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Ovary |
Follicle Biology Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-minimal essential medium (MEM) without (deoxy)ribonucleosides at a lower concentration of fetal bovine serum (FBS; 1%) for 6 days with mechanical prohibition of thecal cell attachment. Growing follicles reaching at least 100 µm were transferred to -MEM medium enriched with a higher concentration (5%) of FBS to allow attachment and were cultured subsequently for an additional 12 days. Overall, more than 85% of the follicles survived the first culture step, and oocyte growth and granulosa cell proliferation had increased by 25% (P < 0.05). Follicle survival at Day 18 was related to initial follicle diameters at isolation. Average meiotic maturation rates and estrogen secretion were lower compared to those of cultures starting with early preantral follicles of 100–130 µm. Although reverse transcription-polymerase chain reaction analysis revealed the presence of LH-receptor mRNA in thecal cells, an exogenous androstenedione replacement resulted in an increase of estrogen production, suggesting substrate insufficiency. The time needed to grow from early preantral stages to in vitro ovulation is strongly dependent on the initial follicle diameter at isolation. Morphological characteristics of cultured follicles were suggestive for combined transforming growth factor ß deficiencies during in vitro culture.
follicle, follicular development, gamete biology, oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The timing of follicle growth has been determined in rodents. In mice, once follicles have entered the growing follicle population (type 3b) [14], approximately 16 days are required to reach the large antral stage (types 6–7). Follicles reaching this stage in vivo still need to pass through an additional estrous cycle before an ovulatory stimulus is able to cause resumption of meiosis and ovulation [14–16]. Of interest is that the first few waves of growing follicles might not require the long transition phase (existing in adult animals) for transformation of the resting primordial follicles into a growing primary follicle [17, 18]. In vivo, follicles from the first wave never reach the preovulatory stage because of atresia by lack of FSH [17]. In vitro experiments have shown the production of live offspring from follicles (types 3b and 4) isolated from prepubertal animals (14-day-old mice) [9]. Because the mouse primordial follicle is formed between Days 1 and 4 after birth [19], these experiments, together with the ones of O'Brien et al. [12], prove that developmentally competent oocytes can be generated within 3–4 wk after the formation of the follicle.
The culture system used for type 3b and 4 follicles was reported to be inadequate for growing follicles that were smaller than 100 µm [13]. The objective of the present study was to design a reproducible culture system for isolated follicle structures smaller than 100 µm (type 3a follicles).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female F1 hybrid (C57BL/6j x CBA/Ca) mice housed and bred according to national standards were used. The present study was approved by the Institutional Ethical Committee for Animal Experiments (project no. 01-395-1).
Eight- and 14-day-old mice were killed by cervical dislocation, and the ovaries (n = 45) were aseptically removed and collected in 2 ml of L15 Leibovitz-glutamax medium supplemented with 10% fetal bovine serum (FBS), 100 µg/ml of streptomycin, and 100 IU/ml of penicillin (L15* medium; all obtained from NV Invitrogen SA, Merelbeke, Belgium). Every step was carried out at 37°C.
Under a normal stereomicroscope, the ovaries were freed from connective tissues, and each ovary was placed in an organ culture dish containing 1 ml of fresh L15* medium. Follicles were mechanically released from the ovaries with fine 25
-gauge needles (Becton Dickinson, Erembodegem, Belgium).
The follicles to be cultured were selected in two washing steps according to the following criteria: 1) follicles having a diameter of 80–100 µm; 2) intact, round follicle structure with one (primary follicles) up to a maximum of two (early secondary follicles) layers of cuboidal granulosa cells; 3) intact basal membrane with some attached, fibroblast-like thecal cells; and 4) a visible, immature oocyte, round and centrally located within the follicle. All selected follicles were pooled and randomly divided over different culture plates.
Culture Media and Hormones
Three different media were used throughout culture. Medium 1 (nonattaching phase) consisted of -minimal essential medium (MEM)-glutamax without (deoxy)ribonucleosides enriched with 1% heat-inactivated FBS (NV Invitrogen SA), 100 mIU/ml of FSH, 10 mIU/ml of LH (kindly donated by Ares Serono, Geneva, Switzerland), and insulin-transferrin-selenium mix (ITS-mix; 5 µg/ml, 5 µg/ml, and 5 ng/ml, respectively; Sigma, Bornem, Belgium). Medium 2 (attaching phase) consisted of -MEM-glutamax with (deoxy)ribonucleosides enriched with the same components as medium 1 except for the concentration of the FBS, which was used at a dose of 5%. Medium 3 (induction of ovulation) consisted of -MEM-glutamax with (deoxy)ribonucleosides supplemented with 1.5 IU/ml of rhCG (kindly donated by Ares Serono) and 5 ng/ml of recombinant epidermal growth factor (rEGF; Roche, Switzerland) [20].
Experimental Setup: In Vitro Growth of Primary and Early Secondary Follicles
First step: In vitro growth of primary and early secondary follicles in 1% FBS Culture dishes (60-mm Petri dish; Falcon; Becton Dickinson) containing twenty 10-µl culture droplets and two 10-µl wash droplets, covered with 5 ml of mineral oil (Sigma) to prevent evaporation and severe pH and temperature fluctuations, were preincubated overnight at 37°C, 100% humidity, and 5% CO2 in air. Selected follicles were washed and placed individually in the culture droplets (medium 1). At Day 4 of culture, 10 µl of fresh medium 1 were added to each droplet. Follicles were cultured at 37°C, 100% humidity, and 5% CO2 in air.
Second step: In vitro growth of early preantral follicles in 5% FBS At Day 6 of culture, those follicles reaching a diameter of 100 µm or more were transferred to a new dish containing medium 2. At Day 8 of culture, 10 µl of fresh medium 2 were added to each droplet. From Day 10 onward, the medium was refreshed by exchanging 10 µl of conditioned medium with 10 µl of fresh medium 2 every other day.
Ovulation At Day 18 of culture, 10 µl of conditioned medium were collected and pooled per plate and replaced by 10 µl of medium 3 to induce final oocyte maturation. After 18 h, mucification or cumulus expansion of the cumulus-oocyte complexes was assessed. Denuded oocytes were measured and scored for nuclear maturation (germinal vesicle [GV], GV breakdown [GVBD], or polar body).
Histological Evaluation of Follicles During the First Nonattaching Phase
At Days 0, 1, 4, and 6 of culture, follicles were fixed in 25% glutardialdehyde diluted 1:10 in cacodylate buffer at 4°C. After fixation, they were washed three times with Milli-Q water, postfixated in 1% osmium tetroxide (OsO4; in water) for 30 min, stained with 2% uranyl acetate for 60 min, and dehydrated in successive three 5-min washes of 50% (v/v), 70%, and 90% ethanol. The last three washes, in 100% ethanol, were carried out for 30 min. Follicles were embedded in 50% (v/v) spurr/ethanol solution (ERL 4206, DER 736, NSA, and S-1; all purchased from SA Laborimpex NV, Brussels, Belgium) for 20 min, twice in 100% spurr solution for 30 min, and polymerized overnight at 70°C. Semithin sections were made with an ultramicrotome (Reichert Ultracut S/FC S; Leica) and stained with methylene blue.
Observation of Follicle Growth and Differentiation
On each refreshment day, a morphological observation of each follicle was done and recorded. At the beginning of culture, the intactness of the follicle was assessed under the inverted microscope, equipped with a Hoffman contrast-modulation system, by examining the apposition of the oocyte and granulosa cells surrounded by the basal membrane. Presence of fibroblast-like thecal cells was noted. The follicle diameter was measured between the basal membrane with a calibrated ocular micrometer. At Day 2 of culture, possible attachment of follicles was recorded when the follicles formed a monolayer of fibroblast-like cells over the bottom, but they were again detached using a fine, mouth-controlled Pasteur pipette. Follicles were individually classified into three stages for their growth and differentiation profile, as explained in detail elsewhere [13]. Briefly, these classifications were as follows: "follicular" when follicles had kept their spherical structure (i.e., oocyte covered by granulosa cells surrounded by a basal membrane), "diffuse" when follicles showed pronounced outgrowth of the granulosa cells through the basal membrane, and "antral-like" when follicles showed translucent areas within the granulosa cell mass around the oocyte, indicating the formation of antral-like cavities. Follicle survival was regarded as surviving the culture conditions as long as the oocyte was in contact with granulosa cells and the complex remained attached to the culture dish. Follicle degeneration was characterized by failure of the granulosa cells to multiply, oocyte release, or collapse. These follicles were recorded as nonsurviving and were discarded from the experiment.
Assessment of Steroid Production In Vitro
From Day 10, culture medium was refreshed every 2 days by retrieving and adding 10 µl. From the surviving follicles, conditioned medium was pooled per culture dish and stored at –20°C for hormone analysis. Estradiol (E2) and progesterone were measured using RIAs; E2 was measured using a very sensitive (<4 pg/L) and reproducible (total coefficient of variation [CV], <10%) RIA from Clinical Assays (Estradiol-2; Sorin Fueter, Belgium) with a measurement range for E2 from 4 to 500 ng/L. Progesterone secretion was measured using an RIA from CIS Biointernational (Gif-sur-Yvette, France) with an analytical sensitivity of 40 ng/L and a precision of 8% or less (CV %). Both assays have been validated for use of culture medium as sample.
Characterization of Follicle Functionality
LH-receptor expression Ovaries from 8- and 14-day-old mice were snap-frozen in liquid nitrogen and then crushed with a fine, sterile micropestle while submerged in ice-cold lysis buffer. Follicles were first isolated before being snap-frozen in liquid nitrogen. Immature oocytes were harvested from early preantral follicles (diameter, 100–130 µm). Total RNA was isolated using the RNeasy kit (Qiagen, Germany) according to the manufacturer's procedure. Total RNA was reverse transcribed into first-strand cDNA (Pharmacia, The Netherlands) using random hexamer primers and stored at –20°C. Two polymerase chain reaction (PCR) primer pairs specific for the LH receptor were designed to be complementary to the human and mouse sequence with GenBank accession numbers M63108 and M81310, respectively. The first primer pair amplifies a fragmentation of 102 base pairs (bp) containing exons 7 and 8 (forward, 5'-GAAATGGATTTGAAGAAGTACAAA-3'; reverse, 5'-CCATTGTGCATCTTCTCCAG-3'), and the second primer pair amplifies a carboxyterminal part of exon 11 of 216 bp (forward, 5'-AATCTCTCCTTTGCAGACTTTTG-3'; reverse, 5'-AGCATAGGTGATGGTGTGCCA-3'). Two microliters of cDNA were applied in a 25-µl PCR reaction containing 10 mM Tris-HCl (pH 7.4), 50 mM KCl (pH 8.3), 1.5 mM MgCl2, 16 µM of each deoxy-NTP, and 1 U of AmpliTaq (Applied Biosynthesis; Roche). After an initial denaturation process of 3 min at 94°C, 35 cycles were carried out by denaturing for 30 sec at 94°C, annealing for 30 sec at 60°C, extension for 30 sec at 72°C, and a final extension for 5 min at 72°C. The 18S ribosomal mRNA was evaluated to ensure the extraction procedure and reverse transcription (RT) were complete. As a positive control for LH-receptor expression, mouse mural granulosa cells were included.
Aromatase enzyme induction In two separate experiments, androstenedione (Sigma-Aldrich NV/SA, Bornem, Belgium) was added to the medium-2 phase (10 µg/ml) to determine indirectly whether aromatase enzyme activity was functional in granulosa cells from cultured type 3a follicles.
Statistical Analysis
Results were obtained from eight repeated, independent experimental replicates. For analysis, follicles were divided into two groups according to their initial diameter: group 1 (diameter, 80–89.5 µm), and group 2 (diameter, 90–100 µm). Follicle and oocyte growth and development are presented as notched box-and-whiskers plots. The notches define the 95% confidence interval around the median (50th percentile); groups that display nonoverlapping notches can be considered as statistically different (P < 0.05). Values are mean ± SEM throughout.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Small follicles (diameter, 80–100 µm) are very vulnerable to mechanical damage during isolation. Although ovaries from 8-day-old animals had a higher prevalence of follicles in this size range, ovaries from 14-day-old animals, which also contain type 3a follicles, are slightly larger and less rigid. Follicles from older mice were loosened more easily, and damage was minimized. Of a single ovary, approximately 15 follicles of good quality and appropriate size were isolated.
Initial Growth Characteristics of Follicles in 1% FBS (Days 0–6)
Morphological observation All follicles had one to a maximum of two layers of granulosa cells surrounded by an intact basal membrane and a few surrounding fibroblast-like cells (thecal cells) (Fig. 1A). During the first 6 days of culture, all follicles remained "follicular," and the basal membrane, which was clearly visible under the inverted microscope, was kept intact. The fibroblast-like cells at the outer side of the membrane started to attach from the second day of culture, but these cells were gently detached with a mouth-controlled Pasteur pipette. The zona pellucida enlarged as the follicle diameter increased. Follicles that did not reach 100 µm or more at Day 6 inevitably underwent degeneration when transferred to the next culture phase. During the nonadherent phase, the follicles increased in diameter because of oocyte growth and granulosa cell proliferation.
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Histological observation Histological observations showed that follicles within the narrow, selected class of 80–100 µm in diameter were in a transitory state from the primary to the secondary stage, showing a partially formed, double layer of granulosa cells (Fig. 1B). Oocyte diameters within this class were highly variable. Two types of growth patterns became evident. In the first case, granulosa cell proliferation was very slow. In some of these follicles, only two layers of granulosa cells were present after 6 days of culture, although follicle diameter had increased because of oocyte growth. The granulosa cells had rather tight intercellular contacts. In the second case, granulosa cells grew very rapidly, and multiple layers surrounded the oocyte. This rapid growth may have compromised contacts between granulosa cells, because the contacts had become extremely loose (Fig. 1Bd). However, contacts with the oocyte seemed to be maintained, which provides a basis for the observed increased oocyte diameter. At Day 6 of culture, when follicles were transferred to the next step, fibroblast-like interstitial cells (possibly thecal cell precursors) were still present surrounding the basal membrane.
Evaluation of growth In total, 555 follicles were cultured in eight independent experiments. For evaluation, follicles were divided into two groups based on their initial diameter. Group 1 (n = 266) was composed of follicles with a diameter of 80–89.5 µm, and group 2 (n = 289) was composed of follicles with a diameter of 90–100 µm (Table 1).
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Regarding follicle survival, 89.0% ± 4.8% of group 1 and 93.7% ± 3.2% of group 2 follicles survived the first 6 days of culture. Follicles that did not survive this first culture episode demonstrated oocyte release or degenerated and were characterized by oocyte collapse or follicle diameter decrease.
Follicles were measured at days 1, 4, and 6 to evaluate growth. For both groups, a significant increase in diameter (P < 0.05) was measured (Fig. 2). For group 1 (n = 189), the mean diameter was 84.8 ± 3.0 µm at Day 1, 96.7 ± 6.9 µm at Day 4, and 103.5 ± 7.5 µm at Day 6 of culture. For group 2 (n = 230), the mean diameter was 94.4 ± 3.2 µm at Day 1, 105.8 ± 7.1 µm at Day 4, and 113.1 ± 8.4 µm at Day 6 of culture.
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Growth Characteristics During the Second Attached Phase (Days 6–18)
Morphological observations
At Day 6, follicles were transferred to the second step with -MEM plus 5% FBS (Fig. 1C). In this phase, the follicles were allowed to attach to the bottom of the culture dish. Thecal cells attached first. After a period of slow growth during the first 6 days, granulosa cells started to divide more rapidly, beginning to break through the basal membrane and overgrowing the thecal cells to form a so-called "diffuse" follicle. Follicle degeneration occurred at any time of culture and was characterized by either failure of the granulosa cells to multiply (Fig. 3A) or oocyte release. However, as long as the oocyte stayed (partially) in contact with the granulosa cells, the oocyte survived (Fig. 3B). However, when this occurred at Day 6 of culture (first step), this connection repair was no longer operational because of mechanical damage during transfer.
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Group 1 Fifty-eight percent of the transferred follicles survived the 18-day culture period (Fig. 4A and Table 1). As illustrated in Figure 4, follicles progressively grow from a spherical to a diffuse structure and, ultimately, start to display antral-like cavity formation. A high proportion (87%) of the surviving follicles kept their spherical structure (basal membrane intact) up to Day 10 of culture, after which follicles started to become diffuse. The diffusion reflected the proliferation of granulosa cells breaking through the basal membrane, with the highest percentage of follicles (87%) showing this morphology at Day 14. From this day onward, a first antral-like cavity formation was observed.
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Group 2 Seventy-six percent of the transferred follicles survived the second step (Fig. 4B and Table 1). When compared to group 1 follicles (Fig. 4A), group 2 follicles progress through the different morphological stages much more quickly, because they are already in a more advanced stage of follicle growth at the beginning of culture. Follicles already started to become diffuse (5%) from Day 8 onward, and at Day 12 of culture, only 28% of the follicles still had their original follicle structure. Although a comparable percentage of the follicles (85%) were diffuse at Day 14, 13% already displayed a clear, well-developed, antral-like cavity at that time, and many (35%) of the follicles were antral-like by Day 16.
Survival Survival became significantly different between follicle groups from Day 10 of culture onward, with a better survival rate for group 2 (Fig. 4). The survival rate of group 1 follicles was lower because of their initial follicle diameter. When group 1 follicles that did not reach 100 µm in diameter at Day 6 were omitted, survival was no longer significantly different from that of group 2 (data not shown). This shows, again, that achieving the critical barrier diameter of 100 µm is essential for survival in the second culture step.
Growth and Maturation of the Oocyte In Vitro
After 18 days of culture, oocytes were stimulated with rhCG and rEGF. Eighteen hours after the stimulation, mucification of the cumulus-oocyte complexes was assessed under the stereomicroscope. All surviving follicles showed induction of mucification after the hCG/EGF stimulus. A total of 96 oocytes were harvested (Table 1). The denuded oocytes were evaluated under the inverted microscope for diameter (zona excluded) and stage of nuclear maturation.
Oocyte diameter At the end of culture, oocyte diameters of both follicle classes were comparable and not significantly different (P > 0.05) (Fig. 5). For group 1, the mean diameter of the oocytes (n = 26) was 73.3 ± 2.4 µm. For group 2, the mean diameter of the oocytes (n = 70) was 72.2 ± 3.0 µm.
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Oocyte maturation Sixty-seven percent of the oocytes from group 1 underwent GVBD, and 34% of them extruded the first polar body. Seventy-one percent of the oocytes from group 2 underwent GVBD, and 20% of them extruded the first polar body. No significant differences were found between the two groups (P > 0.05).
Steroid Production In Vitro
The production of E2 progressively increased during culture (Fig. 6A) but stayed approximately 50- to 100-fold lower than that from historical cultures of early preantral follicles (diameter, 100–130 µm) [13] despite similar gonadotroph and growth factor supplements. The basal progesterone production (before hCG stimulus) stayed low in these cultures. The production of progesterone increased dramatically 18 h after stimulation with hCG/EGF at Day 18 of culture to induce ovulation (Fig. 6B).
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Characterization of Follicle Functionality
LH-receptor expression To investigate the presence of LH receptors on thecal cells of follicles with a diameter of less than 100 µm, we performed RT-PCR analysis on both whole ovaries and isolated follicles. In the first experiment, whole ovaries of 4-, 8-, and 14-day-old mice were used. The LH-receptor mRNA was detected in all three ages (data not shown). In mice, follicular development has, at most, reached the preantral stage by Day 14, so no corpora lutea are present [13], which means that LH-receptor mRNA expression would be most important in thecal cells. To exclude the possible contribution to LH-receptor mRNA by blood vessels and oocytes [21, 22], the early preantral follicles were isolated from ovaries of 8-day-old (diameter, 80–100 µm) and 14-day-old mice (diameter, 100–130 µm), and their expression was compared to that of immature oocytes (retrieved from early preantral follicles). Figure 7 clearly shows that expression of LH receptor was found in follicles of both diameter classes, whereas no expression was found in the oocytes.
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Aromatase enzyme induction To find out if the low estrogen production resulted from a deficient aromatase capacity of the granulosa cells, two independent experiments were carried out in which 10 µg/ml of androstenedione were added only to the culture of the medium-2 step. A total of 103 follicles were cultured under these conditions, 90.3% of which survived the first step (Days 0–6) and 73.1% of which had reached a follicle diameter of 100 µm or more and were transferred to the second culture step. Follicles became more rapidly diffuse (from 2 days after transfer) but displayed a markedly darker aspect (Fig. 8) compared to control follicles (no androstenedione added), indicating atresia. Also, antral-like cavity formation appeared earlier in culture and was more pronounced than in control follicles. Taking these data together, androgens seemed to stimulate growth and differentiation of preantral follicles in vitro. Because of this rapid differentiation, surviving follicles (n = 67) were stimulated with hCG on Day 16 (vs. Day 18 for control follicles). After denudation, however, oocytes displayed a smaller diameter of 68.8 ± 2.2 µm, which could be the result of either too short a culture period or an adverse influence of androgens on oocyte growth. In comparison to control follicles, E2 production had increased by 250- to 500-fold (Table 2), providing evidence for normal aromatase function in cultured granulosa cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A two-step culture system was devised. In the first step, follicles with diameters of 80–100 µm were cultured singly in 1% FBS for 6 days to develop two to four layers of granulosa cells. To keep the three-dimensional structure intact during the first step, follicles were detached mechanically should attachment of the thecal cells (fibroblast-like cells) occur. Mechanical detachment is easy and avoids the use of more sophisticated techniques to inhibit attachment, such as membrane inserts [25] and coated wells [26]. Detachment kept thecal cells surrounding the follicle, perhaps providing essential biophysical factors to support the basal membrane and, thereby, ensuring functional integrity to the follicle mass [27].
The -MEM without (deoxy)ribonucleosides was chosen as the first basal medium, because provision of nucleosides becomes essential only when the aim is to culture rapidly dividing cell types [1]. Knowing that during early folliculogenesis the growth of the oocyte and proliferation of the granulosa cells are extremely slow processes [28], these DNA precursors were left out of the first medium composition. Prohibition of rapid proliferation of granulosa cells in small preantral follicles avoided rupture of the basal membrane and preserved the spherical structure. Adverse effects of proliferation enhancers had been experienced previously by adding activin, a proliferation enhancer, to types 3b and 4 follicles in the same culture system [29]. It was easy to transfer those grown follicles out of this first culture step to the well-established culture system of Cortvrindt et al. [13] that used -MEM with (deoxy)ribonucleosides] and 5% FBS.
During evaluation, it became clear that the initially chosen class of follicles (diameter, 80–100 µm) behaved as two subgroups. Further analysis of follicles was then done for the two groups, which were divided according to their initial follicle diameter on the day of isolation (group 1: diameter, 80–89.5 µm; group 2: diameter, 90–100 µm).
During the first culture step, follicles of both classes showed a progressive increase in follicle diameter because of oocyte growth and granulosa cell proliferation. On the condition that the basal membrane was intact at the beginning of the culture, all follicles could be kept spherical for at least 6 days.
The follicles of group 2 had already reached a follicle diameter of 100 µm or more at Day 4 of culture; in retrospect, these follicles could have been transferred at that time to the next culture step. Aiming for a single manipulation step, however, we allowed these follicles to grow for two additional days, because the preset inclusion criteria by Cortvrindt et al. [13] for the second culture step is a follicle diameter with an upper limit of 130 µm.
Follicles of group 1 needed 6 days to reach a minimum diameter of 100 µm. Approximately 13% of the follicles of group 1 did not reach the lower limit of 100 µm for inclusion into the second step of culture. A minority (5.8%) of the smallest follicles showed a growth arrest during step 1: Granulosa cells failed to multiply any further but had no visible signs of apoptosis or necrosis. These follicles showed a progressively narrowing granulosa cell layer, as if, because of oocyte growth, the limited number of granulosa cells were stretched over the surface of the oocyte. Similar defects have been described in growth differentiation factor (GDF-9)-null mice, in which ovarian folliculogenesis is blocked in the primary, one-layered follicle stage by the up-regulation of inhibin [30–33]. The granulosa cells of these type 3b follicles in the knock-outs also failed to proliferate, but did not die, as they required GDF-9 both for growth and to become competent to undergo apoptosis [30–32]. Recently, a double knock-out for GDF-9 and inhibin revealed that the absence of inhibin , which normally is upregulated (in case of GDF-9 deficiency), is responsible for uncontrolled granulosa cell proliferation [33]. These experiments underlined that GDF-9 is only an indirect regulator of granulosa cell proliferation. Recombinant GDF-9 supplementation of early preantral follicles enhanced growth and differentiation of cultured early preantral rat follicles [34].
Growing follicles were transferred to the second culture step in medium with a higher concentration of FBS (5%) that allowed the follicle to attach to the culture dish [13]. Thecal cells attached first, and it was 4–6 days before granulosa cells started to break through the basal membrane to proliferate over the already formed thecal cell monolayer. We hypothesized that this period of enclosed granulosa cell culture was a necessity, because an earlier outgrowth of granulosa cells resulted, most of the time, in oocyte release and degeneration.
A significant difference in follicle survival between the follicle-size subgroups became clear from Day 10 onward, with group 2 surviving better than group 1. Because the smallest class was still in an earlier stage of follicular development, not reaching the prerequisite diameter at the day of transfer was shown to be responsible for this difference. At the end of culture, follicles grown from initial diameters of 80–100 µm showed a clear antral-like cavity less frequently than did cultures of early preantral follicles (diameter, 100–130 µm). This could be the result of a globally lower granulosa cell proliferation seen in cultures starting with primary follicles.
After the ovulatory stimulus at the end of culture, the oocytes were able to mature to metaphase II, although in a smaller proportion compared to early preantral follicle cultures (diameter, 100–130 µm), which allows us to believe that somatic cell-to-oocyte communications, which are essential for oocyte growth and the regulation of meiotic maturation [6, 23, 24], were functional. In vitro fertilization experiments showed a low fertilization rate (
35%) compared to that of in vivo-grown oocytes (89%) (data not shown). Only 13% of the resultant two-cell stages developed to the blastocyst stage. Reduced meiotic and developmental competence after fertilization might be caused by insufficient cytoplasmic maturation, because normal oocyte diameters critical to nuclear maturation [7, 35, 36] had been achieved.
During the 18-day culture period, only small amounts of estrogens were produced (50- to 100-fold lower than cultures of early preantral follicles [13]). According to the two-cell, two-gonadotroph model, the FSH-stimulated estrogen synthesis in granulosa cells is dependent on the LH-stimulated supply of androgens from thecal cells [37]. The decreased production of estrogens can then be the result either of an inadequate production of androgens by thecal cells or of a deficient aromatization capacity by the granulosa cells. Both options were examined. A first possibility was that during transfer of the surviving follicles to the second culture step, some thecal cells, which had already attached to the culture plate, were not transferred. Because it was shown previously in cultures of early preantral follicles (diameter, 100–130 µm) that a critical mass of thecal cells is essential for normal estrogen production, this might explain the lower concentrations of estrogens found in the primary follicle cultures during the second growth step. However, after transfer, thecal cells were still morphologically and histologically visible around the basal membrane and proliferated into a monolayer, although perhaps to a lower extent than the one formed in early preantral cultures. Another possibility was that the fibroblast-like interstitial cells had not yet fully differentiated into thecal cells. This could also explain the reduced granulosa cell proliferation seen during the second phase of in vitro culture. Thecal cells have been shown to produce transforming growth factors (TGFs) and ß, hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) to regulate granulosa cell growth and function [38, 39]. Granulosa cells, in turn, produce kit ligand that can feed back on the thecal cells to regulate thecal cell growth and stimulate production of the thecal cell factors (TGF, KGF, and HGF) [39–41]. Therefore, a positive-feedback loop between theca and granulosa cells appears to exist to sustain the dramatic cell growth during folliculogenesis. Thecal cell markers, such as 17-hydroxylase, LH receptor, and c-kit receptor, were not detectable around GDF-9-deficient follicles [31] or in the double-knock-out GDF-9/inhibin model [33]. Also, GDF-9 was shown to stimulate androgen synthesis in theca and interstitial cells [42]. The RT-PCR analysis of the LH receptor on both immature whole ovaries (of 8- and 14-day-old mice) as well as isolated follicles revealed the presence of the LH-receptor mRNA. Whether this mRNA was translated into the functional protein at this stage was not examined. Other etiologies for a possible androgen deficiency are, first, that certain enzymes downstream (of the receptor) in the androgen production pathway are not expressed in vitro and, second, that the thecal cells had dedifferentiated under present culture medium conditions.
Finally, estrogen production could be low because of an incomplete aromatization of androgens in granulosa cells. This was tested by administering androstenedione to the culture medium during the second culture step. Morphologically this had a clear effect on the antral-like cavity formation, which appeared at a more advanced stage and was more pronounced, as also shown previously [43]. Murray et al. [43] showed that androgens could stimulate follicular development by exerting a direct effect on granulosa cells. Addition of androstenedione into the culture medium increased the production of E2 by a factor of 250- to 500-fold (Table 2). Moreover, we could deduce from the increasing amount of progesterone in response to the hCG stimulus that granulosa cells had, indeed, undergone normal differentiation.
This report demonstrates that it is possible to culture primary mouse follicles as single functional units using a biphasic culture system. The initial follicle diameter strongly determined the culture time that was needed to reach maturation: Every 10 µm in diameter under 100 µm requires an extension of the culture period by 2 days. Morphological and functional analysis of follicle growth and hormone secretion patterns suggests that deficiency of oocyte-specific factors might be the basis of poor oocyte cytoplasmic maturation. In vitro culture needs improvement: The extended culture period demands very tightly controlled physicochemical (temperature and pH consistency) parameters. Whether the morphological and functional signs of specific growth factor deficiencies could be reversed by stage-specific addition of growth factors needs further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Sandy Lenie, Follicle Biology Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium. FAX: 32 02 477 5060; sandy.lenie{at}vub.ac.be
Received: 25 February 2004.
First decision: 23 March 2004.
Accepted: 28 June 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  I. Adriaens, J. Smitz, and P. Jacquet The current knowledge on radiosensitivity of ovarian follicle development stages Hum. Reprod. Update, May 1, 2009; 15(3): 359 - 377. [Abstract] [Full Text] [PDF]
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 J C Sadeu, T Adriaenssens, and J Smitz Expression of growth differentiation factor 9, bone morphogenetic protein 15, and anti-Mullerian hormone in cultured mouse primary follicles Reproduction, August 1, 2008; 136(2): 195 - 203. [Abstract] [Full Text] [PDF]
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W. Shen, L. Li, Z. Bai, Q. Pan, M. Ding, and H. Deng In vitro development of mouse fetal germ cells into mature oocytes Reproduction, August 1, 2007; 134(2): 223 - 231. [Abstract] [Full Text] [PDF]
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 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]
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W. Shen, D. Zhang, T. Qing, J. Cheng, Z. Bai, Y. Shi, M. Ding, and H. Deng Live Offspring Produced by Mouse Oocytes Derived from Premeiotic Fetal Germ Cells Biol Reprod, October 1, 2006; 75(4): 615 - 623. [Abstract] [Full Text] [PDF]
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