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a Gamete Biology Group, Department of Reproductive Biology, German Primate Center, D-37077 Göttingen, Germany
b Endocrinology Department, Research and Development Group, N.V. Organon, Oss, Netherlands
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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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Similar to that of other sialylated glycoproteins, the circulatory clearance rate of FSH isoforms is influenced by charge differences, with less sialylation resulting in a shorter circulatory half-life [2]. Less acidic human FSH isoforms from pituitary [3], as well as from similar preparations of recombinant FSH used in the present study [4], have been found to have a lower in vivo bioactivity than acidic isoforms, as measured by increase of ovarian weight. This type of in vivo bioassay, however, does not take species differences in clearance rates into account nor the lack of relevance of bolus applications to the natural pulsatile secretion pattern of FSH [5, 6]. Therefore it is unlikely that such studies reflect the true bioactivity of naturally secreted less acidic FSH isoforms. In contrast to results with in vivo bioassays, isohormones with less acidic pI ranges have been shown to have a higher receptor-binding activity and in vitro bioactivity as assessed by various single-cell bioassays. The contradiction between in vivo and in vitro bioactivity was discussed by Ulloa-Aguirre et al. [1].
In vitro bioassays measure either the aromatization of androgen by cultured rat granulosa [7] or Sertoli cells [8, 9] or the amount of cAMP produced by cell lines expressing human FSH receptors [10, 11]. Results obtained with in vitro bioassays have the advantage of not being complicated by half-life differences. However, each of these assays can measure only a single parameter of response. Since folliculogenesis entails a synchronized development of different cells and compartments over time, a single-parameter response is inadequate to characterize the complexity of response to FSH that the follicles undergo. Important examples of specific follicular developmental patterns include the synergistic differentiation of granulosa and thecal cells and antral development with secretion of antral fluid components. This highly complex developmental sequence requires FSH for its progression and completion [12]. The possible roles of FSH isoforms in this process are to date unknown and require investigation. The follicle culture system introduced by Nayudu and Osborn [13], in which isolated intact follicles can be grown to full maturation, provides a suitable model system by which clarification of FSH isoform actions may be achieved. The aim of the present study was to investigate whether or not three distinct isohormone fractions of human recombinant FSH have different effects on follicular development in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were collected from 18- to 20-day-old mice: F1 female offspring from C57Bl/6J females and CBA/J males from Harlan Winkelmann GmbH (Borhen, Germany). A total of 53 animals with a mean weight (± SD) of 7.92 ± 0.85 g were used for this study in a total of 12 cultures. Mice used for hypogonadotrophic (hpg) serum collection were mature (40–45 days old) offspring of hpg/bm mice (The Jackson Laboratory, Bar Harbor, ME). Homozygous hypogonadotrophic (hpg/hpg) mice were selected by visual inspection of outer and inner reproductive organs (barely visible testes and hairline-thin uteri in comparison to those of littermates). Both mice strains were kept under specific pathogen-free conditions with controlled humidity, temperature, and light regimen and fed ad libitum on a standard mouse pelleted breeding diet. Experiments were conducted according to German animal protection laws and the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. The animals were anesthetized with diethyl ether and killed by heart bleeding and subsequent cervical dislocation.
Follicle Collection
For each culture, the ovaries of at least 4 animals were collected aseptically and transferred into L-15 Leibovitz medium (Gibco 90523; Life Technologies, Eggenstein, Germany) supplemented with 3 mg/ml BSA (Sigma A9647, fraction V, Deisenhofen, Germany), 2.5 µg/ml insulin (Sigma I 1882), 0.5 µM L-glutamine (Gibco 15039), and 5 µg/ml transferrin (Sigma T5391). Follicles were isolated by needle dissection (insulin needles, 29-gauge 1/2) of one ovary pair at a time by two workers to avoid tissue anorexia. The best available follicles with centrally located oocyte, homogenous granulosa layers, and thin monolayered theca in the size range between either 110 and 210 µm or 150 and 180 µm were collected into 4-well multidishes (Nunclon; Nunc, Naperville, IL) with 
MEM (Gibco 90044). On average, 12 follicles from one mouse fulfilled these quality criteria. The MEM was prepared freshly from powder every 2 wk and stored at 4°C in glass bottles. The collection medium was supplemented with 3 mg/ml BSA, 5 µg/ml insulin (Sigma I 1882), 1 µM L-glutamine (Gibco 15039), 10 µg/ml transferrin (Sigma T5391), and 50 µg/ml L-ascorbic acid (Sigma A 4544). All supplements were stored in Eppendorf cups as stock solutions at -80°C except for the stock solution of L-ascorbic acid, which was dissolved new every 24 h. The follicles from different ovaries were pooled in the collection medium. Media vessels were maintained at 37°C during all procedures.
Culture Conditions and Media
After collection, the follicles were transferred to the final culture medium, MEM supplemented with 5 µg/ml insulin (Sigma I 1882), 1 µM L-glutamine (Gibco 15039), 0.01 mg/ml transferrin (Sigma T5391), 50 µg/ml L-ascorbic acid (Sigma A 4544), and 5% hypogonadotrophic (HPG) mouse serum. The HPG serum [14] collected from hpg/hpg mice was pooled (female and male separately), and aliquots of 100 µl were stored at -80°C in Eppendorf cups. The samples were thawed on Day 0 of the culture, pooled (50% male and 50% female), and stored during culture at 4°C, so that serum composition was identical for each day and treatment group. Except for control groups without FSH addition, the culture medium was also supplemented either with unfractionated recombinant human FSH or with one of three isoform fractions in different concentrations according to the experimental design. Follicles within the size ranges mentioned above were randomly distributed to the different treatment groups. The diameter of each follicle was measured again after transfer into the culture medium. The follicles were individually cultured for 5 days in 96-well hydrogel-coated flat-bottomed plates (Costar, Bodenheim, Germany), whereby 8 follicles were cultured on one plate. The outer wells of the plates were filled with sterile water. Each follicle was cultured in 40 µl culture medium without an oil cover and was transferred every 24 h into a well filled with freshly prepared culture medium. After the follicle had been removed, the spent culture medium was collected from each well and frozen at -80°C in Eppendorf cups. The culture was carried out in humidified incubators at 37°C and gassed with 5.5% CO2 in air.
FSH Source
Recombinant human FSH and three different isohormone fractions were donated by Organon (Oss, The Netherlands). The molecular biology and biochemistry of this FSH were described by Olijve et al. [15]. The FSH sample used had a purity of more than 95% and a biological potency of 9380 IU/mg. The fractionation of FSH was done using ion exchange chromatography. Approximately 80 mg FSH was dialyzed against 10 mM Tris-HCl (pH 7.4, buffer A) and loaded on a column of Q-Sepharose FF (25 x 1.6 cm; Pharmacia, Roosendaal, The Netherlands) equilibrated in buffer A. After application of the sample, the column was washed with buffer A, and FSH was eluted using a gradient of 30 column volumes to buffer A containing 0.2 M NaCl (flow rate 1 ml/min, 4°C). Elution was monitored at 280 nm and FSH was determined by an enzyme immunoassay specific for FSH [8]. The obtained fractions were analyzed by isoelectric focusing as described previously [4]. The isoelectric focusing profile of the FSH preparations employed in this study is shown in Figure 1. The most acidic fraction was designated acid (3.6–4.6, main peak at 3.8 = 48%), the mid acidic was designated mid (4.5–5.0, main peak at 4.8 = 50%), and the isoform with the highest pI range was designated least acidic (5.0–5.6, main peak at 5.5 = 63%) FSH. The unfractionated human recombinant FSH (unfractionated FSH) consisted mainly (63%) of fractions present in mid FSH (pI range of 4.5–4.8), whereby approximately 22% and 15% comprised the pI ranges of least acidic and acid FSH, respectively. The amount of protein in the samples was measured by UV spectroscopy (extinction = 
· conc · l = 1.05 at 1 mg/ml; l = 1; extinction = A280 nm) independently of carbohydrate (no A280 absorption). The single isoform fractions were desalted using PD10 columns (Pharmacia) equilibrated in ultrapure water (Millipore Corp., Bedford, MA) and lyophilized. The lyophilized hormone fractions were diluted in L-15 Leibovitz with 3% BSA to give 50 ng/8 µl, and 20 µl aliquots were stored at -80°C in 500-µl Eppendorf cups. The required number of aliquots was thawed immediately before use each day of the culture.
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Experimental Design
The treatment group conditions and follicle starting sizes for the FSH isoform supplementation trials were decided upon in a preliminary experiment using unfractionated human recombinant FSH. In this experiment, follicles of three different size groups (small: 110–140 µm; medium: 150–180 µm; large: 190–210 µm) were cultured at concentrations of 0, 0.5, 2.5, 10, and 50 ng/ml unfractionated FSH. Eight follicles were cultured in each treatment group (size and FSH concentration) in 4 cultures (total of 120 follicles). In each culture all treatment groups were represented. Each follicle was cultured in a constant concentration of FSH with daily changes of medium for 5 days.
In the main experiment, the three distinct isoforms (acid, mid, least acidic) were added each at three different concentrations (2.5, 10, and 50 ng/ml), giving a total number of 9 treatment groups. Each treatment group consisted of a minimum of 20 follicles (medium size: 150–180 µm), distributed over at least three different cultures. In every culture, all three isoforms were supplemented in parallel. In supplementary trials, follicles were cultured with 0.5 and 1.5 ng/ml of least acidic, 5 and 7.5 ng/ml of mid, and 100 ng/ml of acid FSH. In these experiments at least 8 follicles were cultured in every treatment group. All follicles were substituted with the same isoform and the same concentration for 5 days with fresh medium every 24 h. A total of 301 follicles were cultured in 8 different cultures.
Quantification of Follicle Parameters
The size of each follicle was measured every 24 h using a Leica (Milton Keynes, Buckinghamshire, UK) stereomicroscope ocular scale at a magnification of x50. Two different diameters were measured: 1) the diameter between the outer limits of the theca, referred to as outer follicle diameter, and 2) the diameter between the basement membrane, referred to as inner follicle diameter. For each diameter the length and width were measured and the mean was calculated. The growth profile of each treatment group was determined from values for mean inner and outer follicle diameter of all follicles on each culture day. Antral formation was observed with the stereomicroscope and photographed (Fig. 2, A and B) with an inverted microscope (Axiovert; Zeiss, Oberkochen, Germany) at a magnification of x100. The percentage of follicles showing antral formation for each culture group was calculated.
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The spent culture media samples were assayed in duplicate enzyme immunoassays for estradiol-17ß (E2). Samples were assayed without chromatography using an anti-17ß-estradiol-6 (carboxymethyloxime-BSA) antibody (E2 002; Steranti Res. Ltd., St. Albans, Hertfordshire, UK), cross-reacting 2% with estrone, 1.6% with estronesulfate, and < 0.01% with all other estrogens and progestins tested. Measurements are reported as immuno E2/ml medium. The sensitivity of the assay was 0.5 ng/ml. The intra- and interassay coefficients of variation were below 10% and 15%, respectively.
Data Analysis
Only follicles that grew and remained intact for the whole culture period (5 days) were considered for analysis. The distributions of follicle starting sizes between the treatment groups of the main experiment were not significantly different as assessed by one-way ANOVA. The growth profiles of follicles in each treatment group were assessed by calculation of the mean (± SEM) sizes of the follicles for each day of the culture. Additionally, the mean (± SEM) daily increase in size was assessed for each treatment group as an appropriate method for serial biological measurements [16]. Comparison of daily follicular sizes, daily increase in size, and the secreted amount of E2 between the treatment groups was analyzed using one-way ANOVA. Significant differences in number of follicles showing antral formation and E2 production between the culture groups were assessed with chi-square test, and for low total numbers with Fisher's exact test. Values of p < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a preliminary experiment, three different size groups of follicles were cultured with 0, 0.5, 2.5, 10, or 50 ng/ml of unfractionated FSH. Small follicles (starting size, 124 ± 2.39 µm) showed a dose-related increase in growth, but even at the highest tested concentration the follicles did not surpass preantral sizes (final size, 258 ± 7.65 µm). In contrast, large follicles (starting size, 196 ± 3.13 µm) grew rapidly for the first 3 days of culture, achieving around 90% of their maximal growth during this time period and thereafter growing only slowly to a final size of 397 ± 9.96 µm. These follicles, though, also reached final sizes of 306 ± 5.42 µm without any FSH addition.
Medium-sized follicles (starting size, 161 ± 3.42 µm) grew more linearly and at a rate intermediate between those for the small and large groups. Without FSH addition these follicles reached final sizes smaller (274 ± 2.35 µm) than the large follicles, suggesting stronger dependence on FSH addition. With FSH addition, though, these medium-sized follicles surpassed preantral sizes and with increasing doses reached final sizes that were comparable to those of large-sized follicles. It was therefore concluded that medium-sized follicles were the most appropriate for the main experiment. Follicles cultured without FSH had significantly smaller sizes from Day 2 of the culture than did those of all FSH treatment groups except for 0.5 ng/ml unfractionated FSH, which did not induce significantly larger final sizes than cultures without FSH (Fig. 3). This contrasted with the growth at higher concentrations (2.5, 10, and 50 ng/ml). Therefore these higher concentrations were used in the main experiment to compare the three FSH isoforms, with additional concentrations used where necessary to estimate threshold concentrations.
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Comparison of Follicular Growth Patterns (Outer Follicle Diameter) among the FSH Isoforms
Least acidic FSH at a concentration of 2.5 ng/ml produced a final follicle size that was significantly larger (373 ± 5.80 µm) than those achieved with acid or mid FSH (< 325 µm) (Fig. 4, A1). This difference was related to a significantly higher growth rate between Days 2 and 3 seen in the presence of least acidic FSH (57 ± 2.08 µm/day) compared to the growth rate of the other two isoforms (24 ± 2.3 and 28 ± 4.92 µm/day for mid and acid FSH, respectively) (Fig. 4, A2). As a result of this growth enhancement between Days 2 and 3, the follicles cultured with least acidic FSH were significantly larger from Day 3 of the culture until Day 5, although the rate of growth on all other culture days was not significantly different from that achieved with the other two isoforms. No significant differences in rate of growth or final size reached could be detected between the acid and mid isoform treatments. Follicles cultured with 2.5 ng/ml unfractionated FSH showed a growth profile intermediate between those of least acidic FSH and the other two isoforms, reaching sizes similar to those with least acidic FSH on the last day of the culture.
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At a concentration of 10 ng/ml, least acidic FSH induced a significantly larger final size of follicles (385 ± 4.64 µm) than the other two isoforms (Fig. 4, B1). This was due to a specific increase in growth (62 ± 4.08 µm/day) between Days 1 and 2 of the culture, one day earlier than at 2.5 ng/ml. During this period (Days 1–2), both acid and mid FSH induced significantly less increase in diameter (both approximately 43 µm/day) than least acidic FSH (Fig. 4, B2). At all other days of the culture, the daily growth rate was not significantly different between least acidic FSH and the other two isoforms. Thus, follicles cultured with 10 ng/ml least acidic FSH remained significantly larger from Day 2 until Day 5 of the culture due to a rapid increase in growth over a single 24-h period. Mid and acid FSH induced similar growth rates during the first 4 days of culture; however, in cultures with acid FSH the growth rate was not sustained during the last 24 h, and the increase of follicle diameter was significantly less (11 ± 4.93 µm) than with mid FSH (36 ± 3.48 µm). Therefore the final size obtained with mid FSH was significantly larger (350 ± 6.49 µm) than for follicles cultured with acid FSH (326 ± 8.03 µm). The unfractionated FSH produced growth patterns intermediate between those with least acidic FSH and the other two isoforms. In contrast to observations with lower concentrations, the growth patterns and mean final sizes induced by the highest concentration tested (50 ng/ml FSH) were similar for all isoforms and unfractionated FSH (final size approximately 360 µm). This effect was produced by an increase in growth of follicles cultured with acid and mid FSH and a reduction of growth with least acidic FSH in comparison to the lower concentrations.
Threshold Concentration and Proportion of Antral Formation for Each FSH Isoform
Antral formation could be observed only in living follicles with a size above 340 µm (outer follicle diameter). As shown in Table 1, the concentration of FSH required to induce an increase in mean follicle diameter beyond 340 µm (threshold concentration) differed according to the FSH isoform. At 2.5 ng/ml, only follicles cultured with least acidic FSH reached mean final sizes above 340 µm (Fig. 4, A1). In a supplementary series, follicles were also cultured with lower doses (1.5 and 0.5 ng/ml) of least acidic FSH. The threshold size for antral formation was surpassed only in cultures with 1.5 ng/ml (final size, 346 ± 9.7 µm), the threshold concentration for least acidic FSH. Follicles cultured with 0.5 ng/ml reached significantly smaller final sizes (300 ± 7.32 µm). At 2.5 ng/ml least acidic FSH, a significant increase in the final size of follicles compared to that in cultures with 1.5 ng/ml was observed; however, the addition of 10 ng/ml least acidic FSH did not induce further increase in final size. At 50 ng/ml of least acidic FSH, the final size reached (358 ± 6.55 µm) was significantly smaller than the final sizes obtained with 2.5 and 10 ng/ml, suggesting that this concentration exceeded the optimal range.
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Trials with two additional concentrations of mid FSH (5 and 7.5 ng/ml) revealed that the final size of follicles grown with 7.5 ng/ml mid FSH was significantly larger than with 5 ng/ml and reached the critical size for antral formation, while follicles grown with 5 ng/ml did not. Therefore the threshold concentration for mid FSH was 7.5 ng/ml and was higher than for least acidic FSH. Higher doses of mid FSH (10 and 50 ng/ml) did not induce growth of follicles to significantly larger final sizes. The threshold concentration for acid FSH with 50 ng/ml was the highest for the three isoforms. In supplementary trials with 100 ng/ml acid FSH, the final sizes obtained (368 ± 4.62 µm) were not significantly larger than those with 50 ng/ml acid FSH (355 ± 7.1 µm).
Apart from different threshold concentrations, the proportion of follicles forming antra also differed among the isoforms (Table 1). At all concentrations above 1.5 ng/ml, least acidic FSH induced antral formation in more than 70% of follicles that grew larger than 340 µm. Mid FSH could only induce comparable proportions of antral formation in these larger follicles at and above 7.5 ng/ml, whereas acid FSH even at its threshold concentration of 50 ng/ml induced antral formation in only 56% of the follicles larger than 340 µm.
Comparison of Increase of Inner Follicle Diameter (Granulosa Compartment) among the FSH Isoforms
For the results described in the previous sections, follicle growth was calculated from the whole diameter measured (outer follicle diameter). However, separate evaluation of the inner follicle (Fig. 5) revealed differences in early growth that were not evident when follicle growth was considered as a whole. During the first 24 h of culture with mid and acid FSH, the inner follicle diameters did not increase significantly regardless of concentration. This contrasted with the significant increase of the outer follicle diameter during this time; therefore follicle growth was due mainly to enhancement of the theca. In contrast, the inner follicle diameter did increase significantly (21 ± 2.7 µm) during the first 24 h in cultures with 2.5 ng/ml of least acidic FSH. As a result of this rapid initial growth, the inner follicle diameters obtained with least acidic FSH on Day 1 (189 ± 4.73 µm) and Day 2 (216 ± 7.94 µm) of the culture were significantly larger than those produced by acid FSH (170 ± 3.53 and 191 ± 6.36 µm on Days 1 and 2, respectively). With mid FSH, intermediate sizes of the inner follicle diameter were produced. This differential inner growth contrasted with the lack of difference in outer follicle diameter among the isoforms on these days (compare Fig. 4, A1). The difference between acid and least acidic FSH in the early development of outer and inner follicle diameter is illustrated in Figure 6.
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E2 Secretion Onset and the Proportion of Secreting Follicles Induced by the FSH Isoforms
The secretion of E2 by each follicle was assessed at three different concentrations (2.5, 10, and 50 ng/ml) for the three isoforms. At 2.5 ng/ml, the amount of E2 secreted on Day 5 of the culture was significantly higher in follicles cultured with least acidic FSH (2.53 ± 0.42 ng/ml) than in those with mid (0.74 ± 0.01 ng/ml) and acid FSH (1.6 ± 0.16 ng/ml). The amount of E2 secreted after 5 days of culture with the same concentration of unfractionated FSH was 1.44 ± 0.87 ng/ml. Follicles cultured without FSH supplementation did not secrete detectable amounts of E2. The mean average amount of estrogen produced increased with higher concentrations for all isoforms, but as a result of high intragroup variation of E2 secretion there were no significant differences between the isoforms at the two higher doses. Apart from the amount of estrogen secreted, the follicle culture system also allowed the assessment of the onset day of E2 secretion. During the first 24 h of the culture, none of the follicles secreted measurable amounts of E2. From Day 2 until end of the culture, E2 was secreted by a variable proportion of follicles according to isoform and dose. The most striking difference between the isoforms was obtained with cultures at 2.5 ng/ml (Fig. 7A). At this concentration, starting on Day 2 with 14% of the follicles, least acidic FSH induced a progressively increasing proportion of follicles to secrete E2. In contrast, follicles cultured with mid or acid FSH at a dose of 2.5 ng/ml did not secrete E2 until Day 5, and even then the percentage of secreting follicles was significantly lower with both mid (25%) and acid (13%) than with least acidic FSH (86%).
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At a concentration of 10 ng/ml mid FSH, E2 secretion was induced in an increasing proportion of follicles similar to that with least acidic FSH, both initiating E2 secretion on Day 2 (Fig. 7B). The proportion of follicles secreting E2 increased significantly between Day 3 and Day 4 of the culture for both least acidic and mid FSH. In contrast to the other two isoforms, acid FSH induced E2 secretion at this concentration only on Day 4 in 47% of the follicles, increasing to proportions similar to those for the other two isoforms on Day 5 of the culture. At the highest concentration tested (50 ng/ml), the day of initial E2 secretion was delayed to Day 3 for least acidic FSH (Fig. 7C). The pattern of E2 secretion of mid and acid FSH at 50 ng/ml was similar to the pattern produced at 10 ng/ml. Acid FSH was unique in that no follicle secreted E2 before Day 4 independent of dose, although at 10 ng/ml the growth trajectories of acid FSH were similar to that of mid FSH (Fig. 4, B1) and to those for both other isoforms at 50 ng/ml. Therefore the difference was not primarily due to smaller follicular size.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effect of FSH on follicular growth at the cellular level is expressed as coordinated alterations in cell functions and organization over time. Follicular growth arises from an increase in cell number as a result of granulosa and theca cell proliferation as well as from the formation of the antrum filled with antral fluid, a critical follicular differentiation stage. The larger final sizes of follicles reached with least acidic FSH were due mainly to a specific increase in growth rate during Day 2 or 3 of the culture, depending on dose. Such specific growth during preantral follicle development could not be induced in follicles cultured with the other two isoforms at any dose. This rapid follicular growth induced by least acidic FSH most probably consisted mainly of granulosa cell proliferation, as indicated by the specific increase of the inner follicle diameter. In contrast, 10 ng/ml mid FSH induced more follicular growth during the last 24 h of culture than did the other two isoforms, resulting in follicles with larger final sizes than those cultured with acid FSH. Since 10 and 50 ng/ml of mid FSH also induced higher percentages of antral formation than acid FSH, it is proposed that this last increase in diameter was due mainly to the antrum-forming potency of this isoform. This stage-specific effect on preovulatory development, however, was not as marked as the growth surge induced earlier by the least acidic FSH. In contrast to the other two isoforms, acid FSH did not have a developmental stage-specific effect. Although the possibility exists that this isoform may act preferentially on earlier stages of follicle development, it revealed a lower bioactivity on the late preantral and antral follicle stages supported by this culture system. This finding suggests a functional explanation for the reported in vivo reduction of comparable FSH subfractions occurring during midcycle [18].
Antral formation in this study could be detected only in follicles larger than a critical threshold size. Once antral formation has commenced, growth is no longer a function of cell proliferation alone, as antral formation becomes an important factor. Regarding only those follicles that had surpassed the threshold size, differences were also found among the capacities of the isoforms to induce antral formation. Acid FSH had a lower capacity to induce antral formation within 5 days of culture than had the other two isoforms even at the highest concentration tested. This indicates that size is not the only factor influencing antral formation and that the cellular signal necessary for antral fluid secretion and formation of organized space between antral granulosa cells and cumulus cells is weaker with acid FSH.
As variable thecal width contributed to total follicular size and therefore blurred the specific effect of the FSH isoforms on the granulosa cells, the inner follicle diameter was also analyzed separately. During the first 2 days of culture, a large proportion of the increase in outer follicle diameter was found to be due to thecal growth for all isoforms. Comparison among the isoforms indicated that the application of acid FSH resulted in less inner follicle growth and more enlargement of the theca during the first culture period than did least acidic FSH. Since the growth of the granulosa cell compartment and that of the theca were found to be inversely related, growth of the whole follicles during this early culture period was not different among the isoforms. The same relationship, broad thecal structures in follicles with smaller inner follicle diameters, also occurred in cultures with suboptimal doses of least acidic FSH, indicating a general relationship between lack of inner follicle development and thecal growth. Thecal overgrowth has been previously seen by Nayudu (unpublished results) to be associated with poor in vitro follicle growth and has been suggested to be related to atresia in morphology studies [19, 20], but the phenomenon has not yet been critically evaluated. Since no evidence of FSH receptors on thecal cells has been reported, the influence of FSH on the follicular theca must therefore be assumed to be indirect. Paracrine factor(s) secreted by granulosa cells are known to influence theca cell differentiation [21]. FSH subfractions that have a low capacity to induce growth of the granulosa compartment (i.e., acid FSH) may therefore produce a physiological balance permissive of thecal broadening.
Findings on the effect of isoforms on steroid production are to date confined to investigations with isolated cells [22, 23]. Less acidic FSH isohormones have been shown to induce more estrogen production by isolated granulosa cells than the more acidic ones [24]. However, these studies could not assess onset of E2 secretion and relate it to other parameters of follicle development. In the present study, least acidic FSH was shown to induce E2 secretion earlier and at a lower dose than the other isoforms, with a proportion of follicles commencing E2 production before antral formation. Mid FSH produced a similar effect but required a higher concentration. In contrast, follicles cultured with acid FSH, regardless of concentration, secreted estrogen only on the last 2 days of culture. At the highest dose, however, least acidic FSH induced E2 secretion onset 1 day later than at lower doses. This could be related to the lower growth potential of the follicles at this dose, as the final size obtained was smaller than at lower doses. Both these parameters could be indicators for an overdosing effect obtained with increasing concentrations of least acidic FSH.
The preantral estrogen production induced by least acidic and mid FSH may in fact be a stimulating factor in inducing granulosa cell differentiation and earlier antral formation, as estrogen is known to act as a mitogen on granulosa cells [25, 26] and to augment the action of FSH on antrum formation [27]. The possibility therefore exists that the timing of initial E2 production may be of greater importance for the productive follicle than the absolute amounts produced during antral development.
In summary, using in vitro murine follicle development as a model system, we have provided the first evidence that FSH isohormones can induce different follicular development. Least acidic FSH was shown to have the highest bioactivity by production of rapid preantral follicle growth, antral formation, and initiation of E2 secretion in the preantral follicle at lower concentrations than mid and acid FSH. Mid FSH required higher concentrations in order to produce an effect similar to that with least acidic FSH, thereby demonstrating lower bioactivity. Mid FSH had an additional effect on later antral growth, shown as a selective increase in growth during the last day of culture. Acid FSH was shown to have the lowest bioactivity of the three FSH subfractions, as indicated by the highest threshold concentration for antral formation and E2 secretion that commenced only at the end of culture. These results are expected to provide a basis for further fundamental study of the role of FSH isoforms in the modulation and regulation of follicle growth and differentiation.
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FOOTNOTES |
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2 Correspondence: P.L. Nayudu, Gamete Biology Group, Department of Reproductive Biology, German Primate Center, Kellnerweg 4, D-37077 Göttingen, Germany. FAX: (49)551–3851–288; pnayudu{at}gwdg.de
Accepted: May 26, 1998.
Received: March 9, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 C. R. West, N. E. Carlson, J. S. Lee, A. S. McNeilly, T. P. Sharma, W. Ye, and V. Padmanabhan Acidic Mix of FSH Isoforms Are Better Facilitators of Ovarian Follicular Maturation and E2 Production than the Less Acidic Endocrinology, January 1, 2002; 143(1): 107 - 116. [Abstract] [Full Text] [PDF]
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U. A. Vitt, P. L. Nayudu, U. M. Rose, and H. J. Kloosterboer Embryonic Development after Follicle Culture Is Influenced by Follicle-Stimulating Hormone Isoelectric Point Range Biol Reprod, November 1, 2001; 65(5): 1542 - 1547. [Abstract] [Full Text] [PDF]
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 C.Y. Andersen, L. Leonardsen, A. Ulloa-Aguirre, J. Barrios-De-Tomasi, K.S. Kristensen, and A.G. Byskov Effect of different FSH isoforms on cyclic-AMP production by mouse cumulus-oocyte-complexes: a time course study Mol. Hum. Reprod., February 1, 2001; 7(2): 129 - 135. [Abstract] [Full Text] [PDF]
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J. Wu, B. R. Emery, and D. T. Carrell In Vitro Growth, Maturation, Fertilization, and Embryonic Development of Oocytes from Porcine Preantral Follicles Biol Reprod, January 1, 2001; 64(1): 375 - 381. [Abstract] [Full Text]
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G. Horsman, J.A. Talbot, J.D. McLoughlin, A. Lambert, and W.R. Robertson A biological, immunological and physico-chemical comparison of the current clinical batches of the recombinant FSH preparations Gonal-F and Puregon Hum. Reprod., September 1, 2000; 15(9): 1898 - 1902. [Abstract] [Full Text] [PDF]
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J. Wu, P. L. Nayudu, P. S. Kiesel, and H. W. Michelmann Luteinizing Hormone Has a Stage-Limited Effect on Preantral Follicle Development In Vitro Biol Reprod, July 1, 2000; 63(1): 320 - 327. [Abstract] [Full Text]
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 M. P. Rose, R. E. Gaines Das, and A. H. Balen Definition and Measurement of Follicle Stimulating Hormone Endocr. Rev., February 1, 2000; 21(1): 5 - 22. [Abstract] [Full Text]
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G. Bédécarrats, D. Guémené, C. Morvan, U. Kühnlein, and D. Zadworny Quantification of Prolactin Messenger Ribonucleic Acid, Pituitary Content and Plasma Levels of Prolactin, and Detection of Immunoreactive Isoforms of Prolactin in Pituitaries from Turkey Embryos During Ontogeny Biol Reprod, September 1, 1999; 61(3): 757 - 763. [Abstract] [Full Text]
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C. Y. Andersen, L. Leonardsen, A. Ulloa-Aguirre, J. Barrios-De-Tomasi, L. Moore, and A.G. Byskov FSH-induced resumption of meiosis in mouse oocytes: effect of different isoforms Mol. Hum. Reprod., August 1, 1999; 5(8): 726 - 731. [Abstract] [Full Text] [PDF]
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