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a Leland J. and Dorothy H. Olson Center for Women's Health, Division of Reproductive Endocrinology and Infertility, Department of Ob/Gyn and Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4515
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the presence of 0.2 µg/ml insulin, primary follicles developed after 8 days, and ~4% secondary follicles with 2–3 layers of granulosa cells appeared after 16 days of culture. However, higher dosages (> 0.2 µg/ml) of insulin retarded primary follicle formation and induced the formation of primordial follicles with larger oocytes. An increased number of larger oocytes with a few granulosa cells accumulated at the periphery of the ovary. The results indicate that although primordial and primary follicles can develop after 12 days in vitro in the absence of exogenous insulin, the latter is required for timely progression of follicular development through primary and secondary stages.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because perinatal ovarian development in vivo is regulated by hitherto unknown complex processes, understanding cellular interaction during early ovarian development in vitro appears to be a suitable alternative. Blandau et al. [4, 5] were the first to observe mouse follicular development in organ culture. Eppig and O'Brien [6] have successfully developed the neonatal mouse ovary in an organ culture and have shown that oocytes developed in the cultured ovary are capable of undergoing in vitro fertilization and producing offspring upon transfer to a foster female. Their results suggest that oocyte-somatic cell interaction can apparently continue in vitro in the absence of the conventional endocrine milieu that exists in vivo. In both the rat and mouse, the formation of primordial follicles is completed by birth [1, 6], while in the hamster, germ cell mitosis continues for a few days after birth; therefore, ovaries of a newborn hamster contain germ cells, primordial oocytes, and somatic cells [7]. Somatic cells begin organizing around the oocyte clusters by the 3rd and 4th postnatal day, forming cord-like structures. Primordial follicles do not appear until 4–5 days of postnatal life. Therefore, hamster ovaries provide the opportunity to study events during early ovarian morphogenesis and the onset of primordial follicle formation.
The objectives of the present study were to establish an organ culture for fetal hamster ovaries and to evaluate the possibility of primordial and primary follicle formation in vitro. We also tested the supporting role of insulin that had been used in the culture of preantral follicles from adult ovaries [8, 9] and during mouse neonatal ovary culture [6]. We used 15-day-old fetal hamster ovaries to ensure that absolutely no morphological evidence of follicle formation was present.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pregnant golden hamsters (Sasco, Madison, WI) at the 15th day of gestation were anesthetized with an i.p. injection of pentobarbital sodium (Nembutal, 8 mg/100 g BW; Sigma Chemical Co., St. Louis, MO), and the abdomen was rinsed with 70% ethanol followed by sterile PBS, pH 7.4. Uterine horns containing fetuses were taken out of the abdomen, one at a time, and placed between two PBS-soaked gauges to keep the fetal sacs moist. Uterine blood supply was kept intact to maintain fetal viability until removal from the amniotic sac. Fetuses were removed through an incision in the implantation chamber; fetal ovaries were dissected out along with the oviduct and placed in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Bethesda, MD), pH 7.4, containing antibiotics (100 U/ml of penicillin G, 100 µg/ml of streptomycin sulfate, 0.25 µg/ml amphotericin B; Gibco-BRL), 0.5% BSA, and 0.3 µg/ml phenol red at room temperature in a 30-mm plastic in vitro fertilization-embryo culture dish (9, Falcon; Fisher Scientific, Pittsburgh, PA). Ovaries were dissected free of the oviduct under a Nikon (Garden City, NY) stereozoom microscope; they were then rinsed four times with DMEM without any supplement and twice in DMEM containing transferrin, selenium, linoleic acid, and hydrocortisone ([9]; DMEM-complete) and cultured essentially as described by Eppig and O'Brien [6]. Briefly, ovaries were placed on a Falcon tissue culture insert with 3-µm porous nontissue culture-coated polystyrene membrane and covered with 10 µl of DMEM-complete. The inserts were placed in a 6-well Falcon tissue culture cluster containing 2 ml of DMEM-complete in each well. Ovaries were cultured in a Heraeus (Scientific Instruments, Greenwood, IN) incubator under 5% CO2 in air for up to 16 days. Medium was changed every 48 h with replacement of 1.5 ml of the spent medium with fresh medium. Ovaries were removed every 4th day and used for light microscope morphometry and electron microscopic evaluation of follicular development.
Evaluation of the Effect of Insulin on Ovarian Development In Vitro
Because insulin is an important supplement for in vitro culture of hamster preantral follicles [9] and mouse ovaries [6], the effect of various dosages of insulin was tested on hamster preantral ovary development in vitro. Fetal hamster ovaries were cultured as described previously in the presence of 0.1, 0.2, 0.4, and 0.5 µg/ml insulin (bovine insulin; Mallinckrodt, Phillipsburg, NJ). As a control for ITS+ (insulin, transferrin, selenium, and linoleic acid; Collaborative Research, Bedford, MA), we also tested a dose of 6.25 µg/ml insulin, which is routinely used in hamster preantral follicle culture [9].
Evaluation of DNA Synthesis During Ovarian Development In Situ
Ovaries were cultured as described above in the absence or presence of 0.2 µg/ml insulin and exposed to 1 µCi/ml of [3H]thymidine (Amersham; now Amersham Pharmacia Biotech, Piscataway, NJ; specific activity 40 Ci/mmol) for 24 h before termination. The objective was to evaluate DNA synthesis as an index of cell proliferation during somatic cell rearrangement. Ovaries were removed every 4th day and fixed in Bouin's fixative for 10 h for autoradiographic evaluation of the labeling index.
Ultrastructural Morphology
Ovaries were processed for ultrastructural morphology as described by Luft [10] with minor modifications. Briefly, ovaries were fixed in 2.5% glutaraldehyde in 0.1 M potassium phosphate buffer, pH 7.4, for 2 h at 4°C; they were then rinsed three times in the same buffer (10 min each time), postfixed in 1% osmium tetroxide for 1 h, rinsed in potassium phosphate buffer for 10 min, dehydrated in ascending grades of ethanol (10 min each), and cleared in propylene oxide 3 times (10 minutes) each followed by infusion overnight with propylene oxide-resin. Finally, ovaries were embedded in Araldite 502 (Ladd Research, Burlington, VT) epoxy resin. Ultrathin sections at 60–80 nm were cut in an RMC-ET-7 ultramicrotome (Research Manufacturing Company, Tucson, AZ), placed on a 200 mesh grid, stained with uranyl nitrate and lead acetate, and examined under a Philips 300 electron microscope (EM Core Facility, Cell Biology and Anatomy, University of Nebraska Medical Center).
Histology
Plastic-embedded ovaries were cut at 0.5 µm using the RMC-ET-7 ultramicrotome and placed on aminopropyl-triethoxysilane (Sigma) slides, dried, and stained with toluidine blue for light microscopic morphometric analysis of follicle development.
Autoradiography
Bouin's-fixed ovaries were processed for routine paraffin embedding, and 7-µm-thick sections were coated with Kodak NTB2 (Eastman Kodak, Rochester, NY) liquid photographic emulsion. Sections were exposed for 7 days, developed in Dektol, and stained with hematoxylin and eosin as described previously [9].
Morphometric Analysis of Oocyte Growth and Follicle Development
Oocyte diameter and the percentage of follicles at each stage of development were assessed under x100 magnification with a Leica DMR research microscope equipped with a Sony video camera (North Central Instruments, Minneapolis, MN) and Image Pro-Plus image analysis software (Media Cybernetics, Silver Spring, MD). Three hundred follicles from each ovary for each group were evaluated for oocyte diameter and percentage of follicles in primordial (presence of flattened granulosa cells), primary (unilaminar cuboidal granulosa cells), and secondary (
2 layers of granulosa cells) follicle classes [11]. Oocyte diameter was calculated from the average of two perpendicular diameters. Oocytes showing the nucleoli were selected to avoid duplicate counting of follicles and oocytes. The average diameter of the oocyte in each class of follicles from each ovary was considered as one replicate (n = 1). Values from at least three different ovaries (three replicates, n = 3) cultured at different times were used to obtain the mean ± SEM. The percentage of follicles in each class was calculated to assess the rate of follicular development. The results were examined by two-way ANOVA with Scheffe's test with level of significance at 5%.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphologically fetal ovaries at the 15th day of gestation contained clusters of primordial oocytes surrounded by a few undifferentiated somatic cells; however, no distinct follicular structure was noted (Fig. 1). Ultrastructurally, primordial oocytes had a large nucleus and scanty cytoplasm with relatively fewer tubular mitochondria, which were primarily located at one pole of the nucleus (Fig. 2). Somatic cells around the oocyte had a few spherical mitochondria and had vacuoles reminiscent of lipid droplets. Both oocytes and somatic cells had rough endoplasmic reticulum (RER). The presence of microfilaments in undifferentiated somatic cells, which were undeveloped, was also evident (Fig. 2). After 4 days of culture in vitro in the presence of 0.2 µg/ml insulin, all oocytes had dispersed chromosomes, but a large majority of oocytes were still in clusters (Fig. 3A). Flattened somatic cells were in the process of aligning themselves around many oocytes as a prelude to forming primordial follicles; however, such follicular structures were few in number (Fig. 3A). The morphology of fetal ovaries developed in vitro for 4 days was more or less similar to that of ovaries of 4-day-old hamsters, except that the pachytene type chromosome was visible in many oocytes (Fig. 3B). Ultrastructurally, nuclear and cytoplasmic development was evident for the oocytes and adjacent somatic cells (Fig. 4). The extension of somatic cell processes between two adjacent oocytes was evident, and usually a pair of oocytes was found to share 2–3 somatic cells. Distinct filamentous structures were present exclusively in somatic cells (Fig. 4). The number of mitochondria appeared to increase in the oocytes as well as in adjacent somatic cells, and distinct intercytoplasmic bridges between two adjacent but partially separated oocytes were clearly visible (Fig. 4). Moreover, round mitochondria with developing cristae and numerous RER were visible in the oocytes (Fig. 4). Distinct filamentous and membrane-like structures and fat vacuoles were found encapsulated (Fig. 4). No degenerative change in the oocytes or somatic cells was noted after 4 days of culture. There was virtually no ultrastructural difference between oocytes or somatic cells developed with and without insulin; for the sake of brevity, therefore, the morphology of follicles developed in the presence of 0.2 µg/ml insulin has been furnished.
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After 8 days of culture in the presence of insulin, distinct primordial follicles were visible (Fig. 5A), and occasional primary follicles with unilaminar cuboidal granulosa cells started appearing (not shown in the figure); however, no primary follicle was visible in ovaries cultured in insulin-free medium (see Fig. 13). The morphology of the ovary developed in vitro for 8 days was more or less similar to that of an age-matched ovary collected from an 8-day-old hamster (Fig. 5B). Distinct primordial follicles were visible in the in vivo-developed ovaries (Fig. 5B); however, ovarian architecture was more compact than that of the in vitro-developed ovary. Ultrastructurally, follicular oocyte and granulosa cells had developed further, and the number of well-developed spherical mitochondria had increased noticeably (data not shown). After 12 days of culture in the presence of 0.2 µg/ml insulin, numerous well-developed primary follicles with cuboidal granulosa cells were visible (Fig. 6A), and oocytes grew significantly in volume. The number of interfollicular somatic cells also increased noticeably. However, when compared with the morphology of an ovary of a 12-day-old hamster, ovary development in vitro appeared to be slower, as many growing secondary follicles with 2–4 layers of granulosa cells were visible in the in vivo-developed ovary (Fig. 6B). After 16 days of culture in the absence of insulin, ovaries were flat without any apparent sign of degeneration, and follicular development up to the primary follicle stage continued. Secondary follicles with 2–3 layers of granulosa cells, however, were visible only in ovaries exposed to 0.2 and 0.4 µg/ml insulin (Fig. 7A). Follicular development in vitro, however, was significantly slower than that of a 16-day-old in vivo-developed ovary (Fig. 7B). In vivo-developed ovaries had numerous large preantral follicles with 5–6 layers of granulosa cells (Fig. 7B). Ultrastructurally, oocytes contained numerous well-developed mitochondria that were distributed more or less evenly around the nucleus. Oocyte cytoplasm contained distinct RER, lysosome-like structures, and microvilli protruding from the oocyte plasma membrane (Fig. 8). Granulosa cells of primordial follicles were well organized around the oocyte and had numerous RER, distinct nucleus, and mitochondria (Fig. 9). The mitochondria of the oocytes were mainly round, whereas those of somatic cells were both round and spherical with well-developed tubular cristae (Fig. 9). Although gap junctions between the oocyte and granulosa cell membrane became prominent after 16 days of culture (Fig. 10), the structures appeared as early as 8 days in vitro (data not shown). The microvilli projected from the oocyte plasma membrane extended into an amorphous structure resembling the zona pellucida (Fig. 11).
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Autoradiographic studies of ovaries cultured for 16 days revealed that granulosa cells of primary (stage 1) and secondary (stages 2 and 3) classes of follicles incorporated [3H]thymidine; however, very few interstitial cells showed signs of DNA synthesis by the 16th day of culture (Fig. 12). No significant change in the labeling index was noted when insulin was absent (data not shown). Moreover, the labeling index of ovarian cells did not differ appreciably throughout the culture period (data not shown).
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Morphometric analyses revealed that ovaries grown for 4 days in vitro had a few primordial follicle-like structures regardless of the treatment condition (Fig. 13A). According to our classification, true primordial follicles possess only flattened granulosa cells [11]. In the absence of insulin, the formation of primary follicles did not occur before 12 days of culture (Fig. 13C) and increased slightly after 16 days (Fig. 13D). Insulin showed a biphasic effect on primordial and primary follicle formation from Day 8 through 16 (Fig. 13, B–D). The formation of primary follicles increased significantly (P < 0.05) up to Day 16 following exposure to 0.1 and 0.2 µg/ml insulin. Furthermore, 0.2 and 0.4 µg/ml insulin induced the formation of secondary follicles with 2–3 layers of granulosa cells only after 16 days of culture (Fig. 13D). Insulin dose-dependently inhibited the percentage of primary follicle formation by Day 12 of culture, even though the percentage of primary follicles had increased noticeably compared to that at Day 8. A similar trend was also evident for both primary and secondary follicles on Day 16 when the insulin doses were more than 0.2 µg/ml (Fig. 13D). Increasing the dosage further up to 0.5 µg/ml resulted in a significant reduction in primary follicle formation and a complete failure in secondary follicle formation (Fig. 13D). Insulin at the dose of 6.25 µg/ml caused significant retardation in ovarian growth and induced ovarian degeneration (pyknosis), especially in the medullary region (data not shown).
The average diameter of the oocyte in 15-day-old fetal ovaries was 10.2 ± 0.01 µm. Oocyte diameter increased progressively with the advancement of culture regardless of insulin exposure (Fig. 14, A and B); however, an increasing number of oocytes became part of primordial and primary follicles in the presence of insulin. The diameter of the oocyte of primordial follicles increased significantly (P < 0.05) with up to 0.2 µg/ml insulin by Day 12 of culture (Fig. 14A). A further increase in the oocyte diameter was noted by Day 16 when ovaries were exposed to 0.5 µg/ml insulin (Fig. 14A). In the absence of insulin, distinct primary follicles were not visible until Day 12, and thereafter the diameter of the oocyte of primary follicles did not change appreciably. In the presence of 0.1 µg/ml insulin, the diameter of the oocyte of primary follicles had increased sharply by the 12th day as compared to the 8th day (Fig. 14B). The diameter of the oocyte of primary follicles increased progressively in the presence of 0.2 µg/ml insulin, and significant increases, compared to values with 0.1 µg/ml insulin, were noted after 8 and 16 days of culture (Fig. 14B). Although oocyte diameter increased throughout the culture in the presence of > 0.2 µg/ml insulin, the growth rate was generally sluggish (P < 0.05) by the 12th day compared to that in the non-insulin and low-dose insulin groups. A gradual decline in the oocyte diameter was noted after 12 days when insulin concentration was increased beyond 0.1 µg/ml (Fig. 14B). A similar trend was observed for the oocytes of primordial follicles developed in the presence of 0.2 µg/ml insulin doses (Fig. 14A). Moreover, the growth of the oocytes was also altered by the 8th day for primary follicles developed in the presence of 0.5 µg/ml insulin. Eventually, the oocyte diameter reached the control level after 16 days of culture (Fig. 14B). Because there were only a few secondary follicles for some groups, no attempt was made to measure oocyte diameter for those follicles.
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Follicular development beyond 2–3 layers of granulosa cells did not occur despite extension of the culture or supplementation of the culture with 5% or 10% of hamster or bovine serum.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The gradual increase in number and the maturation of mitochondria during the 16 days of culture suggest that oocyte growth occurs throughout the culture period. The formation of gap junctions between the oocyte plasma membrane and granulosa cell processes as early as 8 days of development in vitro clearly indicates that oocyte-somatic cell interaction is important for follicle formation. The importance of gap junctions in oocyte-somatic cell communication has been recognized [24, 25], and gap junctions play an important role in inducing developmental competence and fertilizability in the oocyte [3]. Null mutation of cx37 gene that is responsible for gap-junctional protein synthesis in the mouse results in meiotic incompetence after follicles enter the antral stage [26]. The appreciable development of the RER both in the oocyte and in somatic cells also suggests a marked increase in protein synthesis, which is a hallmark of cellular growth process.
The increase in the percentage of primary follicles up to Day 16 suggests that a constant transition of primordial follicles to primary stage occurs throughout the culture, although the rate of primary follicle formation undergoes significant alteration between 12 and 16 days of culture, especially in the presence of a moderate dose of insulin. This sudden acceleration of follicular transition from the primordial to the primary stage suggests that the insulin effect may depend on the maturation status of follicular cells. An insulin overdose, on the other hand, adversely affects granulosa cell differentiation, thereby reducing the recruitment of primordial follicles to the primary stage. Because the major morphological difference between primordial and primary follicles is the transition of flattened epithelial cells to cuboidal granulosa cells, the first part of follicular growth may significantly depend on the development of somatic cells. This is evident from the size of the oocytes of primordial and primary follicles after 8 days of culture. It is noteworthy that although follicles remain in the primordial stage, the diameter of the oocytes continues to increase, suggesting that part of the oocyte growth during early follicular development is independent of somatic cell development. Conversely, it is also possible that the slower rate of somatic cell development in vitro may have altered the synchrony between oocyte and somatic cell development. It is, however, evident that no significant oocyte growth occurs for the primary follicle class beyond 12 days in the absence of insulin.
Despite the formation of primary follicles in the absence of insulin, insulin appears to act as a maintenance factor for the growth of primordial and primary follicles, and it can improve the formation of primary follicles at lower dosages. The increases in oocyte diameter and the percentage of primordial follicles in response to higher dosages of insulin suggest that the differentiation of flattened granulosa cells to a cuboidal shape may be compromised by higher dosages of insulin, resulting in asynchronous development of the oocyte and somatic cells and the formation of primordial follicles with a larger oocyte. This response is quite different from that found in the adult hamster, where preantral follicle growth is supported by a higher dose of insulin (6.25 µg/ml), which forms a part of the culture supplement ITS+ [8, 9]. Moreover, primordial follicle development in vitro in the presence of high-dose (6.25 µg/ml) insulin has been reported for mice [6] and cattle [12]. High-dose insulin also does not influence glucose metabolic activity of human preantral follicles in vitro [27]. Insulin induces the proliferation of immature rat granulosa cells [28] and the maturation of porcine oocytes [29]. Moreover, insulin influences granulosa cell steroidogenic activity [30–32] and induces LH receptors in porcine granulosa cells in culture [33]. Therefore, the insulin effect appears to depend on the maturational status of ovarian cells and the dosage. The unique stimulation of oocyte development, especially in the primary follicle class, suggests that a moderate dose of insulin either may modulate oocyte development directly or may do so indirectly via regulating granulosa cell functions.
In summary, the results of the present study furnish evidence that early onset of follicle formation can occur in vitro from undifferentiated somatic cells and oocytes in ovary organ culture. Although follicle formation beyond 2–3 layers of granulosa cells requires further improvement of the culture system, the usefulness of this model for study of the mechanisms of primordial and primary follicle formation, and the beneficial role of a low dose of insulin on follicle and oocyte development, are apparent.
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FOOTNOTES |
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2 Correspondence: Shyamal K. Roy, Departments of OB/GYN and Physiology and Biophysics, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}mail.unmc.edu
3 Current address: Department of OB/GYN, Division of Reproductive Biology, Stanford University School of Medicine, 300 Pasteur Drive, Room A344, Stanford, CA 94305-5317.
Accepted: August 10, 1999.
Received: May 17, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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