Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation

The objectives of the present study were to achieve 1) oocyte maturation, 2) oocyte competence of fertilization, and 3) oocyte competence of embryogenesis with oocytes from primordial follicles obtained from cryopreserved newborn mouse ovaries by using a two-step method. In the first step, frozen-thawed newborn mouse ovaries were transplanted under the kidney capsule of recipients for the initiation of growth from the primordial follicle stage on. In the second step, growing preantral follicles in the ovarian grafts were recovered and cultured. The results demonstrated that primordial follicles were able to be recruited to preantral follicles during the period of transplantation, and preantral follicles could be mechanically isolated from ovarian grafts. Under the present in vitro culture conditions, 85.8% of the isolated follicles (n = 332) from ovarian grafts survived the 12-day in vitro culture process, 84.9% of the recovered oocytes (n = 285) were germinal vesicle breakdown (GVBD)-competent, and 76% of the oocytes that underwent GVBD (n = 242) developed to the metaphase II (MII) stage. In the in vitro fertilization experiments, 75.4% of 142 inseminated MII oocytes underwent fertilization and cleavage to the 2-cell stage. Subsequently, 79.7% of the 2-cell-stage embryos (n = 69) progressed to the late morula-early blastocyst stage. Transfer of late morula-early blastocyst embryos resulted in the production of live offspring. From our experiments, it may be concluded that in vivo maturation by grafting followed by in vitro maturation of frozen-thawed primordial follicles can restore fertility in mice. This model could be useful for a similar application in the human.     

Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation

We investigated the effect of meiotic stages and two maturation protocols on bovine oocyte's resistance to cryopreservation. Oocytes at germinal vesicle breakdown (GVBD) and metaphase II (MII) stage as well as oocytes matured for 22 h in media supplemented with FSH or LH were vitrified by the open pulled straw method. After warming, oocytes underwent additional 16 h (GVBD group) or 2 h (MII group) maturation. Then they were subjected to in vitro fertilization and culture. Some oocytes that matured in the medium supplemented with LH were subjected to parthenogenetic activation after vitrification to determine their developmental potential in absence of fertilization. Survival of oocytes after vitrifying/warming was determined after 22 h in fertilization medium. Cleavage and blastocyst formation rates were used to assess their developmental competence. In both experiments, a portion of unvitrified MII oocytes were subjected to in vitro fertilization and culture as control groups. In Experiment 1, similar cleavage rates were obtained for both GVBD and MII oocytes (53.56 versus 58.01%, P > 0.05). However, significantly higher proportion of cleaved embryos from vitrified MII oocytes developed into blastocysts than those from vitrified GVBD oocytes (1.06 versus 8.37%, respectively, P < 0.01). In Experiment 2, vitrified MII oocytes matured in medium supplemented with LH were superior to vitrified MII oocytes matured in FSH supplementation not only in cleavage rates (61.13 versus 50.33%), but in blastocyst formation rates (11.79 versus 5.19%, P < 0.01) as well. Cleavage and blastocyst formation rates of parthenogenetically activated oocytes were similar to those that were fertilized. Nevertheless, the vitrifying/ warming process significantly compromised the oocytes' developmental capacity since the vitrified oocytes showed significant reduction in both cleavage and blastocyst rates compared to those of not vitrified controls in both experiments (P < 0.01). We showed that oocytes at different maturation stages respond to cryopreservation differently and MII stage oocytes have better resistance to cryopreservation than GVBD stage oocytes. The maturation protocols also influence oocyte's ability to survive cryopreservation. Poor developmental potential after vitrification seem to have resulted from the cryodamage to the oocyte itself. These results suggested the importance of maturation on the developmental competence of cryopreserved oocytes.     

In vitro growth, maturation, fertilization, and embryonic development of oocytes from porcine preantral follicles

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.     

Effects of aromatase inhibition on in vitro follicle and oocyte development analyzed by early preantral mouse follicle culture

In vivo studies on folliculogenesis have documented a relation among intrafollicular steroid content, follicle growth, and oocyte development. This study examined how profound changes in androgen/estrogen ratio would affect mouse in vitro follicular development. Arimidex, a potent follicular aromatase inhibitor was used for this purpose. Early preantral follicles were cultured for 12 days up to the preovulatory stage. Oocyte's meiotic maturation, spindle and chromosome configurations, in vitro fertilization and preimplantation embryo development were evaluated. Compared to controls, Arimidex reduced E2 concentration in follicle culture medium by a factor 1000, and an expected simultaneous accumulation of testosterone was measured in the conditioned medium. Arimidex treatment provoked a dose-dependent earlier differentiation of the granulosa cells as judged by an earlier antrallike cavity formation and slightly elevated basal progesterone secretion. Follicle survival exceeded 98% in all groups and all follicles responded normally to HCG/EGF addition on day 12 by cumulus mucification. By the HCG ovulatory challenge, progesterone output was reduced in Arimidex supplemented groups suggesting preovulatory luteinization. These results indicate that in vitro mouse follicles can develop normally under very low levels of estrogens and that a local androgen increase by a factor 3 is not atretogenic. Oocyte growth did not differ among culture conditions. Arimidex treatment induced a dose dependent enhancement of GVBD and polar body formation rate in response to HCG at the end of culture. Spindle and chromosome analyses demonstrated that in all groups, 90% of the oocytes which extruded a polar body had also reached the MII stage. While most of the cultured MII oocytes had a normal spindle and well aligned chromosomes, significantly less oocytes were fertilized in the groups cultured in the presence of Arimidex. Once fertilized, however, there was found to be no difference for preimplantation embryo development between controls and Arimidex treatment. These data suggest that in mice a pronounced estrogenic environment is not essential for in vitro folliculogenesis. Drastic changes in the intrafollicular steroid concentrations do not disrupt meiotic maturation nor compromise early preimplantation development, but adversely affect fertilization of in vitro grown oocytes.     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence

Changes in MPF and MAPK activities during meiotic maturation of goat oocytes were investigated. Detection of MPF activity occurred concomitantly with GVBD, increased at MI, decreased during anaphase-telophase I transition, and increased thereafter in MII oocytes. The appearance of MAPK activity was delayed compared to MPF activity. MAPK activity increased after GVBD and persisted during the MI-MII transition. Whether MAPK was implicated in goat oocyte meiotic competence was also investigated by using oocytes from different follicle size categories that arrest at specific stages of the maturation process (GV, GVBD, MI, and MII). Results indicate that the ability of goat oocytes to resume meiosis is not directly related to the presence of Erk2. The ability to phosphorylate MAPK is acquired by the oocyte during follicular growth after the ability to resume meiosis. GVBD-arrested oocytes exhibited a high level of MPF activity after 27 hr of culture. However, 28% of oocytes from this group contained inactive MAPK, and 72% exhibited high MAPK activity. In addition, 29% of GVBD-arrested oocytes contained a residual interphasic network without recruitment of microtubules around the condensed chromosomes; 71% of GVBD-arrested oocytes displayed recruitment of microtubules near the condensed chromosomes and contained asters of microtubules distributed throughout the cytoplasm. These results indicate that oocytes arrested at GVBD were not exactly at the same point in the meiotic cell cycle progression, and suggest that MAPK could be implicated in the regulation of microtubule organization. The data presented here suggest that in goat oocytes, MAPK is not implicated in the early events of meiosis resumption, but rather in post-GVBD events such as spindle formation and MII arrest. © 1996 Wiley-Liss Inc.     

Vitrification of calf oocytes: effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.     

In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.     

Changes in the 3-dimensional distribution of mitochondria during meiotic divisions of mouse oocytes

Mitochondrial reorganization during meiotic maturation and parthenogenetic activation was studied in mouse oocytes using a laser scanning confocal microscope and a transmission electron microscope. Mitochondria were mainly distributed perinuclearly in the germinal vesicle (GV) stage oocytes and were dispersed throughout ooplasm after germinal vesicle breakdown (GVBD), except for a slightly higher occurrence in one hemisphere of oocytes, from which the first polar body (PbI) would become extruded. Mitochondria reaggregated around the metaphae II (MII) spindle and pronuclear region after alcohol activatation at the MII stage. The mitochondrial distribution may correspond to the Ca(2+) changes during meiotic maturation and parthenogenetic activation.     

Studies on Ca2+-channel distribution in maturation arrested mouse oocyte

The present study was carried out to identify the existence of voltage-dependent Ca2+-channels (P/Q-, N-, and L-type) and their distributional differences in germinal vesicle (GV) and GV breakdown (GVBD)-arrested mouse oocytes which includes GVBD to telophase I of meiosis I and matured oocytes (MII, metaphase of meiosis II) by using the immunocytochemical method and a confocal laser scanning microscope. (1) Comparison between follicular oocytes (GV) and GV-arrested oocytes after 17 hr of in vitro culture. In follicular oocytes, P/Q-, N-, L (anti-alpha1C anti-alpha1D)-type Ca2+-channels showed both localized and uniform staining. In contrast, GV-arrested oocytes, after in vitro culture for 17 hr, showed no presence of Ca2+-channels in most oocytes. (2) Comparison between GVBD oocytes after culture in vitro for 3 hr and GVBD-arrested oocytes after culture in vitro for 17 hr. In GVBD oocytes, P/Q-, N-, L (anti-1C, anti-alpha1D)-type Ca2+-channels showed both localized and uniform staining. In contrast, in GVBD-arrested oocytes, none of the three types of Ca2+-channels were identified in 72-86% of oocytes. The present study demonstrates that in most GVBD-arrested oocytes that do not mature to MII, there is no Ca2+-channel identified. Therefore, most of the GVBD-arrested oocytes seem to have defects in Ca2+-channel expression/translation. Also, distributional changes of Ca2+-channels take place depending on the maturation progress in GV oocytes and MII stage oocytes (ovulated and 17 hr cultured MII stage oocytes). In addition, we found evidence that a functional voltage-dependent Ca2+-channel (L-type) exists in mouse oocytes (ovulated and cultured MII staged oocytes by a confocal laser scanning microscope).     

Early preantral mouse follicle in vitro maturation: oocyte growth, meiotic maturation and granulosa-cell proliferation

Mechanically isolated early preantral mouse follicles were cultured singly for 16 d and fully grown oocytes were obtained from these follicles. We then compared in vitro and in vivo follicle growth by trypsinising the follicles and counting their cell numbers in a Neubauer-counting chamber and recording the diameter and meiotic status of oocytes under an inverted microscope. As long as the granulosa cells were within the basal membrane, proliferation was slow. From Day 6, when granulosa cells had broken through the basal membrane, the proliferation rate progressed up to Day 10 and decreased thereafter to approximately 12,000 cells per culture droplet. Incorporation of BrdU revealed that proliferating cells were evenly distributed throughout the follicle until antrum formation. As granulosa cell differentiation progressed, proliferation of mural-granulosa cells ceased, while cells around the oocytes continued dividing. Oocyte diameter increased discontinuously in relation to follicle remodelling. During the first growth phase, diameters increased from 56.5 (+/- 4.4 microns) to 67 (+/- 4.1 microns) until the onset of antral-like cavity formation. The last growth phase started after Day 10, and by Day 14 oocyte diameters were not significantly different from those of 26-d-old in vivo control oocytes. The potential to resume meiosis after mechanical removal of granulosa cells was first reached on Day 8; thereafter, removal of the corona showed that all oocytes cultured with FSH remained arrested at the GV stage up to Day 16. After Day 8, approximately 70% of all oocytes underwent GVBD as a result of granulosa-cell removal, but only 23% of these reached MII after 24 h. The in vivo controls reached a comparable GVBD rate (66%) when the granulosa was removed, but most of the oocytes (82%) underwent first polar body extrusion 24 h later. These results suggest that although oocyte diameters after IVM are not different from those of the controls, culture conditions are not yet adequate to support complete meiotic maturation.     

Cumulus cells suppress meiotic progression in pig oocytes cultured in vitro

The objective of this study was to examine the effect of cumulus cell removal from cumulusoocyte complexes (COCs) on meiotic progression. In Experiments 1, 2 and 3, pig COCs were cultured for 16, 20 and 24 h, respectively. The cumulus cells were then removed, and the denuded oocytes were incubated in fresh medium for another 32 h in Experiment 1, for 28 h in Experiment 2 and for 24 h in Experiment 3. In Experiment 4, the denuded oocytes and COCs were co-cultured in a drop of fresh medium from 24 h of cultivation to the end of the culture period (48 h). Removal of the cumulus cells after 16 h of cultivation had no effect on the proportions of oocytes both undergoing germinal vesicle breakdown (GVBD) and reaching MII. When the denuded oocytes were further cultured for 24 h, following the removal of their cumulus cells after 24 h of cultivation, the proportion of oocytes undergoing GVBD was significantly higher (90%, P < 0.05) than that of oocytes that were continuously cultured for 48 h without removing the cumulus cells (80%). Removal of the cumulus cells after 20 and 24 h of incubation produced a significant increase in the proportion of oocytes reaching the MII stage (84%, P < 0.05 and 76%, P < 0.01, respectively) as compared with COCs cultured continuously for 48 h without removing cumulus cells (71% and 55%, respectively). The maturation rate of denuded oocytes co-cultured with COCs for the second 24 h of cultivation was comparable to that of denuded oocytes cultured without COCs (77 and 74%, respectively). From these results, it was concluded that cumulus cells surrounding oocytes suppressed meiosis of both the GVBD process and progression from GVBD to MII in pig oocytes cultured in vitro, and that the suppressive factor in meiotic progression produced by the cumulus cells might be transferred to the oocytes through gap junctions rather than through the medium.     

A co-culture system with preantral follicular granulosa cells in vitro induces meiotic maturation of immature oocytes

Development of technologies to mature oocytes in vitro is important for in vitro fertilization research. Here, we investigated the ability of preantral follicular granulosa cells (PAGCs) to restrain apoptosis and to promote the growth and meiotic resumption of immature murine oocytes in vitro. The oocytes of 55–65 μm derived from 12 to 14 days old juvenile mice were co-cultured with PAGCs in vitro. The results showed that the oocytes co-cultured with PAGCs for 7 days grew faster and 14.6% of immature oocytes were able to complete the first meiotic division and arrive at the MII stage. 71 oocytes co-cultured with PAGCs were fertilized and 16 embryos were able to form morula-blastocysts. Following the co-culture of immature oocytes with/without PAGCs for 7 days, the percentage of apoptotic oocytes were 33.5 and 51.4%, respectively (p < 0.01). Furthermore, the inhibition of apoptosis was communicated between oocytes and PAGCs through the GDF9-PI3 K-Akt signaling pathway. In conclusion, the co-culture with PAGCs has a beneficial effect on the growth and maturation of immature oocytes.     

Chromatin, microtubules, and kinases activities during meiotic resumption in bitch oocytes

In contrast to the majority of mammals, canine oocytes are ovulated at immature germinal vesicle (GV) stage and complete meiotic maturation to metaphase II during 48-72 hr within the oviducts. This study aims to characterize meiotic maturation process in bitch oocytes, with both morphological and biochemical approaches. The follow-up of chromatin and microtubules during maturation was described, and MPF and MAP kinase activities were quantified at different stages of maturation. Since bitch oocyte cytoplasm is darkly pigmented, the first step was to setup an appropriate staining method for DNA. We thus compared the efficiency of two visualization techniques and demonstrated that propidium iodide coupled to confocal microscopy was a better method than Hoechst/fluorescence microscopy for nuclear stage observation (determination rates: 98.6 vs. 69.5%, respectively; P < 0.01, n = 1622 oocytes). Microtubule organization, evaluated by tubulin immunodetection, revealed subcortical and perinuclear alpha-tubulin and asters in GV oocytes and a clear network of microtubules in GVBD oocytes. In MI and MII oocytes, a symmetrical, barrel-shaped, and radially located spindle was observed. MPF and MAP kinase activities were assayed concomitantly using histone H1 and MBP as substrates. Kinase activities were detected at low levels in oocytes at GV and GVBD stages and were significantly higher at MI and MII stages. In conclusion, despite the particular pattern of meiotic resumption in canine oocytes (ovulated at GV stage), cytoskeleton/chromatin organization and kinase activities follow a similar pattern to those observed in other mammalian species.     

Comparison of three in vitro culture systems for maturation of early preantral mouse ovarian follicles

The aim of this study was to compare three different culture systems for in vitro follicular growth and oocyte maturation in ovarian follicles of mice in order to assess the technique with the optimal growth and improved rate of meiotic maturation. The three systems tested were culture under oil, on a hydrophobic membrane and on agar respectively. Early preantral follicles were cultured for 12 days in alpha-MEM GlutaMAX medium. Follicular growth, oocyte meiotic maturation, oocyte extrusion, atresia and estradiol production were analysed. Follicular development showed two phases in the three systems, with slow growth before day 5 and subsequent acceleration. The percentage of follicles transferred into oocyte maturation medium was significantly higher after culture under oil. The proportion of oocytes that achieved nuclear maturation (metaphase II) was higher when follicles were cultured under oil or on a hydrophobic membrane than on agar. Our results support the use of culture under oil for in vitro follicular growth from the early preantral stage in order to obtain metaphase II oocytes. Fertilization ability of these oocytes and the capacity to obtain healthy mice in a reproducible manner warrants further investigation.     

Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA

Despite many attempts to improve the in vitro maturation (IVM) of canine oocytes using various culture conditions, the efficiency of canine IVM remains very low compared with that of other domestic animals. In the present study we examined the effect of ovarian estrus stage on oocyte quality, and the effect of epidermal growth factor (EGF) in the presence and absence of macromolecules on the IVM of canine oocytes. More oocytes >or=100 microm in diameter were obtained from follicular ovaries than from ovaries at other estrus stages. After 72 h of culture, significantly more oocytes recovered from follicular ovaries than from anestrous and luteal ovaries were in germinal vesicle break down (GVBD). Bovine serum albumin (BSA) or fetal bovine serum (FBS) supplementation improved meiotic resumption as compared to polyvinyl alcohol (PVA) supplementation; however, there was no difference between the BSA and FBS supplements. The oocytes matured in North Carolina State University (NCSU) 37 medium containing 0.4% BSA and 100 ng/ml EGF showed the highest rates of development to the metaphase II (MII) stage when compared with the control treatment (P < 0.05). These results suggest that the estrous cycle of bitches influences the meiotic resumption of oocytes cultured in vitro, and EGF increases the meiotic resumption of canine oocytes in the presence of BSA in vitro.     

Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro

The perfection of in vitro maturation in the bitch has yet to be achieved, and is an essential prerequisite for gamete salvage programmes in endangered canine species. In contrast to most mammals, the bitch ovulates an immature oocyte which undergoes meiotic maturation within the oviduct. A model of the oviductal environment may therefore be useful for performing in vitro maturation. This study was performed to investigate the effect of introducing an oviductal element to the culture environment, first with the use of a synthetic oviductal fluid (SOF), and secondly, using coculture with isolated canine oviductal epithelial cells, upon the rate of oocyte maturation in vitro. It was found that there was no difference in the proportion of oocytes undergoing germinal vesicle breakdown (GVBD) after 48 h in culture between SOF containing 0.3% bovine serum albumin (BSA, 45%), containing 4% BSA (36%) and control medium 199 (27%). There was also no difference in oocyte nuclear maturation to metaphase I/anaphase I/metaphase II (MI/AI/MII) after 48 h in culture between SOF containing 0.3% BSA (5%), containing 4% BSA (7%) and control medium 199 (6%). In addition, there was no difference in oocyte nuclear maturation to MI/AI/MII after 96 h between SOF containing 0.3% BSA (0), containing 4% BSA (7%) and control medium 199 (11%). In contrast, the proportion of oocytes undergoing GVBD after 96 h in culture was affected by the treatment used, with 27% in SOF + 0.3% BSA, 62% in SOF + 4% BSA and 63% in medium 199. It was found that there was no difference in the proportion of oocytes undergoing GVBD between the coculture treatments 199 (33%), 199 + cells (37%), coculture medium (30%) and coculture medium + cells (49%), and for oocyte nuclear maturation to MI/AI/MII, between medium 199 (2%), 199 + cells (0), coculture medium (6%) and coculture medium + cells (2%) after 48 h in culture. In addition, there was no difference in oocyte nuclear maturation to GVBD after 96 h between 199 (61%), 199 + cells (59%), coculture medium (65%) and coculture medium + cells (53%). In contrast, the proportion of oocytes maturing to MI/AI/MII after 96 h in culture was affected by the treatment used, with a significant difference between 199 (0), 199 + cells (9%), coculture medium (0) and coculture medium + cells (0). It was shown, therefore, that the culture of oocytes in the SOF improved oocyte nuclear maturation when supplemented with a high concentration of protein and that culture in the presence of oviductal epithelial cells improved oocyte maturation, but only after a prolonged period of time.     

小鼠年龄及卵丘—卵母细胞间联接的解离对卵母细胞体外成熟的影响

实验研究了小鼠卵母细胞体外过程中卵丘－卵母细胞间的相互作用。实验小鼠为雌性Ｂ６Ｄ２杂交一代。激素处理４８小时后分离出卵后天和卵母细胞复合体,并培养在含有次黄嘌呤的培养液中。２４小时后检查卵母细胞核成熟情况。     

Oocyte diameter in relation to meiotic competence and sperm penetration

This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and >120 microm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 +/- 0.4 microm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 microm groups were significantly higher (P<0.01) than in the > or = 110 microm groups. None of the oocytes <110 microm reached metaphase II (MU), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 microm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 microm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes > or = 120 microm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 microm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.