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Regular Article |
a Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands
ABSTRACT
Preantral follicles (140–160 µm) were isolated mechanically from the ovaries of 10-day-old rats and cultured in groups of four to six for 6 days in medium containing 0, 1, 10, or 100 ng/ml of activin A. Activin stimulated (P < 0.05) the growth of preantral follicles in a dose-dependent fashion and enhanced the proliferation of preantral, oocyte-free follicular cells. Furthermore, treatment with activin induced the majority of follicles to form an antrum-like structure and helped to maintain the ultrastructure of these follicles during culture. Activin A also induced further changes characteristic of follicle and oocyte maturation, such as the elongation of granulosa cells contacting the oocyte and migration of the cortical granules to the oocyte cortex. In addition, gene expression for activin and activin receptor type II (ActR II) was demonstrated in both the oocytes and the somatic cells using the reverse transcription-polymerase chain reaction, and immunohistochemical studies demonstrated the presence of activin and ActR II proteins in the somatic tissue and, especially, the oocytes of these follicles. It is concluded that, in vitro, activin A stimulates the growth of rat preantral follicles and promotes antrum formation. Furthermore, because activin A and ActR II are synthesized within preantral follicles, intrafollicular activin likely plays an important role in early follicular development.
activin, follicle, follicular development
INTRODUCTION
It is generally accepted that pituitary gonadotropins play a vital role in stimulating folliculogenesis [1]. However, evidence is increasing that folliculogenesis is also subjected to important paracrine/autocrine regulation exerted by a variety of local intraovarian factors. Among these proposed intraovarian regulators, inhibin and activin are structurally related proteins that belong to the multifunctional transforming growth factor ß family. Inhibins are heterodimers consisting of an 
subunit and one of the two ß subunits (ßA or ßB), whereas the activins consist of two ß subunits and include the ß subunit homodimers (ßAßA and ßBßB) and the heterodimer (ßAßB). Classically, inhibins have been described as ovarian feedback regulators of pituitary gonadotropin release; however, they are now known to play important paracrine/autocrine roles within the reproductive system [2, 3]. Ovarian expression of mRNA for the inhibin subunits and for their related ligands changes with the stage of the estrous cycle and, indeed, is regulated by both gonadotropins and reproductive steroid hormones [4]. Furthermore, the exact inhibin-family molecules that are produced may depend on the stage of follicular development, because in primates [5] and rodents [6], ß subunit mRNA is relatively more abundant in the granulosa cells of immature antral follicles whereas subunit mRNA predominates in the granulosa cells of preovulatory follicles [7]. On the other side of the equation are two types of activin receptors, designated as type I (ActR I and ActR IB) and type II (ActR II and ActR IIB), and both are serine/threonine kinases. However, whereas type II receptors can bind activins directly, type I receptors cannot. Instead, the activin must first be phosphorylated within a type II receptor-activin complex. Furthermore, the association of activin with both a type I and a type II receptor is necessary for signal transduction [3, 8].
In vitro, activin enhances aromatase activity and suppresses FSH-induced progesterone synthesis in rat granulosa cells. Therefore, it has been proposed as a local modulator of granulosa cell steroidogenesis [9]. Some inconsistencies, however, have been found with regard to the effects of activin A on the development of preantral follicles in vitro. For example, Woodruff et al. [10] reported that the direct injection of activin A into isolated, immature rat ovaries resulted in atresia of preantral follicles, whereas Li et al. [11] reported that activin promoted the in vitro development of theca-free granulosa-oocyte complexes isolated from similar follicles. Similarly, Yokota et al. [12] reported that activin A had a stimulatory effect on cultured, intact preantral follicles recovered from immature mice but an inhibitory effect on similar follicles recovered from adult animals.
In the present study, the effect of activin A on the growth and development of immature rat preantral follicles was investigated. To assess the quality of cultured follicles, emphasis was put on detailing ultrastructural differences in follicles cultured in the presence or absence of activin A. In addition, to determine how activin A exerts its effects and to explore the existence of an endogenous activin A regulation system within preantral follicles, mRNA expression and protein localization studies were performed for both activin A and ActR II.
MATERIALS AND METHODS
Isolation of Preantral Follicles
Ten-day-old female Wistar rats-CPB ("specific pathogen free"; supplied by the Department of Laboratory Animal Science, Utrecht University, and approved for use by the University's Ethical Committee) were killed by decapitation, and their ovaries were removed and transferred, at 37°C, to a Petri dish (Costar, Corning Life Sciences BV, Badhoevedorp, The Netherlands) containing Dulbecco modified Eagle medium and Hepes/Ham F12 (Gibco Life Technologies, Breda, The Netherlands) supplemented with 0.3% (w/v) BSA (Sigma Chemical Co., St. Louis, MO) and a mixture of antibiotics and antimycotics (penicillin, 100 IU/ml; streptomycin, 100 µg/ml; and fungizone, 250 ng/ml; all from Gibco). Preantral follicles (defined as follicles in which definite antrum formation was not yet detectable) were dissected out under stereomicroscopic control using fine needles and collected with micropipettes. The preantral follicles selected for further analysis were those between 140 and 160 µm in diameter, with more than two layers of granulosa cells, and with visible theca cells.
Culture Media and Coating of Culture Wells
To ensure better follicle attachment, the surfaces of 48-well cell-culture plates (Costar) were coated with collagen as described previously [13]. For this, type I collagen [14] (a gift from the Laboratory of Experimental Dermatology, University of Liege, Belgium) was prepared using the method described by Figueiredo et al. [15]. Briefly, collagen type I was dissolved in 0.1 M acetic acid at a concentration of 4.2 mg/ml and, immediately before use, mixed with an equal volume of 2x concentrated Medium 199 (Gibco). The pH of the resulting solution was adjusted to 7.2 before coating the culture plates, which were incubated at 37°C for 10–15 min before any culture medium was added to the wells.
The basic culture medium used was alpha minimal essential medium supplemented with 0.3% BSA, antibiotics (penicillin, 100 µg/ml; streptomycin, 100 µg/ml; and fungizone, 250 ng/ml), ITS (insulin, 6.25 µg/ml; transferrin, 6.25 µg/ml; and sodium-selenite, 6.25 ng/ml), 0.23 mM pyruvate, and 2 mM glutamine. In addition, recombinant human FSH (Org 32489; Organon, Akzo-Nobel, Oss, The Netherlands) was added to the medium at a concentration of 0.05 IU/ml, because we had shown previously that FSH is necessary for follicle survival in this system [13] and that 0.05 IU/ml to be the optimal FSH concentration for bovine preantral follicle culture [15].
Experimental Conditions
Preantral follicles between 140 and 160 µm in diameter were cultured in groups of four to six in 400-µl aliquots of culture medium containing 0, 1, 10, or 100 ng/ml of activin A (recombinant bovine activin A; Innogenetics, Ghent, Belgium) [16]. As described previously [13], follicles within a group were spaced evenly throughout the well, with an approximate distance of three to six follicular diameters (
700 µm) between adjacent follicles. The medium was then covered with a 75-µl layer of mineral oil, and the follicles were incubated for 6 days at 37°C in an atmosphere of 5% CO2 in air. On Days 2 and 4 of each incubation, half the culture medium was removed and replaced by fresh medium. Three replicates were performed for each concentration of activin A.
Follicle Growth and Cell Proliferation
Follicle growth was investigated by comparing follicular diameter before and after the 6-day incubation using an inverted microscope (Olympus IMT-2; Olympus, Hamburg, Germany) with an eyepiece micrometer at a magnification of 40x. Both oocyte and follicular diameters were recorded on Day 0, and the measurements were repeated on Day 6 in the surviving follicles. The follicular diameter was defined as the maximum diameter measured within the basal membrane. Follicle survival was determined using an inverted microscope as described previously [13]. Briefly, follicles with a bright appearance were considered to be viable, and those with obvious, dark clusters of granulosa cells were considered to be degenerate (dead).
Follicle cell proliferation was investigated by quantifying the DNA content of preantral follicles using Hoechst 33258 staining (bisbenzimidazole; Sigma) and a Hoefer DyNA Quant 200 fluorometer (excitation, 365 nm; emission, 460 nm; Pharmacia Biotech, Roosendaal, The Netherlands) in a modification of the technique described by Boland and Gosden [17]. The analysis was performed on large preantral follicles that were either freshly isolated or had been cultured for 6 days with or without 1 ng/ml of activin A added to the culture medium. Freshly isolated follicles were analyzed in groups of 10 and precultured follicles in groups of 5. To release the DNA, follicles were first immersed in 50 µl of 10x trypsin solution (Sigma) in a V-well assay plate (Costar) and incubated at 37°C. After 30 min of digestion, the follicles in each well were crudely teased apart and reimmersed in the trypsin solution for a further 30 min at 37°C. Next, the contents of each well were mixed by pipetting three times, and the plate was incubated for a further 15 min. At the end of this time, the digestion was stopped by adding 50 µl of a trypsin inhibitor (4.4 mg/ml in double-distilled deionized water; Sigma), and the contents of the wells were neutralized using 100 µl of alkaline double-distilled deionized water (pH 11.2). Finally, the contents of each well were mixed thoroughly, and the DNA determination was carried out. As a negative control, wells containing 100 µl of double-distilled water, 50 µl of trypsin, and 50 µl of trypsin inhibitor were similarly analyzed with the fluorometer, where the latter was calibrated using a method described previously [13].
Follicle Morphology
Follicle quality was evaluated using transmission electron microscopy (TEM) to examine the ultrastructure of follicles cultured for 6 days in media containing 0 or 100 ng/ml of activin A (n = 10 randomly selected follicles in each group). In addition, follicles harvested from 10-day-old rats cultured for 6 days were compared with those freshly isolated from 17-day-old rats (n = 10 randomly selected follicles). Follicles intended for TEM were fixed by immersion for 2 h at room temperature in Karnovsky fixative and then incubation for 30 min at 4°C in 1% osmium tetraoxide in a 0.1 M cacodylate buffer. After routine dehydration in alcohol, the follicles were embedded in epoxy resin in beem capsules. Thin sections of the fixed follicles were cut with glass knives and stained with 1% toluidine blue. Ultrathin sections were double-stained with 2% uranyl acectate and 0.4% lead citrate before being examined under a Philips CM10 electron microscope (Philips, Eindhoven, The Netherlands) at 80 kV.
Extraction of RNA from Isolated Intact and Oocyte-Free Preantral Follicles
One rat ovary, 50 complete rat preantral follicles, and 50 oocyte-free preantral follicles (defined as somatic follicular tissue) were isolated and washed in PBS. Each of these three different categories of tissue were then placed in separate Eppendorf tubes, to which 500 µl of Ultraspec (Biotecx Laboratories, Inc., Houston, TX) were added. All three samples were homogenized by briefly vortexing, and the suspensions were stored at -20°C until the RNA was extracted.
The RNA extraction was performed in the manner described previously by Izadyar et al. [18]. The samples were thawed slowly on ice, and 15 µg (5 µg/ml) of polyinosonic acid (Boehringer, Almere, The Netherlands) were added to each as a carrier. The total RNA isolation was then performed essentially as recommended by Biotecx (Biotecx, bulletin 27, 1992). Briefly, 100 µl of chloroform were added to each thawed sample, and the samples were homogenized by vortexing for 10 sec. Next, the homogenate was incubated for 5 min at 4°C (on ice) before being centrifuged at 13 000 x g for 15 min. Approximately 80% of the supernatant was then transferred to a fresh Eppendorf tube, and the RNA was precipitated by adding an equal volume of isopropanol. The new suspension was then incubated for at least 30 min at -80°C and centrifuged for 30 min at 13 000 x g and 4°C. The supernatant was discarded, and the pellet was washed once with 0.5 ml of ice-cold 75% ethanol. After gentle inversion of the tube, the suspension was once again centrifuged for 15 min at 13 000 x g. The resulting supernatant was removed completely, and the pellet containing the RNA was resuspended in 50 µl of RNAse-free water containing 80 U/ml of nuclease inhibitor (Promega, Leiden, The Netherlands) and stored at -20°C.
Extraction of mRNA from Oocytes Isolated from Rat Preantral Follicles
Preantral follicles isolated mechanically from rat ovaries were crudely torn apart to release the oocyte, which usually retained a small number of granulosa cells on its surface. The oocytes were then vortexed for 1 min to remove any remaining granulosa cells, and thus denuded, the oocytes were collected and rinsed in PBS before being stored at -80°C (50 oocytes/Eppendorf tube). The Dynabeads mRNA Direct Micro kit (Dynal ASA, Oslo, Norway) was used to extract mRNA from these oocytes using the protocol described in the product handbook. In short, 100 µl of Lysis/binding buffer (100 mM Tris-HCl [pH 8.0], 500 mM LiCl, 10 mM EDTA, 1% [w/v] lithium dodecysulfate [LiDS], and 5 mM dithithreitol [DTT]) were added to the frozen oocyte sample, and the mixture was pipetted repeatedly to ensure complete lysis of the oocytes. Next, 20 µl of prewashed Dynabeads oligo(dT)25 were added to each tube, and the two solutions were mixed thoroughly. After a 5-min incubation at room temperature to allow the binding of poly(A)+ to the oligo(dT) Dynabeads, the beads were removed using a Dynal MPC-E-1 magnetic separator and washed twice in 100 µl of washing buffer 1 (10 mM Tris-HCl [pH 8.0], 0.15 M LiCl, 1 mM EDTA, and 0.1% [w/v] LiDS) and a further two times in 100 µl of washing buffer 2 (10 mM Tris-HCl [pH 8.0], 0.15 M LiCl, and 1 mM EDTA). Poly(A)+ RNAs were then eluted from the beads by incubating them in 10 ml of Tris-HCl buffer at 65°C. Aliquots of the supernatant were immediately subjected to reverse transcription (RT).
Reverse Transcription
In preparation for the RT reaction, 10 µl of an RNA sample were incubated for 5 min at 70°C and then chilled on ice. Reverse transcription was performed in a total volume of 20 µl, made of 10 µl of sample RNA, 4 µl of 5x reverse transcriptase buffer (Gibco BRL, Breda, The Netherlands), 8 U of RNAsin (Promega), 150 U of superscript II reverse transcriptase (Gibco BRL), and 0.5 µg of oligo(dT)12–18 primer and containing DTT and each deoxynucleoside 5'-triphosphate (dNTP) at final concentrations of 0.01 M and 0.5 mM, respectively. The mixture was incubated for at least 1 h at 42°C and then for 5 min at 95°C before being stored at -20°C.
Amplification of Activin A and ActR II cDNAs by Polymerase Chain Reaction
Before carrying out RT-polymerase chain reaction (RT-PCR) with rat activin A and ActR II specific primers, all samples were first amplified with ß-actin specific primers to confirm the success of RNA isolation and cDNA production. Only samples that demonstrated a clear ß-actin product of the expected size were used for RT-PCR with the specific primers
The oligonucleotide primers used for the amplification of activin A and ActR II are described in Table 1. Primers were designed according to the base of the sequence that crosses the exon-intron boundary to avoid the possible influence of genomic DNA contamination. Amplification was performed in two steps, and after the first round of 40 cycles, heminesting of all samples was performed to increase the yield of the final PCR product and to exclude the possible genomic DNA contamination. The heminesting was performed using the same 5'-primers used in the first round in combination with new 3'-primers.
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The PCR reaction was carried out in 200-µl tubes (Eurogentec, Seraing, Belgium) and used 2 µl of cDNA as a template in a total reaction volume of 50 µl, which also contained PCR buffer (final concentration: 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, and 0.5 µM of each primer) and 1.25 U of TaKaRa Taq polymerase (Boehringer Ingelheim, Alkmaar, The Netherlands). The thermal cycling for the first round consisted of an initial denaturating period of 5 min at 94°C, followed by 40 cycles of 15 sec at 94 °C, then 30 sec at 57°C (for activin A) or 52°C (for ActR II), and finally, 45 sec at 72°C. Chain extension was allowed to occur during a subsequent 10-min incubation at 72°C. For heminesting, 2 µl of the first-round products were transferred to another 200-µl tube containing 48 µl of PCR buffer, and this was amplified for 30 cycles using the above profile, with the exception that the annealing temperatures were changed to 56°C for activin A and 51°C for ActR II, respectively. All PCRs were performed in a 24-well thermocycler (Perkin-Elmer, Gouda, The Netherlands), and thereafter, 10 µl of the second-round product were resolved by electrophoresis (Eurogentec) on a 1% agarose gel at 90 mA for 45 min. A negative control was performed by using MilliQ water as a template for the PCR, and rat liver was used as a positive control for activin A. A 100-base pair (bp) ladder (Gibco BRL) was also run on the agarose gel to act as a reference for fragment size. After electrophoresis, the DNA was stained with 1 µg/ml of ethidium bromide for 5 min and then visualized by exposing the gel to ultraviolet illumination for 4 sec. An image of the gel was taken using a CCD camera (Appligene; B & L Systems, Zoetermeer, The Netherlands) and stored in digitized form.
Restriction Enzyme Analysis
To verify the identity of the RT-PCR products, an analysis of restriction endonuclease fragments was performed. The amplified sequences for activin A and ActR II have single restriction endonuclease digest sites for AluI and PstI, respectively (Eurogentec). For activin A, the expected restriction fragments are 244 and 133 bp, and for ActR II, they are 295 and 151 bp. The digestion was performed by mixing 10 µl of the second PCR product with 1 µl (12 U) of enzyme, 2 µl of buffer, and 7 µl of water to yield 20 µl of reaction mix containing 33 mM Tris-acetate (pH 7.9 at 37°C), 10 mM Mg-acetate, 66 mM K-acetate, and 0.1 mg/ml of BSA for AluI or 10 mM Tris-HCl (pH 8.0 at 37°C), 5 mM MgCl2, 100 mM NaCl, and 0.1 mg/ml of BSA for PstI. The mixture was incubated for 1 h at 37°C, and finally, 10 µl of the mixture were resolved by electrophoresis on a 1% Agarose gel, as described previously.
Immunohistochemical Localization of Activin A and ActR II
To confirm that the mRNA for activin A and ActR II was actually translated into protein within preantral follicles and to determine the location of these proteins, immunohistochemical staining was performed. Ovaries were collected from 10-day-old rats, immersion-fixed for 24 h in 4% buffered formaldehyde (pH 6.8–7.2; Klinipath BV, Duiven, The Netherlands), dehydrated, and embedded in paraffin (Stemcowax; Adams Instruments BV, Amerongen, The Netherlands).
For each of activin A and ActR II, protein localization was performed on 30 sections selected randomly from serial 5-µm sections of 10 ovaries collected from five rats. These sections were mounted on 3-aminopropyl triethoxysilane (TESPA; Sigma)-coated slides and dried overnight at 37°C. Immunohistochemistry was performed using a modified version of the method described by Schrans-Stassen et al. [21]. Briefly, after removing the paraffin and washing the slides in PBS (pH 7.4), endogenous peroxidase was blocked by a 10-min incubation in 0.35% H2O2 (Merck, Darmstadt, Germany) in PBS. The slides were then rinsed twice in PBS buffer and once in PBS/0.2% Tween (Merck) before being incubated for 1 h in PBS supplemented with 5% normal serum and 5% BSA fraction V to minimize any background signals. Next, the slides were incubated overnight with the primary antisera (Table 2) in PBS supplemented with 1% BSA at room temperature in a moist chamber. The following day, the slides were rinsed three times for 15 min in PBS and then incubated with the secondary antisera (Table 2) for 90 min. Subsequently, the slides were washed twice in PBS and incubated with an avidin-biotin complex (1:100, Vectastain Elite ABC kits; Vector Laboratories, Inc., Burlingame, CA) for 90 min, after which they were washed further twice in PBS and twice in 0.05 M Tris-HCl buffer (pH 7.6) supplemented with 0.3 M NaCl and 0.1% (v/v) Tween. Finally, protein presence was visualized using 0.5 mg/ml of 3,3'-diaminobenzidine tetrachloride (Dako Corp., Carpenteria, CA) in 0.05 M Tris-HCl (pH 7.6) containing 0.03% (v/v) H2O2. The slides were washed in aquadest and counterstained with Mayer hematoxylin (Sigma). After counterstaining, the slides were washed for a final time in aquadest, and then tap water, before being mounted with Pertex (Cellpath Ltd., Hemel Hempstead, UK). The specificity of immunostaining was confirmed by replacing the primary antiserum with nonimmune serum from the species in which the former had been raised.
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Statistical Analysis of Data
The increase in follicle diameter between Days 0 and 6 of culture and the DNA content of follicles before and after culture were analyzed using Bonferroni corrected t-tests, and differences in the percentage changes were examined by chi-square analysis. Differences were considered to be statistically significant when P < 0.05.
RESULTS
Growth and Survival Rates
The increase in the diameter of preantral follicles during a 6-day culture is shown in Figure 1a. Follicles cultured in a medium containing 10 or 100 ng/ml, but not 1 ng/ml, of activin A showed a significantly greater increase in diameter than those cultured without activin A. Furthermore, the rate of follicle growth in the presence of 100 ng/ml of activin A was significantly higher than that in the presence of 10 ng/ml of activin A. However, activin A treatment did not significantly affect follicle survival rates or oocyte growth (Fig. 1b). At the onset of culture, oocyte diameter varied between 47 and 52 µm, and during the 6-day culture, it increased by an average of 8 µm, irrespective of the activin A concentration in the medium.
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The DNA content of freshly isolated, large preantral follicles was 128 ± 11 ng/follicle (mean ± SEM; n = 12). After 6 days of culture, the DNA content of preantral follicles incubated in medium containing 100 ng/ml of activin A had increased to 1525 ± 26 ng/follicle (n = 12), whereas that of follicles incubated in control medium had risen only to 1148 ± 58 ng/follicle (n = 12) (Fig. 1c).
Follicle Morphology
Light microscopic examination of semi-thin sections of preantral follicles cultured for 6 days in activin A-containing medium revealed an antrum-like structure in 8 of the 10 follicles investigated (Fig. 2). Similarly, TEM examination demonstrated an elongation of the granulosa cells surrounding the oocyte in 8 of these 10 follicles (Fig. 3a). The TEM examination also demonstrated that by the end of the culture period, the oocyte cortical granules were located just beneath the oolemma (Fig. 3b), and that heterologous gap junctions had developed between the microvilli of the oocyte and protrusions extending from the granulosa cells (Fig. 3b). The granulosa cells themselves were seen to contain rough endoplasmic reticulum, a highly developed smooth endoplasmic reticular system, and long mitochondria with well-developed cristae (Fig. 3c). Numerous gap junctions were present between neighboring granulosa cells and between adjacent theca cells (Fig. 3, c and d), and the theca cells contained both smooth and rough endoplasmic reticulum and mitochondria with vesicular to tubular shaped cristae (Fig. 3d).
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No apparent elongation of granulosa cells was found in follicles cultured in serum- and activin A-free medium (Fig. 4). In these culture conditions, the granulosa cells showed markedly less cell-cell contact, and gap junctions were rarely seen. Furthermore, approximately 20% of the granulosa cells in follicles cultured in serum- and activin A-free medium were degenerate by the end of the 6-day culture period. Occasionally, the nuclear material of these degenerate granulosa cells appeared to be intensely electron dense (Fig. 4a), and vacuoles and/or lipid droplets were frequently observed in both degenerate granulosa cells and in most theca cells (Fig. 4, a and c). Furthermore, in many cases, oocytes from follicles cultured in activin-free medium had relatively poorly developed microvilli and very few detectable cortical granules (Fig. 4b).
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The morphology of preantral follicles isolated from 17-day-old rats (photos not shown) resembled that of follicles cultured in the presence of activin A. However, no obvious elongation of the granulosa cells was observed in the former follicles, and the cortical granules had not migrated to the oocyte cortex.
Reverse Transcription-Polymerase Chain Reaction
The RT-PCR amplification of RNA isolated from somatic follicular cells and oocytes from preantral follicles using cDNA primers specific for rat activin A and ActR II resulted in PCR products with the expected sizes for activin A and ActR II (377 and 446 bp, respectively) (Fig. 5). No PCR product was detected when MilliQ water was used as a template for PCR. Digestion of the putative activin A PCR products with the restriction endonuclease AluI resulted in two fragments of the expected size (244 and 133 bp), and digestion of the products from ActR II amplification with the restriction endonuclease PstI also resulted in two fragments of the expected size (295 and 151 bp; data not shown), thereby confirming the identity of the PCR products.
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Immunohistochemistry
With regard to activin A immunoreactivity, in every ovarian follicle (primordial, primary, preantral, or early antral) present within the ovaries of 10-day-old rats, the oocyte was the most strongly stained cell. In addition, moderate to strong immunoreactivity was detected in granulosa and theca cells of preantral (Fig. 6a) and early antral follicles (Fig. 6b), and moderate staining was detected in the granulosa cells of primordial/primary follicles (Fig. 6c). Similarly, for ActR II, the oocyte was the most strongly immunoreactive cell within follicles of all developmental stages (Fig. 7a). The granulosa and theca cells of preantral (Fig. 7b) and early antral follicles (Fig. 7c) showed weak to moderate ActR II staining, and the granulosa cells of primordial/primary follicles had only a weak immunosignal for this receptor protein (Fig. 7c). Interstitial tissue and ovarian surface epithelial cells also stained positively for activin and ActR II presence, and when, for control purposes, the primary antisera were replaced by nonimmune sera from the appropriate species, no positive staining was observed.
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DISCUSSION
In this study, activin A clearly enhanced the growth of cultured preantral follicles in a dose-dependent manner. Furthermore, activin A not only stimulated proliferation of follicular cells but also induced formation of an antrum-like structure within the follicles. Such a proliferative effect of activin A on cultured follicular cells has been reported previously for granulosa cells recovered from antral follicles of immature rats [23] and for granulosa-oocyte complexes from 14-day-old rats [11]. However, whereas activin A has also been reported to have a stimulatory effect on preantral follicles from mice [12, 24] and cows [16], Woodruff et al. [10] reported that it induced atresia in preantral follicles recovered from immature rats.
In the present study, 10-day old rats were used, because their ovaries yield high numbers of preantral follicles in which the granulosa cells have yet to be subjected to gonadotropin-induced differentiation [25]. In rats, this differentiation begins at approximately Day 16 postpartum, when a dramatic increase in systemic FSH concentrations occurs [26]. In the current study, FSH was included in control culture medium to sustain the survival of preantral follicles [13]. However, antrum formation did not occur when activin A was omitted from the medium. This suggests that activin A has an important effect, over and above that of FSH, on the development of preantral follicles in immature rats. In vitro antrum formation has been reported previously in preantral follicles recovered from 25-day-old mice and cultured in the presence of serum and FSH [27], and this may, presumably, be possible because of activin A present in the serum. Activin A also induced a larger increase in follicular diameter but a smaller rise in the DNA content of cultured follicles than was seen previously with growth hormone (GH) [28] or insulin-like growth factor I (IGF-I) [29] under similar conditions, and it is, therefore, tempting to speculate that a major effect of activin A is the induction of antrum formation. Furthermore, because early preantral follicles were used in the current study, it is possible that activin A exerted its effect by promoting the responsiveness of follicular cells to FSH stimulation, such as by up-regulating FSH receptors in the granulosa cells [30–33].
It is widely accepted that folliculogenesis is a process characterized by profound morphological and functional changes of the follicular cells, and it is, therefore, logical that ultrastructural criteria should be considered when assessing the quality of cultured follicles [34]. However, to our knowledge, only limited attention has been paid to the morphological aspects of follicular development in vitro [11, 35]. In the present study, we were able to show that most (80%) preantral follicles cultured in activin-containing medium developed an almost perfectly normal ultrastructure, including an antrum and an area of elongated granulosa cells surrounding the oocyte. These follicular characteristics were not detected in previous studies when other factors, such as GH [28] and IGF-I [29], were included in the culture media, again suggesting a specific stimulatory effect of activin A on follicular cell differentiation.
With respect to the oocytes of cultured follicles, those cultured in the presence of activin A developed relatively large microvilli, which extended out to contact the granulosa cells via gap junctions. Furthermore, the cortical granules migrated to the outer region of the ooplasma, a change that is classically associated with oocyte maturation [35] and suggests that activin A, like GH [28], promotes cytoplasmic maturation of in vitro cultured oocytes. A beneficial effect of activin A on the cytoplasmic maturation of cultured oocytes was previously suggested by Alak et al. [36], who found that adding activin A to the culture medium for in vitro maturation (IVM) of primate oocytes led to a higher blastocyst yield after in vitro fertilization. On the other hand, activin A was reported not to positively influence IVM of bovine oocytes [37].
Previously, activin A gene expression has been found in ovulated mouse oocytes [38] and in immature/cultured oocytes from bovine antral follicles [39], but not in oocytes from human or mouse preantral follicles [40, 41]. Similarly, the expression of activin A mRNA by the somatic follicular tissue has hitherto been reported only for granulosa/cumulus cells recovered from antral follicles of various species (rat [19], mouse [38, 40, 41], cow [39, 42], sheep [43], primate [5], and human [41]). Thus, the current detection of activin A mRNA expression and immunoreactivity within rat preantral follicles constitutes, to our knowledge, the first confirmation of activin A biosynthesis by oocyte or somatic cells from such follicles. On the other hand, a role for activin A in early follicular development has been proposed in women [44–46], rats [47], and cows [16] following immunohistochemical studies. Similarly, whereas ActR II mRNA expression has been reported previously in oocytes recovered from antral follicles of mice [41, 48] and rats [49], the present study is, to our knowledge, the first description of such expression in preantral follicles, and again, detection of the ActR II protein in the same location confirmed that ActR II is biosynthesized by preantral follicles. The findings of activin A and ActR II expression and presence, and the effects of the former factor on cultured follicles, strongly suggest that activin A plays an important role in cell proliferation and differentiation in rat preantral follicles, probably via a local regulatory system.
In conclusion, activin A was found to enhance the growth of, and to induce the formation of an antrum-like structure in, preantral follicles recovered from immature rats and incubated in vitro, and it appears that in the presence of FSH, activin A promotes the development of preantral follicles into antral follicles. In addition, the demonstration of the local biosynthesis of both activin A and ActR II in oocytes and somatic follicular tissue of preantral follicles further suggests a significant role for activin A as a local modulator of early follicular development.
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ACKNOWLEDGMENTS
The authors would like to thank Mr. H. van de Kant of the Department of Cell Biology, University Medical Center, Utrecht, The Netherlands, for his assistance in immunohistochemical work and Dr. T. Stout of the Faculty of Veterinary Medicine, University of Utrecht, The Netherlands, for revising the English language of this manuscript. We would also like to acknowledge the generous gift of vital reagents by Mrs. B. Nusgens of the Laboratory of Experimental Dermatology, University of Liège, Belgium (type I collagen); Dr. D. Huylebroeck of Celgen, Belgium (antiserum against ActR II); and Dr. R.G. Hanssen of the Department of Pharmacology, N.V. Organon, Oss, The Netherlands (recombinant human FSH). Finally, we thank the Department of Biochemistry, Cell Biology and Histology and the Faculty of Veterinary Medicine, University of Utrecht, The Netherlands, for providing us with the facilities for in vitro culture.
FOOTNOTES
1 Correspondence: J. Zhao, Department of Pharmacology (RE 3132), N.V.
Organon, Molenstraat 110, P.O. Box 20, 5340 BH, Oss, The Netherlands.
FAX: 31 0412 662542; j.zhao{at}organon.oss.akzonobel.nl 
Accepted: April 30, 2001.
Received: June 15, 2000.
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