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.     

Spontaneous oocyte maturation in Rana pipiens: Estrogen and follicle wall involvement

Immature (germinal vesicle stage) Rana pipiens oocytes typically remain arrested in prophase I of meiosis even after extended periods of in-vitro culture, if not stimulated with hormones. We have, however, sporadically observed “spontaneous” occurrences of oocyte maturation in vitro without the addition of hormones. This study documents some of our observations on this phenomenon and presents experimental results concerning the effects and possible involvement of estrogen and follicle wall components in regulating spontaneous oocyte maturation. Estrogen was found to inhibit spontaneous oocyte maturation (GVBD) in a dose-dependent fashion. Follicles in which spontaneous maturation was inhibited by estrogen retained their responsiveness (GVBD) to both frog pituitary homogenate (FPH) and progesterone stimulation. Inhibitory effects of estrogen on spontaneous maturation, however, were not reversed following incubation of washed follicles in plain culture medium without added hormones. Possible involvement of progesterone synthesis in spontaneous oocyte maturation was ascertained by simultaneously monitoring endogenous progesterone synthesis and the occurrence of spontaneous GVBD over the course of the maturation process. In spontaneous maturing follicle there was a gradual increase in basal levels of progesterone synthesis that preceded GVBD. Significantly, addition of estrogen abolished both the spontaneous progesterone production and spontaneous oocyte maturation. When FPH was added to follicles exhibiting spontaneous oocyte maturation, progesterone production was augmented and the time course of oocyte maturation was greatly accelerated. Involvement of ovarian components in the maturation process was investigated by selective removal of various follicle layers by microdissection. Removal of follicle epithelium and theca layer (defolliculation) markedly decreased spontaneous and FPH-induced maturation, whereas removal of the entire follicle wall (denudation) completely blocked it. Our results suggest that both spontaneous and FPH-induced maturation involve an estrogen sensitive process in the follicle wall. Thus, somatic follicle cells appear to serve as a common mediator for both types of maturation, which are linked by some intrafollicular mechanism involving steroidogenesis. Hence, estrogen may play an important role as an endogenous intrafollicular regulator of oocyte meiotic maturation.     

Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.     

Secondary follicle growth and oocyte maturation by culture in alginate hydrogel following cryopreservation of the ovary or individual follicles

An option for fertility preservation for women facing a cancer diagnosis involves the cryopreservation of ovarian tissue for later re‐transplantation or in vitro culture, with in vitro culture preferred to avoid reintroduction of the cancer. Small, immature follicles survive the freeze‐thaw process, and can be matured through in follicle maturation (IFM) that involves an initial growth of the follicle and subsequent maturation of the oocyte. The ovarian tissue can be cryopreserved in two forms: (i) cortical strips consisting of follicles and surrounding stroma (Cryo‐Ov) or (ii) individually isolated follicles (Cryo‐In). The aim of this study was to assess the follicle growth and oocyte maturation for follicles that were cryopreserved either as strips or individually using a slow‐freezing cryopreservation method. The two follicle groups, together with non‐cryopreserved control follicles, were grown in an alginate‐based three‐dimensional culture system for 12 days. The overall survival, size increase and antrum formation rates were comparable among the three groups. At day 12 of culture, Androstenedione levels were decreased in the Cryo‐Ov group relative to the other two, and the ratio of progesterone to estradiol was increased in the two cryopreserved groups relative to the control. Both Gja1 (known as connexin 43) and Gja4 (known as connexin 37) mRNA expression were decreased at day 6 in the cryopreserved groups relative to controls, and by day 12, Gja1 was similar for all three groups. Moreover, Cryo‐In resulted in lower GVBD rate indicating some impaired oocyte development. Overall, the present study demonstrated that mouse preantral follicles, either within ovarian tissues or individually isolated, could be successfully cryopreserved by the slow‐freezing method, as evidenced by post‐thaw follicle development and steroidgenesis, oocyte maturation and molecular markers for oocyte and/or granulosa cells connection. Biotechnol. Bioeng. 2009;103: 378–386. © 2009 Wiley Periodicals, Inc.     

Effects of Kit Ligand and anti-Kit antibody on growth of cultured mouse preantral follicles

Paracrine regulations between the oocyte and granulosa cells are likely to be key regulators of early folliculogenesis. Evidence obtained from genetic mutants as well as in vivo experiments suggest that Kit and Kit Ligand (KL) may regulate early follicular morphogenesis and function. In this study, we used in vitro culture of intact mouse follicles to confirm and extend these findings. Two concentrations of Kit Ligand (20 and 50 ng/ml) or an antibody blocking the Kit-Kit Ligand interactions (SC1494) were added to preantral follicles grown individually for 12 days and which were finally triggered to ovulate. Effects on follicle and oocyte survival, granulosa cell function (antrum formation, cell numbers, steroidogenesis), and oocyte function (growth, survival, nuclear and/or cytoplasmic maturation) were then analyzed. In optimal culture conditions (presence of 5% fetal calf serum), 50 ng/ml of KL significantly improved cytoplasmic maturation of the oocyte and increased follicular testosterone output, but other parameters were not altered. In serum-free culture conditions, KL was mitogenic for granulosa cells at 50 ng/ml, but could not induce antrum formation and no differences were observed between control and treated groups for steroidogenesis or oocyte growth. Blockade of Kit-Kit Ligand interactions by addition of a blocking antibody decreased oocyte survival 6-9 days after addition of the antibody, but did not upset granulosa cell proliferation. Antrum formation was, however, strongly inhibited. In addition, the blocking antibody markedly reduced aromatase activity of granulosa cells. We conclude that Kit/KL interactions are important for antrum formation and follicular steroidogenesis and regulate survival and cytoplasmic maturation of the oocyte.     

Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture

Trichlorfon (TCF), an organophosphate insecticide and potent inhibitor of choline esterases, was previously shown to induce first meiotic nondisjunction and spindle aberrations in isolated, follicle cell-denuded mouse oocytes maturing in vitro. To explore dose-response and direct and indirect, potentially synergistic effects of TCF on the somatic cells and the oocyte within a follicle, we presently employed preantral follicle culture. 100 microg/ml TCF added at the time of hormonally stimulated resumption of meiosis of follicle cell-enclosed mouse oocytes, 16 h before in vitro ovulation, induced significant rises in first meiotic nondisjunction in oocytes from preantral follicle culture. Lower concentrations (6 microg/ml TCF) disturbed polar body formation. Nuclear maturation to meiosis II in absence of cytokinesis resulted in significant increases in polyploidy. Oocytes maturing in follicles in the presence of TCF had aberrant second meiotic spindles. Influences of TCF on somatic cell function were evident by reduced follicular mucification in vitro and deceased progesterone production. In contrast to TCF, acetylcholine (0.1-100 microM) increased progesterone production. The observations therefore suggest that TCF influences oocyte maturation and folliculogenesis directly and indirectly. High TCF is aneugenic at first meiotic division in oocytes, irrespective of the presence or absence of follicle cells. At lower concentrations TCF interferes with spindle formation, chromosome congression at meiosis II, and coordination of nuclear and cytoplasmic maturation, posing risks for second meiotic errors. The observations suggest that chronic TCF exposure during maturation in the follicle may predispose oocytes to the formation of chromosomally unbalanced preimplantation embryos after fertilization.     

Mammalian oocyte growth and development in vitro

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes from preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. © 1996 Wiley-Liss, Inc.     

Retrievable hydrogels for ovarian follicle transplantation and oocyte collection

Cancer survivorship rates have drastically increased due to improved efficacy of oncologic treatments. Consequently, clinical concerns have shifted from solely focusing on survival to quality of life, with fertility preservation as an important consideration. Among fertility preservation strategies for female patients, ovarian tissue cryopreservation and subsequent reimplantation has been the only clinical option available to cancer survivors with cryopreserved tissue. However, follicle atresia after transplantation and risk of reintroducing malignant cells have prevented this procedure from becoming widely adopted in clinics. Herein, we investigated the encapsulation of ovarian follicles in alginate hydrogels that isolate the graft from the host, yet allows for maturation after transplantation at a heterotopic (i.e., subcutaneous) site, a process we termed in vivo follicle maturation. Survival of multiple follicle populations was confirmed via histology, with the notable development of the antral follicles. Collected oocytes (63%) exhibited polar body extrusion and were fertilized by intracytoplasmic sperm injection and standard in vitro fertilization procedures. Successfully fertilized oocytes developed to the pronucleus (14%), two‐cell (36%), and four‐cell (7%) stages. Furthermore, ovarian follicles cotransplanted with metastatic breast cancer cells within the hydrogels allowed for retrieval of the follicles, and no mice developed tumors after removal of the implant, confirming that the hydrogel prevented seeding of disease within the host. Collectively, these findings demonstrate a viable option for safe use of potentially cancer‐laden ovarian donor tissue for in vivo follicle maturation within a retrievable hydrogel and subsequent oocyte collection. Ultimately, this technology may provide novel options to preserve fertility for young female patients with cancer.     

Dichotomous effects of forskolin on somatic and germ cell components of the ovarian follicle: evidence of cAMP involvement in steroid production and action

The role of cyclic AMP (cAMP) in ovarian follicular functions in Rana pipiens was investigated with the use of the adenylate cyclase stimulator, forskolin, which is thought to elevate intracellular level of cAMP. Effects of forskolin on oocyte germinal vesicle breakdown (GVBD) and on progesterone production by the follicles were assessed during the course of in vitro culture. Addition of forskolin to culture medium suppressed both progesterone-and frog pituitary homogenate (FPH)-induced meiotic maturation of the oocytes. Inhibitory effects of forskolin were essentially reversible and forskolin completely inhibited GVBD when added during the first four hours of incubation following exposure to progesterone. Forskolin alone stimulated a low level progesterone production by isolated follicles, but markedly stimulated progesterone production when it was supplemented with a low dose of FPH (0.005 pituitary equivalent/ml). Thus, forskolin acts synergistically with FPH on follicle cells to stimulate progesterone production. A higher dose of FPH (0.05 pitui. eq./ml) produced no additional synergistic effect of forskolin. Therefore, forskolin appears to have two contradictory functions in ovarian follicles: it augments FPH induced follicle secretion of meiosis initiator, progesterone, and simultaneously suppresses the maturation of the oocytes triggered by exogenous progesterone or FPH. The data presented indicate that there are two independent adenylate cyclase systems in the ovarian follicles which have separate functions: one in the follicle cells and the other in the oocyte. The two enzyme systems are thus compartmentalized and regulate different biological functions using the same messenger, cAMP. The data provide evidence that in amphibians, as in mammals, pituitary hormones regulate steroid hormone production by follicle cells via a cyclic AMP system. Thus, control of oocyte maturation induction appears to be determined by the relative levels of cAMP present in the follicle cells and oocytes.     

Maturation, fertilization and complete development of porcine oocytes matured under different systems

This study was designed 1) to determine the effectiveness of 2 in vitro maturation systems commonly employed to produce nuclear and cytoplasmically mature pig oocytes, 2) to assess the effects of boar, sperm concentration and maturation system on oocyte penetrability and male pronucleus formation and 3) to determine the ability of the in vitro matured oocytes to be fertilized in vivo by artificial insemination (AI) of sows. The differences examined between the 2 maturation systems included the culture medium (Waymouth vs TCM199), hormones, additives, culture conditions (static vs gentle agitation) presence or absence of porcine follicular fluid (PFF) and presence or absence of follicular shells. The results showed that nuclear maturation rate was similar in both systems (83.3 +/- 3.5 vs 86.4 +/- 2.5%), and intracellular content of glutathione was 5.21 +/- 0.73 vs 3.5 +/- 0.39 pmol/oocyte, although no correlation between these parameters was observed. The penetration rate and number of sperm cells per oocyte were dependent on the boar, maturation system and sperm concentration, but the rate of male pronuclear formation seemed to be influenced only by the boar and the maturation system but not by sperm concentration. In vivo fertilization of in vitro matured oocytes showed that both maturation systems could yield viable oocytes since 3 of 4 gilts and 2 of 4 gilts, respectively, became pregnant. Failure to become pregnant was not associated with inadequate oocyte maturation since control gilts, which received their own ovulated oocytes rather than in vitro matured oocytes at transfer, also did not become pregnant. We conclude that polyspermy may be an inherent problem in the IVF but not in the IVM systems.     

Meiotic competence in horse oocytes: interactions among chromatin configuration, follicle size, cumulus morphology, and season

Horse oocytes were collected from an abattoir over a 15-mo period. After classification of follicle size and cumulus morphology, oocytes were either fixed immediately (0 h) or matured in vitro (24 h). There was no effect of season on the number of antral follicles present on the ovaries, or on oocyte maturation rate for any class of oocyte. The proportion of oocytes having condensed chromatin at 0 h increased with increasing follicle size. The oocyte maturation rate also increased with follicle size, and for follicles 相似文献   

EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturation

EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus-oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell.     

Effect of IGF-I on pig oocyte maturation,fertilization, and early embryonic development in vitro,and on granulosa and cumulus cell biosynthetic activity

Porcine granulosa cells have been shown previously to both secrete and respond to insulin-like growth factor-I (IGF-I), suggesting an autocrine function of this peptide in the follicle. The present work was undertaken to determine possible effects of IGF-I on in vitro maturation, in vitro fertilization, and early embryonic development in culture. Granulosa and cumulus cell proliferation and differentiation based on ³H-thymidine uptake and progesterone production, respectively, were also assessed. The results showed that the cleavage rate of oocytes was markedly stimulated in a dose-dependent manner by the addition of IGF-I to the oocyte maturation medium (P < 0.05). Embryo development beyond the 8-cell stage was improved by IGF-I, reaching a maximum of 22% at 200 ng/ml IGF-I. Treatment with IGF-I after fertilization increased the percentage of total oocyte cleavage (P < 0.05) to approximately 52%, 43%, and 57% at, respectively, 25, 50, and 100 ng/ml IGF-I. ³H-thymidine incorporation by granulosa cells was significantly increased in cultures treated with FSH (3-fold) or IGF-I (6-fold) compared to the control. For the cumulus cells, FSH caused a similar increase (3-fold) in ³H-thymidine incorporation while IGF-I stimulated a 15-fold increase. Progesterone production by the granulosa cells was increased to the same extent by treatment with FSH or IGF-I (4.7 and 5.1-fold, respectively). However, for the cumulus cells, while FSH caused a marked 16-fold increase in progesterone production, IGF-I caused only a marginal increase of 2.5-fold. These results indicate a beneficial effect of IGF-I on in vitro porcine oocyte maturation and pre-implantation embryo development, suggesting a physiological role for IGF-I in vivo. The in vivo effect of IGF-I may be indirect via autocrine stimulation of cumulus and/or granulosa cells resulting in enhanced oocyte maturation and fertilization. © 1994 Wiley-Liss, Inc.     

Mouse oocyte development in vitro with various culture systems

These experiments were designed to determine whether or not hormones are required for the growth of mouse oocytes and to assess the possible role of companion granulosa cells in oocyte growth. To approach these problems, four systems for the culture of oocytes, either alone or in association with granulosa cells, were utilized: (1) isolated oocyte culture, (2) isolated oocyte-ovarian cell coculture, (3) isolated follicle culture, and (4) ovarian organ culture. Oocytes from 8-day-old B6D2F₁ mice failed to grow in isolated oocyte culture. Addition of follicle-stimulating hormone (FSH), 17β-estradiol (E₂), or serum to the medium failed to prevent oocyte degeneration or to promote oocyte growth. On the other hand, oocytes in isolated follicle culture or in organ culture grew significantly in defined medium. The results showed that oocytes grown in isolated follicle culture under defined conditions and in the absence of gonadotropins resemble oocytes grown in vivo in terms of their ultrastructural characteristics, with the exception of enlarged mitochondria. In addition, these oocytes were shown to exhibit some normal functional characteristics in terms of their increased levels of CO₂ evolution from exogenous pyruvate, and the ability of the fully grown oocytes to initiate meiotic maturation when freed from granulosa cells. It was concluded that gonadotropins are not necessary for oocyte growth and that gonadotropins are not required to potentiate the spontaneous meiotic maturation of oocytes which occurs after their isolation from granulosa cells. The results indicated that association of granulosa cells and oocytes was necessary for oocyte growth. However, isolated oocytes in coculture with ovarian cells failed to grow. Addition of FSH or E₂ to the cocultures failed to promote oocyte growth or delay oocyte degeneration. It was concluded that, under the culture conditions used, granulosa cells must be in contact with the oocyte, perhaps by means of specialized cell junctions, for oocyte growth to occur.     

Induction and inhibition of in vitro oocyte maturation and production of steroids in fish follicles by forskolin

The effects of forskolin (FK) on in vitro oocyte maturation and production of steroids were examined in Oryzias latipes. When oocytes within preovulatory follicles were preincubated in the presence of FK for 2-10 hr, they matured normally after additional incubation for 10-20 hr in plain culture medium. Naked (follicle cell-free) oocytes did not mature under these conditions. FK stimulated dose-dependent production of steroids (estradiol-17 beta, E2, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta-diOHprog) and cAMP in follicle (granulosa) cells. On the other hand, exposure to FK within 2 hr after 17 alpha,20 beta-diOH prog stimulation caused reversible inhibition of gonadotropin (PMS)- or 17 alpha,20 beta-diOH prog-induced maturation of the intrafollicular oocytes in vitro. FK also significantly inhibited the 17 alpha,20 beta-diOHprog-induced maturation of naked oocytes, suggesting the existence of adenylate cyclase in fish oocytes. These data indicate that in Oryzias latipes, FK induces oocyte maturation by stimulating follicular production of maturation-inducing steroid (MIS), probably 17 alpha,20 beta-diOH prog, via an increase in cAMP, and that it may inhibit oocyte maturation by increasing ooplasmic cAMP and some inhibitory interaction between the granulosa cells and the oocyte through intercellular communication.     

Effect of 5-Hydroxytryptamine on Steroidogenesis and Oocyte Maturation in Pre-ovulatory Follicles of the Medaka Oryzias latipes

The effect of 5-hydroxytryptamine (5-HT) on steroidogenesis and oocyte maturation in pre-ovulatory follicles of the medaka Oryzias lalipes was examined using in vitro culture system. The earliest breakdown of the germinal vesicle of intrafollicular oocytes occurred about 17 hr after the beginning of incubation in the presence of 5-HT at concentration of 10 ng/ml or more. 5-HT induced oocyte maturation in a dose-dependent manner. Cyanoketone inhibited this stimulation. The concentration of 5-HT required to induce oocyte maturation corresponded to that required to enhance the production (secretion) of estradiol-17β and 17α,20β-dihydroxy-4-pregnen-3-one by pre-ovulatory follicle cells. At a concentration of 1 μg/ml, the follicle had to be exposed to 5-HT for at least 4 hr for oocyte maturation accompanied by ovulation to occur. These results indicate that 5-HT induces in vitro maturation of medaka oocytes by stimulating 17α,20β-dihydroxy-4-pregnen-3-one production by pre-ovulatory follicular cells.     

Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development

The availability of viable oocytes is the limiting factor in the development of new reproductive techniques. Many attempts have been made to grow immature oocytes in vitro during recent decades. Recently, a modified alginate-based three-dimensional culture system was designed to support the growth and maturation of multilayered secondary follicles. This system was able to produce oocytes that successfully completed meiosis, fertilization, and development to the blastocyst stage. Subsequent attempts to culture two-layered secondary follicles were unsuccessful under the original conditions. Herein, we investigated the effect of alginate consistency on two-layered follicle growth and oocyte developmental competence by encapsulating follicles into alginate scaffolds of various concentrations. Although there were no significant differences in survival rates, 0.25% and 0.5% alginate supported more rapid growth of follicles and antrum formation compared with 1.5% and 1.0% alginate after 8 days of culture. Alginate scaffold concentration also affected the proliferation and differentiation of somatic cells (theca and granulosa cells), measured in terms of morphological changes, steroid profiles (androstenedione, estradiol, and progesterone), and specific molecular markers (Fshr, Lhcgr, and Gja1). Theca cell proliferation and steroid production were hindered in follicles cultured in 1.5% alginate. In vitro fertilization and embryo culture revealed that oocytes obtained from 0.25% alginate retained the highest developmental competence. Overall, the present study showed that the alginate scaffold consistency affects folliculogenesis and oocyte development in vitro and that the alginate culture system can and should be tailored to maximally support follicle growth depending on the size and stage of the follicles selected for culture.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.