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Ovary |
Genetic Diversity Department,3 National institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
Agriculture, Forestry and Fisheries Research Council,4 Ministry of Agriculture, Forestry and Fisheries, Kasumigaseki, Tokyo 100-8950, Japan
Department of Research Planning and Coordination,5 National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0091, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, follicular development, oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protocols for the use of animals were approved by the Animal Care Committee of the National Institute of Agrobiological Sciences. Preliminarily, we collected three ovaries from three piglets (crossbreeds of Landrace x Large White x Duroc) at 10, 20, 30, and 40 days old and sectioned each ovary at 6 µm. Follicles were classified in three sections of each ovary according to the criteria described in the section below on histological analysis examination. In the 20-day-old piglets, primordial follicles accounted for 96% of the total number of follicles classified, and the remaining 4% were almost all primary follicles. In the 10-day-old piglets, the percentage of primary follicles was similar (3.7%) to that at 20 days old, but each ovary contained several germ cell nests. Between 30 and 40 days after birth, the secondary follicles appeared and the percentage of primordial follicles decreased. Therefore, we chose ovaries of 20-day-old piglets for stimulation of growth of primordial follicles by xenografting. Donor ovaries were dissected from 20-day-old piglets of the same crossbreed mentioned above. Immediately after removal, the cortex of each ovary was cut into small pieces, and these pieces were further minced into approximately 1-mm3 pieces in saline supplemented with 668 U/ml of penicillin (Sigma Chemical Co., St. Louis, MO) and 0.2 mg/ml of streptomycin sulfate (Sigma). As recipients, 6- to 8-wk-old female immunodeficient mice (CD1[ICR]-nu/nu; Charles River Japan, Yokohama, Japan) (n = 26) were anesthetized and ovariectomized. Small holes were made in the mouse kidney capsule with a pair of fine forceps, and five or six fragments of ovarian tissue were inserted under the capsule of each kidney. Vaginal smears were taken every day from 2 weeks after grafting. After the first detection of cornified epithelial cells in their vaginal smears, the mice were randomly assigned to the following four experimental groups. The mice were given (intraperitoneally) 5 IU of equine chorionic gonadotropin (eCG) (PMS 1000; Nihon Zenyaku Kogyo, Koriyama, Japan) 10 (eCG-10 group, n = 7), 30 (eCG-30 group, n = 5), or 60 (eCG-60 group, n = 7) days after detection of vaginal cornification. The other recipient mice (n = 7) received no hormone treatment (-eCG group).
Sample Collection
Forty-eight hours after eCG treatment, the mice were anesthetized and bled by heart venipuncture. Serum was stored at -30°C until it was assayed for total inhibin. Immediately after blood sampling, the number of grafts was counted and cumulus-oocyte complexes (COCs) were isolated mechanically with a surgical blade in medium 199 [12] (with Hanks salts; Sigma) from the antral follicles in the tissue grafted under the kidney capsules. The diameters of the recovered oocytes were measured with a caliper under the eyepiece of an inverted microscope. Three grafts were obtained from three mice of the eCG-60 group, fixed in Bouin solution, and embedded in paraffin for histological examination. COCs, blood samples, and grafts were also obtained from the -eCG group 10 days after detection of cornified epithelial cells. To estimate plasma total inhibin levels in mice that received no grafts, we also collected blood samples from five ovariectomized, ungrafted mice 120 days after ovariectomy (OVX group). For comparison with the follicular growth in the xenografted ovarian tissue, three pieces of ovarian tissues measuring approximately 1 mm3 that had been prepared for grafting were processed for histological examination.
In Vitro Maturation and In Vitro Fertilization of Oocytes
Recovered COCs were matured in vitro as described previously [12]. Briefly, the COCs were cultured for 20–22 h in modified North Carolina State University-37 (NCSU-37) solution [13] supplemented with 10% porcine follicular fluid, 0.6 mM cysteine, 50 µM ß-mercaptoethanol, 1 mM dibutyl cAMP (dbcAMP; Sigma), 10 IU/ml of eCG (PMS 1000; Nihon Zenyaku), and 10 IU/ml of hCG (Puberogen 500 U; Sankyo, Tokyo, Japan) for 20–22 h. Subsequently, they were cultured for 24 h in in vitro maturation (IVM) medium without the dbcAMP and hormones for 24 h. Maturation culture was performed under conditions of O2, CO2, and N2 adjusted to 5%, 5%, and 90% (5% O2), respectively, at 39°C.
After IVM, cumulus cells were removed by hyaluronidase treatment (150 IU/ml; Sigma) and gentle pipetting. Twenty oocytes with the first polar body in the eCG-60 group were harvested as matured oocytes and placed in fertilization medium (Pig-FM) [14] supplemented with 2 mM caffeine and 5 mg/ml of BSA (fraction V; Sigma). Frozen epididymal spermatozoa [15] were thawed and preincubated for 15 min at 37°C in medium 199 (with Earle salts; Gibco, Life Technologies Inc., Grand Island, NY) adjusted to pH 7.8 [16]. A portion (10 µl) of the preincubated spermatozoa was introduced into 90 µl of fertilization medium containing 10 matured oocytes. The final sperm concentration was adjusted to 1 x 106/ml. Coincubation was performed at 39°C under 5% O2 for 5 h. After coincubation, the oocytes were freed from the attached spermatozoa and transferred to IVC medium [12] (NCSU-37 solution containing 4 mg/ml of BSA and 50 µM ß-mercaptoethanol, supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactose), then further incubated for 5 h at 38.5°C under 5% O2.
Assessment of Nuclear Status of Oocytes and Fertilization
Oocytes before and after IVM, stripped of their cumulus cells, and in vitro fertilization (IVF) oocytes were whole mounted on glass slides and fixed in acetic alcohol (acetic acid:methanol = 1:3). After the specimens had been stained with 1% aceto-orcein (Sigma), the nuclear status and extrusion of polar bodies were examined by phase-contrast microscopy.
Histological Analysis
The three ovarian tissues prepared for xenografting and the three grafts obtained from the three mice in the -eCG and eCG-60 groups were serially sectioned at 6 µm and stained with hematoxylin and eosin. To ensure that no follicles were counted twice, follicles were counted only when the nucleolus of the oocyte was present and were classified as follows: primordial follicles with one layer of flattened granulosa cells, primary follicles with one layer of cuboidal granulosa cells, secondary follicles with two or more layers of granulosa cells but no antrum, and antral follicles with an antral cavity. Follicles were classified as atretic if they contained granulosa cells with pyknotic nuclei and disrupted granulosa layers.
Fluoroimmunoassay for Total Inhibin
Concentrations of total inhibin in the plasma of the host mice, as a marker of follicular growth, were determined by a competitive fluoroimmunoassay (FIA) using europium (Eu)-labeled inhibin A as a probe [17]. In the FIA of total inhibin, anti-bovine inhibin serum (TNDH-1: [18]) was used as a primary antibody. Bovine 32-kDa inhibin A was used for Eu labeling and as a reference standard. (Anti-inhibin serum was provided by Dr. K. Taya, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; bovine 32-kDa inhibin was provided by Dr. Y Hasegawa, Kitasato University, Towada, Aomori, Japan.) The detection limit of the FIA was 0.078 ng/ml. The intra-assay and interassay coefficients of variation were 7.8% and 11.0%, respectively.
Data Analyses
Oocytes were divided into two groups according to their diameter (<115 µm and 
115 µm) on the basis of the finding that oocytes larger than 115 µm in diameter from the antral follicles of prepubertal gilts acquire meiotic competence [19, 20]. All data were subjected to ANOVA, and the significance of the difference among means was determined by the Duncan multiple range test. The General Linear Models Procedure of Statistical Analysis Systems (SAS Inc., Cary, NC) [21] was used for the analyses. Differences at P < 0.05 were considered statistically significant.
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RESULTS |
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Ovarian grafts grew to more than twice their original size, especially in the mice of the eCG-60 group (Fig. 1). Many antral follicles were visible under the kidney capsule, but the largest follicles were sometimes hemorrhagic. Grafts sometimes fused together because of the way in which they were positioned. The cortex area of a whole ovary obtained from a 20-day-old piglet in the preliminary experiment is shown in Figure 2A. Similarly, minced ovarian cortex obtained from 20-day-old piglets before xenotransplantation contained largely primordial follicles before xenotransplantation (Table 1). Forty-five to 70 days after grafting (mean ± SEM, 63.2 ± 2.5 days; n = 26), the host mice for the first time showed the presence of cornified epithelial cells in their vaginal smears. The absolute time between the initial surgery and graft recovery was 68.6 ± 1.9 days in the -eCG group, 70.0 ± 2.8 days in the eCG-10 group, 92.0 ± 1.3 days in the eCG-30 group, and 122.3 ± 0.3 days in the eCG-60 group. Around the time of the first detection of vaginal cornification, the follicles in their grafts had proceeded to the antral stage in the mice of the -eCG group, but the number of antral follicles was low (Table 1 and Fig. 2B). However, when the grafts were recovered after treatment of host mice with eCG 60 days after vaginal cornification, there were many more growing follicles, especially antral follicles, in the grafts (Table 1 and Fig. 2C), but there was a sharp decline in the number of primordial follicles. Atresia of follicles was also observed in all grafts in the -eCG and eCG-60 groups. The percentage of atresia in the secondary follicles was 10%–30% in each graft in the -eCG group and 30%–60% in the eCG-60 group, and the percentage of atresia in the antral follicles was 0%–80% in the -eCG group and 60%–85% in the eCG-60 group.
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Changes in Concentrations of Total Inhibin in the Circulation of the Host Mice
Total inhibin levels in the circulation in the OVX group were low (Fig. 3). However, inhibin levels increased significantly (P < 0.05) in the mice of the -eCG group 10 days after vaginal cornification and further increased in the eCG-60 group.
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Growth and Maturation of Oocytes
The number of oocytes recovered per mouse or per graft was low in the -eCG and eCG-10 groups but increased as the interval between detection of vaginal cornification and eCG treatment increased (Table 2). The number of full-sized oocytes larger than 115 µm showed no significant increase in the mice of the eCG-10 and eCG-30 groups compared with the -eCG group. However, the number of full-sized oocytes increased dramatically in the eCG-60 group. Many of the oocytes isolated from the eCG-60 mice were surrounded by cumulus cells (Fig. 4). In all the oocytes recovered from all groups, the nucleus was at the germinal vesicle (GV) stage (Fig. 5A). After IVM, only small numbers of oocytes had the ability to resume meiosis to the metaphase II stage in the -eCG, eCG-10, and eCG-30 groups (Table 2), but a greater number of matured oocytes at the metaphase II stage was obtained from mice of the eCG-60 group (Table 2 and Fig. 5B). In the eCG-60 group, the ratio of metaphase II oocytes to the total number of oocytes or full-sized oocytes was 17% or 46%, respectively. When mature oocytes, as determined by extrusion of the first polar body, were fertilized in vitro with frozen-thawed boar spermatozoa, 11 (55%) of the 20 mature oocytes obtained from the eCG-60 group formed a female pronucleus (Fig. 5C) and extruded the second polar body (Fig. 5D). The sperm nucleus(ei) that had penetrated the oocyte were transformed into male pronucleus(ei) (Fig. 5C). Polyspermy was noted in 2 of 11 fertilized oocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the porcine neonatal ovaries that we used for xenografting, primordial follicles accounted for most of the follicles, as described previously [22, 23]. Histological examination revealed that folliculogenesis was initiated and completed in the grafts in the recipient mice. In particular, the number of antral follicles in the grafts of the eCG-60 group was double the number of primary follicles in the grafts before transplantation, which indicates that at least half of the antral follicles at the time of recovery originated from primordial follicles of the neonatal ovaries, not from primary follicles. The length of time between grafting and the first detection of antral follicles was similar to the time taken for primordial follicles to grow to the antral stage in pigs [24, 25] and was consistent with previous findings that follicular development in the grafts is determined by the graft species [6–8]. However, an important finding of our study is the fact that good performance in terms of follicular growth and oocyte recovery after eCG treatment was only achieved when the eCG was given 60 days after the first detection of antral follicles. This result is strikingly different from those of previous studies [5–9, 11], in which the grafts were recovered and follicular growth was examined around the time of the first detection of antral follicles. We obtained few full-sized oocytes from the mice of the -eCG and eCG-10 groups, and these results suggest that the cohort of follicles that contains the first antral follicles is not a suitable target for stimulation. In the mice of the eCG-60 group, the high plasma concentrations of total inhibin provided further evidence of increased formation of antral follicles. The reasons for the greater success achieved with the eCG-60 group are not clear, but it is possible that the grafts in the eCG-60 mice contained a greater number of late preantral and early antral follicles, which are sensitive to eCG [23], than did the other groups. The requirement for such a long period before eCG treatment in xenografting of the porcine ovary is strikingly different to that in autografting [26] and xenografting [3] of the mouse ovary.
After IVM, a marked number of oocytes recovered from the mice of the eCG-60 group had the ability to resume meiosis, whereas oocytes recovered from the other groups barely matured. These results are attributable to the recovery of a greater number of full-sized oocytes from the eCG-60 groups, because oocytes larger than 115 µm in diameter from the antral follicles of prepubertal gilts acquire meiotic competence [19, 20]. In the eCG-60 group, 46% of the full-sized oocytes ( 115 µm) matured. This ratio is lower than our previous results (70%) after IVM of oocytes collected from prepubertal gilts by using the same IVM system [12, 27], although our percentage was still high enough to be useful. The occurrence of atresia in the antral follicles may account for the relatively low maturation rate. We demonstrated that metaphase II oocytes from the eCG-60 group were fertilized (55% of metaphase II oocytes) in an IVF system, as shown by the formation of female and male pronuclei and extrusion of the second polar body. The fertilization ratio was sufficient compared with that obtained in our previous study (44.8% [12]). The incidence of polyspermy (2/11) in the fertilized oocytes in the eCG-60 group was lower than in our previous results (approximately 30%) after IVF of oocytes collected from prepubertal gilts by using the same IVF system [12]. Nuclear transition from the GV stage to the metaphase II stage and successful pronucleus formation reflect the nuclear and cytoplasmic maturation of oocytes, respectively. Suitable xenografting and subsequent in vitro culture endowed porcine oocytes from the primordial follicles with full maturation and fertilization ability.
Recently, a model of xenografting for sperm maturation of large mammals—the grafting of neonatal porcine testes into mice—was successful in producing sperm, and their fertilizing ability was assessed by the formation of a pronucleus after intracytoplasmic injection into mouse oocytes [28]. However, to our knowledge, our study is the first to demonstrate that full folliculogenesis and oocyte growth can be completed in ovarian tissue from a phylogenetically distant species grafted into mice. In addition, fertilization of the oocytes recovered from mice in a standard pig IVF system showed a greater efficiency in the use of oocytes, the number of which is determined before birth. When combined with cryopreservation of ovarian tissue, this approach will be a powerful tool in clinical and field situations. However, the developmental capacity of the fertilized oocytes remains to be determined.
In conclusion, our results have resolved a major limitation to xenotransplantation in that we were able to obtain a small number of fully grown oocytes from the ovarian tissue of a large mammal. Subsequent IVM and IVF resulted in the successful production of fertilized oocytes. This approach is applicable to a diverse range of gene resources of females of large mammalian species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Hiroyuki Kaneko, Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. FAX: 81 29 838 7447; kaneko{at}nias.affrc.go.jp
Received: 13 March 2003.
First decision: 6 April 2003.
Accepted: 30 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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