles. Cahill et al. [10] showed that ovine follicular fluid inhibited follicle development. Subsequently, steroid-free ovine follicular fluid was shown to lower steroid secretion from the ovary, with an associated decrease in P450arom and inhibin-activin ßb subunit [12, 24]. Granulosa cell-inhibitory factor (GCIF), a factor obtained from steroid-free bovine follicular fluid, has been shown to inhibit rat and bovine follicle growth and development [13, 14], whereas immunization of sheep against GCIF increased the ovulation rate [25]. Similar factors have been reported both in the pig [26] and the rat [27]. Mizunuma et al. [15] have also documented that the development of primary mouse follicles in vitro is inhibited by activin secreted by larger secondary preantral follicles in vitro. Production of stimulatory factors has also been reported (e.g., [13, 16, 27]), although they have been investigated less than the inhibitory ones.
Such follicle-follicle interactions are distinct from intrafollicular mechanisms by which follicles regulate their own development. This probably occurs mainly via the action of FSH, which in turn affects levels of, for example, IGF binding proteins and estradiol [3, 4, 28, 29]. These intrafollicular mechanisms help explain how dominant follicles develop toward ovulation while subordinate follicles instead undergo atresia following the selection process (which is as yet largely unexplained). This work is likely to be an important tool in the identification of dominant and subordinate follicles at an early stage. It is also possible that factors found to change in the dominant follicles then act as interfollicular factors, subsequently affecting the development of other follicles. For such factors (or indeed any interfollicular factors) to be effective, however, they need to affect other follicles while having no effect on the follicle that produced them (perhaps due to differences in their gonadotropin dependency).
The secretory nature of the factor(s) produced in the experiments reported here was first established by culturing follicles across membranes with pores too small to permit gap junction contact, with follicles cultured in high levels of FSH throughout the culture period. The change in experimental paradigm in subsequent experiments was made due to the tendency of follicles to migrate when placed on the polycarbonate membranes. This meant that any migrating follicles had to be excluded from the analysis. The first experiment does, however, allow us to compare the difference between the interactions of pairs of follicles allowed to make physical contact and those pairs able to communicate through secretory factors alone. The results from this experiment show that coculture of follicles across a polycarbonate membrane and in high levels of FSH results in one follicle within the pair stimulating the growth of its cocultured follicle, such that the stimulated follicles grew to a larger final size than that of the control, singly cultured follicles. This stimulatory effect could be detected when the polycarbonate membrane separating the cocultured follicles contained large enough pores to allow cellular communication between the follicles (the 10-µm-diameter pore membranes). In this instance, the effect could be due to a contact-mediated mechanism and/or to the presence of a secretory factor. The stimulatory effect could also be detected when the polycarbonate membrane separating the cocultured follicles contained pores not large enough to allow cellular communication between the follicles (the 0.1-µm-diameter pore membranes). In this instance, the stimulatory effect could only be due to the presence of a secretory factor. As coculturing across the membrane with the larger pore size produced more of a stimulatory effect than coculturing across the membrane with the smaller pore size (resulting in a 70 µm, compared with the 44 µm, increase in diameter), it is possible that this larger effect was due to a combination of both a secretory factor and a contact-mediated mechanism.
In the second experimental paradigm, conditioned medium was obtained from a conditioning culture set up 1 day prior to the experimental culture. In these experiments, it was determined that stimulatory factors were secreted by conditioning follicles (stimulating the growth of the experimental follicles grown in the conditioned media) only if high levels of FSH were maintained throughout cultures. It is not yet known if these same stimulatory factors are also produced when FSH levels are lowered during culture, but with their effects masked by the additional secretion of inhibitory factors. In contrast, inhibitory factors were only able to act (or, possibly, were only produced) if levels of FSH in the cultures were lowered during the culture period. The lowering of FSH during Days 2–4 of culture occurs when the follicles are in the antral stage of development and is likely to more precisely mimic conditions in vivo than a constant FSH environment. Similarly, large doses of FSH can prevent the ability of bovine follicular fluid to delay estrus in ewes [30]. In addition, FSH can partially reverse the inhibitory effect of steroid-free ovine follicular fluid mentioned above [24] and can prevent the atresia of subordinate follicles [28]. The lack of any inhibitory action at high levels of FSH would be an expected characteristic of such factors in vivo, since we know that high FSH levels must be able to override any inhibitory follicle-follicle interactions (such as occurs during superovulation). In vivo, it is likely that both stimulatory and inhibitory factors are produced by follicles, their production determined by the level of circulating FSH at the time and their actions modifying the FSH response of other follicles.
When FSH levels are lowered during the culture period, follicles secrete a factor(s) that inhibits the development of other like-sized follicles. This is similar to the results of Mizunuma et al. [15], who worked with preantral follicles and showed the effect of late preantral follicles on primary follicles. Follicles grown in conditioned medium in the experiments reported here were cultured in a mixture of 20 µl of conditioned medium plus 10 µl of fresh medium, the latter being sufficient to support follicle growth in culture [21]. Thus any inhibitory effect of growing follicles in conditioned medium should be due to a diffusible factor in the conditioned medium, and not merely to insufficient fresh medium present to fully support follicle growth. This is supported by the fact that when follicles were cultured in heat-inactivated conditioned medium, no inhibitory effect was seen, confirming that the inhibitory effect described above was not due to a shortage of medium, serum component, or gonadotropins in the conditioned/fresh media mix, but to inhibitory factor(s) that had been secreted into the media by the conditioning follicle. The factor(s) described here is likely to be a protein, as its effect is removed by heat inactivation. In addition, we have shown that the factor is unlikely to be activin, as conditioned media was still inhibitory in the presence of follistatin at 10 ng/ml. Although higher doses were not tried, if the inhibitory factor was activin, it is likely that conditioned media plus follistatin would at least have been less effective at inhibiting follicle growth than conditioned media alone. Our results are in contrast to those of Mizunuma et al. [15] who used coculture at earlier stages of follicle development. In that instance, the inhibitory factor did appear to be activin, and its effect was successfully abolished by the addition of 2 ng/ml of follistatin. Future work is needed to determine what the factor(s) is in the system described here, and whether it is a known factor other than activin, such as GCIF.
In the experiments described here, both the stimulation and the inhibition of growth is small, although statistically significant and highly reproducible. The magnitude of the effect is probably not surprising considering that all follicles are cultured in medium with serum and with sufficient FSH to support growth (i.e., follicles are cultured in a highly supportive environment). In addition, it is possible that there is continued secretion of stimulatory factors when FSH levels are lowered during conditioned cultures, and that the inhibitory factor needs both to overcome the effect of the stimulatory factor and further to produce an additional inhibitory effect. Likewise, when follicles are cocultured or cultured in conditioned medium in high levels of FSH throughout, follicles may also be secreting inhibitory factor(s), and any stimulatory factor produced may have to first override the effect or these inhibitory factor(s). Although the secretory factors have a relatively small effect on the growth of other follicles, that effect could still be of physiological significance: endocrine regulation of follicle dominance depends on some (dominant) follicles edging ahead in their development in comparison with other (subordinate) follicles, such that, for example, they form LH receptors on their granulosa cells and have increased angiogenesis. Even a slight inhibition in growth could delay subordinate follicles from acquiring these characteristics, thus allowing the dominant follicles to produce increased levels of estrogens and inhibins and for the resulting fall in FSH to exert its effect. In vivo, the difference between dominant and subordinate follicles grows slowly, and it is only late in the process that subordinate follicles undergo atresia. The effect, therefore, although small, could well be of physiological importance.
We report here on the effect of secretory factors on the growth of follicles. Earlier work has shown that follicle diameter correlates closely with DNA content and hence cell number when using this culture system [31]. Further experiments are needed to determine precisely what changes are occurring in the follicles as a result of these secretory factors.
When follicles were cocultured across a polycarbonate membrane in high levels of FSH throughout, a stimulatory factor was produced, affecting only one follicle in the cocultured pair. Where follicles were cultured in conditioned medium, a stimulatory effect was also found, but in this instance by the end of the culture the effect was no longer apparent (i.e., there was no difference between the size of experimental and control follicles on the final day of culture). The first experimental paradigm might have been more effective due to the proximity of the two follicles across the polycarbonate membrane. When coculturing follicles across a polycarbonate membrane, it is far from clear what determined which follicle within the pair responded to the shared environment with enhanced growth. This could possibly be the result of pre-existing differences between the apparently similar follicles at the start of culture. Certainly, the follicle producing the stimulatory factor appears not to be stimulated by this factor itself, or there would be no difference between the growth of control and conditioned-medium follicles. In a similar way, in Spears et al. [17] it was not possible to predict which follicle from a pair of follicles cocultured in contact would react with growth inhibition. As with the effect of secretory factors described here, the effect of coculturing follicles in contact is also dependent on the FSH milieu of the culture media: when FSH levels were lowered during the experiment, subordinate follicles exhibited greatly increased levels of apoptosis [32]. For cocultured follicles, whether in contact or not, it would be interesting to investigate what determines which follicle becomes dominant within a cocultured pair. Even more intriguing would be the mechanism by which the follicle producing the factors remains unaffected by them.
In conclusion, FSH can induce the production of local stimulatory factors from follicles, which are able to act on other follicles in culture that are at the same stage of development. In addition, FSH can override either the production of inhibitory factor(s) by one follicle and/or the effect of these inhibitory factor(s) on other follicles in vitro. In normal ovulatory cycles it is likely that the number of follicles that develop each cycle will be determined by a balance between local stimulatory factors, local inhibitory factors, and the level of FSH at that time. The identity of the inhibitory and stimulatory factors described here remains to be determined, as does their action in vivo.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Norah Spears, Genes and Development Group, Biomedical Sciences, Hugh Robson Building, George Square, Edinburgh EH8 9XD, U.K. FAX: 44 131 651 1706; norah.spears{at}ed.ac.uk
3 Current address: Assisted Conception Unit, Simpson Memorial Maternity Pavilion, Royal Infirmary of Edinburgh, Lauriston Place, Edinburgh EH3 9EF, U.K
4 Current address: Molecular Physiology Laboratory, University of Edinburgh, Wilkie Building, Medical School, Teviot Place, Edinburgh EH8 9AG, U.K
Received: 24 January 2002.
First decision: 21 February 2002.
Accepted: 24 May 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Pesty, O. Broca, C. Poirot, and B. Lefevre The Role of PLC{beta}1 in the Control of Oocyte Meiosis During Folliculogenesis Reproductive Sciences, September 1, 2008; 15(7): 661 - 672. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Xu, E. West, L. D. Shea, and T. K. Woodruff Identification of a Stage-Specific Permissive In Vitro Culture Environment for Follicle Growth and Oocyte Development Biol Reprod, December 1, 2006; 75(6): 916 - 923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I Demeestere, J Centner, C Gervy, Y Englert, and A Delbaere Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents Reproduction, August 1, 2005; 130(2): 147 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A Fowler and N. Spears The cultured rodent follicle as a model for investigations of gonadotrophin surge-attenuating factor (GnSAF) production Reproduction, June 1, 2004; 127(6): 679 - 688. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
