BIOLOGY OF REPRODUCTION 61, 503–511 (1999)
Development and Characterization of an In Vitro Ovulation Model Using Mouse Ovarian Follicles U.M. Rose,1 R.G.J.M. Hanssen, and H.J. Kloosterboer N.V. Organon, Department of Pharmacology, 5340 BH Oss, The Netherlands ABSTRACT
thase enzyme (PGS-2) and the progesterone receptor (PR). Previously, it has been demonstrated in vivo that LH or hCG induces expression of PGS-2 and PR genes in the preovulatory follicle [7–9] and that inhibitors of PGS-2 function and compounds that block the PR clearly inhibit ovulation [10–12]. In addition, the importance of both PGS-2 and PR for the ovulatory process has been shown in PGS-2 and PR knockout mice that fail to ovulate [13– 16]. Until now, ovulation has been studied in in vivo models or in in vitro-perfused ovary models [17]. However, these models do not exclude the involvement of extrafollicular factors participating in the ovulatory process. In order to study local factors, an in vitro mouse follicle culture model was developed that is described in the present paper. Previously, it has been shown that folliculogenesis can be mimicked in vitro using nonspherical, attaching [18], spherical, nonattaching [19–25], or gel-embedded [26] follicle culture systems. In these systems, in vitro-cultured follicles grow from the preantral to the preovulatory stage. The nonspherical cultures, however, are not suitable for ovulation studies since no intact follicular wall is present. In the spherical cultures, ovulation has been induced occasionally, but the results obtained to date are not very consistent [22, 24, 27]. In the present study, an in vitro spherical mouse follicle culture system was optimized and characterized to be used as an in vitro ovulation model in comparison with an in vivo ovulation model. Follicular growth rate, the hCG dose required for ovulation, and the relation between follicular size and ovulation rate were determined. To ensure the quality of the in vitro-ovulated oocytes, fertilization and embryo development of the ovulated oocytes were examined. In addition, expression of the ovulation-associated genes PGS-2 and PR in hCG-treated in vitro-cultured follicles in comparison with their expression in an in vivo ovulation model was investigated. Finally, the physiological significance of the in vitro ovulation model was demonstrated by the inhibition of hCG-induced ovulation with the PGS inhibitor indomethacin and the anti-progestogen Org-31710.
To investigate ovulation, an in vitro model with cultured mouse follicles was developed and compared with an in vivo ovulation model. In this model, secondary follicles were grown in vitro with immature mouse serum (5%) and recombinant human FSH. Addition of ascorbic acid and selenium to the medium increased follicular survival (from 29% to 86%) and resulted in the development of healthy preovulatory follicles (. 400 mm) producing estradiol. Depending on the starting size of the follicles, the preovulatory stage was reached after 4–6 days. The ovulatory response to hCG was maximal in follicles exceeding a diameter of 400 mm. The in vitro-ovulated oocytes could be fertilized and were able to develop to the blastocyst stage. Ovulation induced by hCG was dose dependent, reaching a maximum of 80% at 1 IU/ml. Concomitantly, progesterone production increased from 3.6 6 0.5 to 29 6 2 ng/ml. Both in vivo and in vitro, hCG induced expression of the progesterone receptor and the prostaglandin endoperoxide synthase-2 (PGS-2) gene within 3 h. Ovulation could be completely blocked with the anti-progestogen Org-31710 and partially (50%) with the PGS inhibitor indomethacin in vitro and in vivo. Org-31710 and indomethacin did not affect progesterone production. In summary, a physiologically relevant in vitro ovulation model of cultured mouse follicles that can be used to study the process of follicular rupture has been developed.
INTRODUCTION
The preovulatory LH surge induces a series of changes in various follicular compartments that finally leads to the ovulation of a fertilizable oocyte into the oviduct and the transformation of the remaining follicle into the corpus luteum [1]. The process of ovulation has been the subject of intensive studies for many years, and as a result several factors involved in follicular rupture and luteinization have been identified. It has been demonstrated that the cascade of events during ovulation starts with the binding of LH to its receptor and induces the production of intracellular cAMP [2]. Subsequently, cAMP-dependent protein kinase activation leads to stimulation of steroidogenesis, activation of the cyclooxygenase/lipoxygenase pathway and hence increased prostaglandin/leukotriene synthesis, and activation of proteolytic enzymes such as collagenase to allow degradation of the follicle wall [3–5]. Concomitantly, a prostaglandin-mediated increase in vascular permeability causes changes in the regional blood flow of the follicle, which contributes to a sustained positive intrafollicular pressure during the later stages of the ovulatory process [6]. These events finally result in follicular rupture and extrusion of a mature oocyte. In the ovulatory process, two important factors have been identified, i.e., the prostaglandin endoperoxide syn-
MATERIALS AND METHODS
Follicle Isolation
Ovarian follicles were isolated and serum was prepared according to Nayudu and Osborn [20]. Briefly, female immature mice (F1: B6BCA; 21–23 days of age) were anesthetized with ether, and blood was collected by means of eye extraction. After clotting, blood was centrifuged for 15 min at 4000 3 g, and serum was collected and stored at 2208C until use. Ovaries were removed and placed in Leibovitz-L15 medium (Gibco, Paisley, UK; #11415–049) supplemented with glutamine (2 mM: Gibco; #15039–019), transferrin (10 mg/ml; Sigma Chemical Co., St. Louis, MO, T-5391),
Accepted March 15, 1999. Received December 14, 1998. 1 Correspondence: U.M. Rose, N.V. Organon, Department of Pharmacology, Room RE 2118, P.O. Box 20, 5340 BH Oss, The Netherlands. FAX: 31 412 662542; e-mail:
[email protected]
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insulin (5 mg/ml; Sigma, I-1882), BSA (0.3%; Sigma, A9647) with or without ascorbic acid (50 mg/ml; Sigma, A4034), and selenium (2 ng/ml; Sigma, S-9133) at 378C. Preantral follicles with a diameter of 170–200 mm were isolated with two 30-gauge 3 1/2-in. needles attached to 1-ml syringes and collected in aMEM medium (Gibco, #22571–020) supplemented with glutamine (2 mM), transferrin (10 mg/ml), and insulin (5 mg/ml) with or without ascorbic acid (50 mg/ml) and selenium (2 ng/ml) (aMEM culture medium) and with BSA (0.3%). Isolated follicles were incubated in a humidified incubator gassed with 5% CO2 in air at 378C until enough follicles were isolated for the culture. Follicle Culture
Follicles of 170–200 mm with normal morphological appearance, i.e., a central spherical oocyte, high density of granulosa cells, and a theca cell layer enclosing the entire follicle, were selected and individually cultured in Millicell-CM culture plate inserts (Millipore, Bedford, MA, #PICM 01250) with 250 ml aMEM culture medium supplemented with 5% immature mouse serum. Follicles were cultured in a humidified incubator gassed with 5% CO2 in air at 378C. In the first experiment, follicles were cultured in the presence of 100 mIU/ml (10 ng/ml) recombinant human FSH (recFSH; NV Organon, Oss, The Netherlands) to induce follicular growth, whereas in subsequent experiments, 150 ml medium was replaced by aMEM culture medium supplemented with 5% immature mouse serum and 100 mIU/ml recFSH 20 h after initiation of culture. Culture medium was exchanged every other day, and spent medium was collected and frozen at 2208C until estradiol measurements. The diameter of the follicles was measured each day using 3100 magnification and a calibrated micrometer. In addition, the survival rate of the follicles was checked by evaluation of degeneration (blackening of the follicle) and bursting (loss of the oocyte). At the end of the culture period (4 or 5 days), follicles were used for either histology or ovulation induction. In Vitro Ovulation Induction
At the end of the culture period, follicles were induced to ovulate with hCG (Pregnyl; NV Organon) in aMEM culture medium supplemented with 5% immature mouse serum. In order to test the effects of the PGS-2 inhibitor indomethacin or the anti-progestogen Org-31710, these compounds were added to the follicles in combination with hCG. After 15 h of incubation, follicles were checked for ovulation: follicular rupture, oocyte extrusion, and oocyte maturation. The ovulated oocytes were collected for in vitro fertilization (IVF), and spent culture medium was collected 24 h after hCG addition in order to measure progesterone production. Some of the follicles were collected 3 h after hCG or control treatment in order to evaluate PGS-2 and PR mRNA expression. These follicles were washed in PBS on ice and stored at 2808C until RNA extraction. IVF Assay
Ovulated oocytes were used for an IVF assay adapted from Wood et al. [28]. Ovulated oocytes were collected in T6 medium (98 mM NaCl, 1.4 mM KCl, 0.5 mM MgCl2·6H2O, 1.8 mM CaCl2·2H2O, 0.4 mM Na2HPO4·2H2O, 25 mM NaHCO3, 25 mM sodium lactate, 0.5 mM sodium pyruvate, 5.6 mM glucose, 10 mg/L phenol red) and incubated with capacitated
mouse sperm (1 3 106 cells/ml) at 378C and 5% CO2 in air for 4 h. Then fertilized oocytes were transferred to M16 medium containing 95 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4·7H2O, 1.2 mM KH2PO4, 23 mM sodium lactate, 5.5 mM glucose, 25 mM NaHCO3, 0.3 mM sodium pyruvate, 0.2 mM CaCl2·2H2O, 6 g/L BSA, and 10 mg/L phenol red. Subsequently, medium was exchanged every other day until embryos had reached the blastocyst stage. Estradiol and Progesterone Assay
Estradiol and progesterone levels in the spent culture medium were measured with 125I-RIA kits (ICN Biomedicals, Costa Mesa, CA; #07–138102 and #07–17102, respectively) after extraction with methanol over a Sephadex (Pharmacia and Upjohn, Kalamazoo, MI) C-18 column and subsequent evaporation. The evaporated samples were dissolved in serum-zero standard (ICN Biomedicals) and finally used in the RIA kit. In Vivo Ovulation Induction in Mice
Immature female mice (B6D2-F1, strain C57BL 3 DBA), purchased from the Broekman Institute (Charles River, The Netherlands), were housed in a room with controlled light cycle (12L:12D). At the age of 19–21 days, the mice were injected s.c. with 20 IU urinary FSH (Humegon; N.V. Organon, Oss, The Netherlands) in saline at 1000 h. Fortyeight hours after Humegon injection the mice were injected s.c. with hCG (Pregnyl; 2.5–20 IU/mouse) or saline control. The animals were killed by cervical dislocation 24 or 3 h after hCG administration. Animals killed 24 h after hCG were used to assess the number of ovulated ova in the oviducts. From animals killed 3 h after hCG or control injection, the ovaries were removed, freed of extraneous tissue, and placed in a dish containing 2 ml PBS at 48C. Preovulatory follicles, i.e., follicles exceeding 400 mm with an antral space and no dark ‘‘apoptotic’’ spots, were collected using two 27-gauge needles attached to 1-ml syringes. The follicles were washed in PBS and stored at 2808C until RNA extraction. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Follicles collected from in vitro or in vivo experiments before or 3 h after hCG treatment were used for RNA isolation. Isolation of RNA was carried out according to the RNAzol B protocol (cat. no. CS-104; Campro Scientific, Veenendaal, The Netherlands). The RNA concentration was measured with a UV/visible spectrophotometer (Ultrospec 2000; Amersham Pharmacia Biotech, Roosendaal, The Netherlands). For cDNA synthesis, 0.5 mg random hexamer primers (Amersham Pharmacia) was added to 250 ng isolated total RNA in a total volume of 12 ml. After mixing, the samples were incubated at 708C for 10 min and subsequently kept on ice for 5 min. Next, 4 ml of 5-strength first strand buffer (250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl2), 2 ml 0.1 M dithiothreitol (GibcoBRL, Breda, The Netherlands), and 1 ml 10 mM dNTP (Amersham Pharmacia) was added to the samples. After mixing, the samples were kept at 378C for 2 min. Finally 200 U reverse transcriptase (Superscript II; GibcoBRL) was added. First strand cDNA synthesis was carried out at 378C for 1 h. The cDNA was used in a PCR with specific primer sets for PGS-2 or PR (PGS-2: 59-TGT GAC TGT ACC CGG ACT G-39 [sense], 59-GAC CTG ATA TTT CAA TTT TCC ATC C-39 [antisense]; PR: 59-TCG TCT GTA GTC TCG CCT
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FIG. 1. Follicular survival during 5 days of in vitro culture in the presence of 5% immature mouse serum and in the absence or presence of ascorbic acid and selenium, and with addition of 100 mIU/ml recombinant FSH from Day 0 or Day 1. Results are presented as the percentage follicle survival 6 SE. At least 3 individual cultures were performed in the absence or presence of ascorbic acid and selenium, containing a total of 68 and 327 follicles, respectively. In the absence of ascorbic acid and selenium, follicles survived up to culture Days 4 and 5 in only 1 experiment (N 5 4 follicles).
ATA CC-39 [sense], 59-AAG TCG CCG TAA AGA GGG AAC ACG-39 [antisense]). The PCR reaction was carried out in a final volume of 50 ml containing 100 ng of both sense and antisense primer, 5 ml 10-strength PCR buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl2; 0.01% gelatine; Amersham Pharmacia), 0.2 mM dNTP, and 1.25 U Taq polymerase (Amersham Pharmacia; 5 U/ml). Samples were first denaturated at 948C for 5 min, then submitted to 25 PCR cycles (30 sec 948C, 30 sec 608C, 30 sec 728C) or 30 PCR cycles (30 sec 948C, 30 sec 618C, 30 sec 728C) for PGS-2 and PR, respectively, followed by a final extension of 5 min at 728C. PCR was carried out in a Perkin-Elmer (Irvine, CA) Gene Amp PCR system 2400. After the PCR reaction, 5 ml 10-strength loading buffer (50% glucose; 0.1% bromophenol blue) was added to each sample, and 27.5 ml of each sample was analyzed by gel electrophoresis on a 1.5% agarose gel (SeaKem LE agarose; FMC Bioproducts, Rockland, ME) in 0.5-strength Tris-borate-EDTA buffer. Both buffer and gel contained 0.5 mg/ml ethidium bromide (Sigma). Statistical Analyses
Statistical analysis was performed by means of an ANOVA using Statgraphics Plus 2.0 statistical software (Manugistics, Rockville, MD). Data are presented as mean values 6 SE. Results were considered significantly different at a p , 0.05. RESULTS
In Vitro Follicular Growth: Optimization with Ascorbic Acid and Selenium
At standard culture conditions (i.e., with the addition of glutamine, insulin, transferrin, serum, and recFSH from culture Day 0), preantral mouse follicles grew in vitro from 164 6 1 to 354 6 5 mm in 5 days with a survival rate of
FIG. 2. In vitro development of secondary mouse follicles in the presence of 5% immature mouse serum and 100 mIU/ml recFSH and in the presence of ascorbic acid and selenium. During 7 days of culture, follicles grew, reached the preovulatory stage (A), and started to produce estradiol (B). Results are presented as the mean change in follicular diameter (mm) 6 SE and the average estradiol production per follicle (pg/ml) 6 SE (*p , 0.05: compared with estradiol production on Days 1–3). Each data point includes at least 18 individual follicles.
29% at the end of the culture (Fig. 1). As a result, only few follicles reached the preovulatory stage and could be used for ovulation induction. Follicles were lost at the beginning of the culture, probably owing to damage caused during dissection and around the early antral stage (300– 350 mm) due to premature ‘‘bursting.’’ In order to maintain follicular stability and to protect the follicles against free radicals, ascorbic acid and selenium were added. In addition, follicular growth was not induced immediately after isolation but 20 h later by the administration of recFSH. Figure 1 shows that these changes increased follicular survival significantly (p , 0.05) from 29% to 85%, resulting in a large number of follicles growing to the preovulatory stage and exceeding 400 mm. In addition, the culture period could even be extended to 7 days, which led to an increase in follicular size from 174 6 2 mm to 453 6 10 mm (Fig. 2A). This follicular growth was accompanied by an increased estradiol production, from 38 6 8 pg/ml during the first 3 days of culture to 2550 6 855 pg/ml from Day 5 to 7 (Fig. 2B). Follicular Starting Size and Culture Time
In order to investigate whether follicular growth depends on the diameter of the follicle at the start of the culture
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period, cultures were started with follicles between 150 and 200 mm. As shown in Figure 3, the time needed for follicles to reach the preovulatory stage (. 400 mm) in the presence of 100 mIU/ml recFSH decreased with increasing starting size of the follicle. However, the follicular growth rate did not change. Follicles with a starting size between 145 and 165 mm did not reach a diameter of 400 mm after 6 days of culture. In contrast, follicles with a starting size between 170 and 190 mm or 195 and 210 mm reached a diameter . 400 mm after 5 or 4 days of culture, respectively. For practical reasons, ovulation induction experiments were performed with follicles cultured from a starting size between 170 and 190 mm. In Vitro Ovulation Induction and Fertilization
FIG. 3. Follicular size at the start of culture and the culture time needed to reach a size exceeding 400 mm. Follicles were divided into three groups depending on their starting sizes: 145–165 mm, 170–190 mm, and 195–210 mm. Each group tested contained at least 10 follicles.
To determine the relation between follicular size and the capacity to ovulate, follicles with a starting diameter of approximately 170 mm were cultured in vitro for 5 or 6 days. Groups of follicles that had reached a diameter of 300–400 mm, 400–420 mm, 420–440 mm, or 440–470 mm were treated with 5 IU/ml hCG to induce ovulation. Follicles were checked for ovulation 15 h after hCG was added. Follicles were considered to have ovulated when the follicular wall was ruptured as shown in Figure 4A. In addition, the ovulated oocytes should exhibit cumulus expansion, germinal vesicle breakdown, and polar body extrusion (Fig. 4B). Figure 5 shows that the percentage of ovulated folli-
FIG. 4. Photographs present in vitro follicular rupture, oocyte fertilization, and embryo development. Follicular rupture could be induced with 5 IU/ ml hCG when follicles reached 400 mm (A). The ovulated oocyte exhibited cumulus expansion, germinal vesicle breakdown, and polar body extrusion (B), and could subsequently be fertilized and developed to the 2-cell (C) and hatched blastocyst stage (D). (A) 3100; (B-D) 3200 (published at 95%).
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FIG. 5. Relation between follicular size and ovulatory capacity of in vitro-grown secondary mouse follicles. After 5 or 6 days of culture, follicles were divided into 4 groups depending on their size. Each group was incubated with 5 IU/ml hCG and was checked for ovulation 15 h later. Results are presented as the percentage of at least 20 follicles, obtained from 3 individual cultures, that had ovulated.
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FIG. 6. In vitro ovulation, fertilization, and embryogenesis. In vitrogrown preovulatory mouse follicles were ovulated with 5 IU/ml hCG. Ovulated oocytes were subsequently fertilized and grown to the blastocyst stage. Results are shown as the average percentages of hCG-incubated follicles that ovulated or grew to the 2-cell, 4-cell, or blastocyst stage 6 SE (*p , 0.05: 2-cell, 4-cell, and blastocyst versus ovulated oocytes). The total number of follicles incubated with hCG was 152 divided over 6 individual experiments.
cles depended on the size of the follicle at the time hCG was added. Of the hCG-treated follicles with a diameter between 300 and 400 mm, only 15% exhibited ovulation. In contrast, of the follicles with a diameter . 400 mm, at least 60% ovulated, with a maximum of 80% in the 420– 440 mm group. In order to check the quality of the in vitro-ovulated oocytes, IVF was performed. Figure 4, C and D, shows that ovulated oocytes could be fertilized and cultured to the hatched blastocyst stage. The percentage of in vitro-ovulated and in vitro-fertilized oocytes after in vitro follicle culture is shown in Figure 6. Follicles with an average diameter of 170 mm were cultured for 5 days in vitro until they reached a diameter of . 400 mm. After administration of 5 IU/ml hCG, 69 6 8% of these follicles ovulated. After IVF of these ovulated oocytes, 60% and 42% reached the 4-cell and blastocyst stages, respectively. Thus, approximately 27% of the in vitro-cultured follicles treated with hCG resulted in a blastocyst after IVF. Dose Effect of hCG on In Vitro Ovulation and Luteinization: Comparison with In Vivo Ovulation
To estimate the minimal ovulatory dose of hCG needed in vitro, a dose-response curve was made ranging from 0.5 to 5 IU/ml. Isolated preantral follicles of approximately 170 mm were grown in vitro to the preovulatory stage (. 400 mm). Subsequently, follicles were divided, at random, into 5 groups that received 0.5, 1, 2.5, or 5 IU/ml hCG or medium without hCG as a control. The percentages of ovulation, 15 h after hCG administration, are shown in Figure 7A. In this experiment a dose-dependent response was found, with a maximal ovulation percentage of approximately 80% that leveled at an hCG concentration of 1 IU/ ml. In addition, progesterone production, which is indicative of luteinization, was measured 24 h after hCG administration. The average progesterone levels in the spent medium of individual (ruptured or unruptured) follicles are shown in Figure 7B. All follicles (ruptured and unruptured) showed an increased progesterone production after hCG treatment compared to control, irrespective of the hCG dose used. No clear differences were found in progesterone pro-
FIG. 7. In vitro ovulatory dose response with hCG. In vitro-grown follicles exceeding 400 mm were incubated with 0.5, 1, 2.5, or 5 IU/ml hCG. As a control, follicles were incubated in 100 mIU/ml recFSH. After 24 h of incubation, follicles were checked for ovulation (A), and medium was collected for progesterone measurements (B). Ovulation is presented as the percentage of follicles that ruptured 6 SE (n $ 17 follicles). Progesterone levels, presented as the average progesterone production in 24 h of individual follicles 6 SE (n $ 5 follicles), were estimated for the ruptured and unruptured follicles separately.
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TABLE 1. Effect of the PGS-2 inhibitor indomethacin and the anti-progestogen Org-31710 on in vivo ovulation induction in mice.* hCG (5 IU/animal) hCG/mouse 2.5 5 10 20
No. of ova
Indomethacin (mg/mouse)
6 6 6 6
0 80 160 320
29 62 64 74
3 8 8 10
No. of ova
Org-31710 (mg/mouse)
6 6 6 6
0 0.4 0.8 1.6
62 28 20 20
18 8 5 6
No. of ova 57 54 23 0.1
6 6 6 6
6 7 12 0
* Results are presented as the average of 5 individual animals (mean 6 SE).
duction between ruptured and unruptured follicles at different dose levels. However, at the same hCG dose levels, unruptured follicles tended to produce lower levels of progesterone compared to ruptured follicles. In order to evaluate the optimal hCG dose in vivo, ovulation was induced with hCG doses ranging from 2.5 to 20 IU/animal after stimulation of follicular growth with 20 IU Humegon for 2 days. Table 1 shows that 5 IU hCG per animal gave an average number of ovulated oocytes of 60 ova per mouse. Further increase of the hCG dosage did not lead to an increase in the number of ovulated oocytes. In Vitro hCG-Induced PR and PGS-2 Expression: Comparison with In Vivo Expression
Gene expression of PR and PGS-2 was studied in in vitro-cultured follicles of diameter exceeding 400 mm treated with 5 IU/ml hCG or placebo. RT-PCR analysis of gene expression of both PR and PGS-2, 3 h after hCG or placebo treatment, is shown in Figure 8. As shown, expression of both PR and PGS-2 gene was evident 3 h after hCG treatment. Placebo treatment did not result in an up-regulated gene expression in the in vitro-cultured follicles. For comparison, PR and PGS-2 gene expression was studied in preovulatory follicles and ovaries after hCG or placebo treatment in vivo. The expression of PR and PGS-2 3 h after in vivo hCG or placebo treatment is shown in Figure 9. Similar to findings in the in vitro-treated follicles, expression of PR and PGS-2 did not occur 3 h after placebo treatment, but both genes were clearly expressed 3 h after hCG treatment in both isolated follicles and ovaries.
Effect of Indomethacin on hCG-Induced In Vitro Ovulation and Luteinization: Comparison with In Vivo Ovulation
The PGS inhibitor indomethacin was tested at 5, 10, and 50 mg/ml in preovulatory follicles in the presence of 1 IU/ ml hCG. As a control, follicles were incubated with 1 IU/ ml hCG only. Indomethacin reduced ovulation from 79 6 4% to 54 6 14% and 31 6 6% at 5 and 10 mg/ml, respectively. Further increase of indomethacin to 50 mg/ml did not lead to more inhibition of ovulation (Fig. 10A). In addition to the effect on follicular rupture, the effect on luteinization was tested by measuring the progesterone production. Figure 10B shows that follicles ruptured in the presence of 5 or 10 mg/ml indomethacin produced 37 6 4 and 42 6 5 ng/ml progesterone, which was not significantly different (p . 0.05) from the value for ruptured follicles in the absence of indomethacin (30 6 1 ng/ml). Moreover, the progesterone levels produced by unruptured follicles after culture with or without inhibitor were not different (p . 0.05): 20 6 2 ng/ml for follicles incubated with hCG only and 25 6 4 and 29 6 5 ng/ml for the unruptured follicles incubated with hCG and 5 or 10 mg/ml indomethacin. The effect of indomethacin on hCG-induced ovulation was also tested in vivo at doses ranging from 80 to 320 mg/animal. The number of ovulated oocytes was reduced from 62 6 18 to 20 6 5 at 160 mg/animal in animals that received 5 IU hCG (Table 1). Increasing the indomethacin dose to 320 mg/animal did not lead to a further reduction in the number of ovulated oocytes. Effect of Org-31710 on hCG-Induced In Vitro Ovulation and Luteinization: Comparison with In Vivo Ovulation
The anti-progestogen Org-31710 was tested in preovulatory follicles in the presence of 1 IU/ml hCG. Results presented in Figure 11A show that Org-31710 reduced ovulation from 79 6 6% to 69 6 6% at 0.1 mM and finally blocked ovulation completely at 1 mM. Progesterone measurements showed that luteinization was not affected (Fig. 11B). The effect of Org-31710 on ovulation was also tested in vivo at doses ranging from 0.4 to 1.6 mg/animal. The number of ovulated oocytes was reduced from 57 6 6 to 23 6 12 at 0.8 mg/animal and was completely blocked at 1.6 mg/animal (Table 1). DISCUSSION
FIG. 8. Expression of PGS-2 and PR in in vitro-cultured follicles after 3 h of hCG treatment.
Ovulation, induced by the LH surge, is characterized by extrusion of a fertilizable oocyte into the oviduct and luteinization of the remaining follicular cells. In vivo studies and ex vivo studies in perfused ovaries have shown that factors such as prostaglandins, leukotrienes, and progester-
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509 FIG. 9. Expression of PGS-2 and PR 3 h after hCG treatment of follicles and ovaries isolated from in vivo-treated mice.
one are involved in the control of the ovulatory processes [3–5]. However, these in vivo and ex vivo models cannot distinguish between the role of extrafollicular and intrafollicular factors. To study the role of local intrafollicular factors, an in vitro follicle culture system that includes the various follicular compartments is more suitable. In the present study, the use of an in vitro mouse follicle culture system as a model to study ovulation was evaluated. Subsequently this model was used to study the relevance of prostaglandin and progesterone in the in vitro ovulatory process.
FIG. 10. The effect of the PGS-2 inhibitor indomethacin on in vitro follicular rupture (A) and luteinization (B). Indomethacin was tested at doses ranging from 5 to 50 mg/ml. Results are presented as the percentage ovulated follicles 6 SE (n $ 15) and the average progesterone production of individual follicles 6 SE (n . 5) (*p , 0.05: indomethacin versus control).
The results clearly demonstrate that mouse secondary follicles grow and develop to the preovulatory stage in vitro when cultured in the presence of immature mouse serum and recFSH. Especially the inclusion of both ascorbic acid and selenium in the culture medium improved the culture by preventing follicular bursting (i.e., loss of the oocyte). Since the basal lamina is most important for the stability of a follicle, and since degradation of collagen, a component of the basal lamina, will cause loss of stability, prevention of collagen degradation will maintain membrane
FIG. 11. The effect of the anti-progestogen Org-31710 on in vitro follicular rupture (A) and luteinization (B). Org-31710 was tested at doses ranging from 0.1 to 10 mM; each group contained at least 14 follicles. Results are presented as the percentage ovulated follicles (%) 6 SE and the average progesterone production of individual follicles 6 SE (*p , 0.05: Org-31710 versus control).
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stability and follicular structure. The inclusion of ascorbic acid, which has been proven to promote collagen synthesis [29], probably maintains follicular stability by preventing degeneration of the basal lamina. Moreover, selenium and ascorbic acid are oxygen scavengers [29, 30] and will protect follicles against free radicals. As a result, follicular survival is high and ovulation can therefore be induced in intact preovulatory follicles. The culture time needed to reach the preovulatory stage was shown to be dependent on the starting size of the cultured follicles. Follicles with a diameter between 170 and 200 mm needed 4–5 days to reach a diameter of 400 mm, which is the size of a large antral follicle [31]. Follicles smaller than 160 mm did not reach the 400 mm preovulatory stage within this culture period. During culture, follicles did not only grow but also produced estradiol, indicative of normal follicular differentiation. Similar results have been described by others [32]. Next to growth and estrogen production, a normally developed preovulatory follicle should be able to ovulate a healthy fertilizable oocyte. Although in vitro folliculogenesis has been described extensively, less attention has been paid to in vitro ovulation induction and its characteristics. The studies that have been performed until now show varying results: Boland et al. [24], for example, showed an ovulation rate of 50%, whereas Johnson et al. [27] did not observe ovulation at all. Qvist et al. [21] found both spontaneous and hCG-induced extrusion of oocytes from the follicle, but without resumption of meiosis, which is another characteristic of the ovulatory process. In contrast to these studies, in the present study a spherical culture system was used to investigate in vitro follicular rupture, progesterone production, and PR and PGS-2 gene expression. A nonspherical system in which hCG releases cumulus-oocyte-complexes from their surrounding granulosa and theca cells, and induces cumulus mucification and germinal vesicle breakdown in the oocyte, has been used by others [18]. However, in this model no real rupture of the follicular wall can be observed since the follicles have lost their 3-dimensional structure and do not contain a basal membrane. In spherical systems, the 3-dimensional structure remains intact, which makes it a more natural system. In the model presented in this paper, hCG induced ovulation, i.e., follicular rupture, in more then 60% of the cultured follicles once they exceeded the preovulatory diameter of 400 mm. The percentage of ovulation was much smaller, , 20%, after addition of hCG to smaller follicles with a diameter of 300–400 mm. The quality of the ovulated oocytes was assessed through performing IVF with the ovulated oocytes. The results showed that 60% of the in vitro-ovulated oocytes could be fertilized and that 40% of the ovulated oocytes developed up to the blastocyst stage. In comparable mouse IVF experiments with in vivoovulated oocytes, approximately 60–70% reached the blastocyst stage (data not included), which indicates that oocytes obtained in vivo and in vitro are of similar quality. Previously it has been shown that oocytes of in vitro-grown follicles can be fertilized [22, 25]. However, these oocytes were punctured from the follicles and were not obtained through ovulation induction with hCG. A dose-dependent hCG-induced in vitro ovulation was shown that reached a plateau at a concentration of 1 U/ml. Treatment with hCG was accompanied by an increased progesterone production, which is indicative of luteinization of the follicular cells. The progesterone production of unruptured follicles tended to be lower than the progesterone pro-
duction of ruptured follicles at the same dose level of hCG. Similar results were reported after hCG treatment of guinea pigs in vivo [33, 34]. Those authors reported less progesterone production by luteinized unruptured follicles compared to ruptured luteinized follicles. In humans, it was also suggested that women with the luteinized unruptured follicle syndrome showed reduced luteal progesterone levels [35], although this was not associated with menstrual cycle abnormalities or implantation potential [35, 36]. Besides follicular rupture and induction of progesterone production, hCG also induced expression of PGS-2 and PR, crucial factors involved in the ovulatory process [7–9]. PGS-2 stimulates prostaglandin production, which is involved in activation of a proteolytic cascade that increases follicular pressure, causing weakening of the follicular wall and allowing rupture of the follicle. The exact role of progesterone in the ovulatory process is unknown, but it has been shown that this steroid is essential for the ovulatory process since PR knockout mice exhibit no ovulation in response to hCG [15, 16]. In addition, inhibition of progesterone synthesis or blockade of the PR has been shown to reduce ovulation [11, 12]. In our study, hCG induced the expression of PGS-2 and PR in vitro in a way comparable to that in an in vivo model, which emphasizes the similarity between in vitro and in vivo ovulation. The role of prostaglandin and progesterone in in vitro ovulation induction was confirmed by the inhibitory effects of the PGS-2 inhibitor indomethacin and the anti-progestogen Org-31710. With both compounds, hCG-induced ovulation could be reduced (with Org-31710 even abolished) without affecting progesterone production, indicating that follicular rupture and luteinization are controlled separately. In vivo treatment with indomethacin and Org31710 also reduced the number of ovulated oocytes. These results suggest that prostaglandins and progesterone have a direct effect at the follicular level. In summary, these results clearly demonstrate that hCG initiates ovulation in in vitro-developed mouse follicles, exhibiting similar features in hCG-initiated ovulation as in an in vivo model. In vitro ovulation is characterized by follicular rupture, extrusion of a fertilizable oocyte of good quality, and luteinization. In addition it was shown that both in vitro and in vivo ovulation was marked by an induction of PGS-2 and PR gene expression and that the PGS-2 inhibitor indomethacin and the anti-progestogen Org-31710 reduced and abolished ovulation, respectively, without affecting luteinization seriously. Based on these studies it can be concluded that the in vitro ovulation model of cultured mouse follicles is a physiologically relevant model that can be used to study follicular rupture and the factors involved in this process. ACKNOWLEDGMENTS The authors would like to thank C. vd Perk, R. Tijsen, L. Timmer, L. Janssen, J. Koenders, and G. Schuyers for their technical assistance.
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