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.     

Cyclooxygenase-2 is expressed by cumulus cells during oocyte maturation in cattle.

Prostaglandins could be involved in various aspects of final differentiation of ovarian follicles. Prostaglandins are generated by the cyclooxygenase (cox) pathway. Until now, the expression pattern of isoforms cox-1 and cox-2 of cyclooxygenase in bovine cumulus-oocyte complexes (COCs) was unknown. Using immunodetection procedure, we demonstrated in the present study that cox-2 was expressed by cumulus cells during in vivo and in vitro maturation. Time course induction of cox-2 expression was investigated during in vitro maturation using Western blot analysis. Specific signal of cox-2 was markedly evidenced from 6 hr of culture and increased to reach a maximal level at 24 hr of culture. In vitro, cox-2 expression in COCs was associated with increased concentrations of PGE(2) and PGF(2alpha) in the maturation medium. In addition, the effects of culture conditions on cox-2 expression was considered using RT-PCR and Western-blot analysis. We demonstrated that the addition of 10 ng/ml of EGF to TCM199 clearly increased the expression level of cox-2 mRNA and protein. Higher levels of in vitro cox-2 expression was associated with greater rates of cumulus expansion and oocytes at metaphase II at 24 hr of culture. In conclusion, our present results suggest that cox-2 expression in cumulus cells may be involved in differentiation of COCs that occurs during oocyte maturation.     

Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide

We examined the relationship between integrity of cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturational competence of feline oocytes. Feline cumulus-oocyte complexes (COCs) were collected from either small (400-800 μm) or large (≥800 μm) follicles. Immediately after collection, cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI), which penetrates only non-viable cells. Cumulus cells without PI staining were judged as having good membrane integrity. After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89.8%) was higher (p < 0.05) than that from small follicles (60.8%). There was no difference in the maturation rate of oocytes from follicles with the same size regardless of cumulus morphology. In contrast, oocytes that had cumulus cells with good membrane integrity showed a higher maturation rate (93.8%) than oocytes with poor cumulus integrity (76.9%) in large follicles (p < 0.05). We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide staining can be used to predict the maturational competence of oocytes.     

Involvement of steroid hormones on in vitro maturation of pig oocytes

The purpose of this study was to determine if the addition of steroid hormones into the culture medium could influence the in vitro maturation of pig oocytes. The cumulus-oocyte complexes (COCs). collected from follicles of 2-5 mm diameter, were matured in steroid-free medium supplemented with various concentrations of estradiol-17beta (0-3000 ng/ml), progesterone (0-5000 ng/ml) and testosterone (0-300 ng/ml). The COCs were cultured for 42 h, then fertilized in vitro. We analyzed nuclear and cytoplasmic maturation with lacmoid stain 20 h after in vitro insemination. We observed no significant effect (P > 0.05) on the percentage of oocytes completing nuclear or cytoplasmic maturation or the number of sperm penetrating each oocyte for any concentration of progesterone, estradiol-17beta or testosterone. Similarly, adding a combination of those hormones to the medium did not significantly (P > 0.05) affect any of the criteria. In order to determine if there was a possible secretion of steroids during maturation, we added COCs, denuded oocytes and stripped cumulus cells to drops of a steroid-free medium and cultured them for 42 h, after which we analyzed the medium, before and after culture, for the presence of progesterone, estradiol-17beta and testosterone by radioimmunoassay (RIA) analysis. COCs, as well as cumulus cells alone, secreted similar amounts of estradiol (43.3 and 37.5 pg/ml, respectively) and progesterone (4.24 and 4.79 ng/ml, respectively) into the maturation medium. A small amount of estradiol (28.8 pg/ml) was also detected when oocytes were cultured alone. These results indicate that no steroids need to be added to the maturation medium of pig oocytes and that the COCs secrete steroids during maturation. It is possible that the amounts produced by the COCs fulfill any requirement for steroids if these steroids are required for either nuclear or cytoplasmic oocyte maturation.     

Connexin 43 coupling in bovine cumulus cells,during the follicular growth phase,and its relationship to in vitro embryo outcomes

Gap junctional coupling between cumulus cells is required for oocytes to reach developmental competence. Multiple connexins, which form these gap junctions, have been found within the ovarian follicles of several species including bovine. The aim of this study was to determine the role of connexin 43 (CX43) and its relationship to embryo development, after in vitro fertilization (IVF). Cumulus?oocyte complexes (COCs) were obtained from abattoir sourced, mixed breed, bovine ovaries. COCs were isolated from follicles ranging from 2 to 5 mm in size, representing the preselected follicle pool. Immediately after isolation, two cumulus cell biopsies were collected and stored for analysis pending determination of developmental outcomes. Using in vitro procedures, COCs were individually matured, fertilized, and cultured to the blastocyst stage. Biopsies were grouped as originating from COCs that arrested at the two‐cell stage (low developmental competence [LDC]) or having developed to the late morula/blastocyst stage (high developmental competence [HDC]), after IVF and embryo culture. The expression level of CX43 was found to be significantly higher in cumulus cells from COCs that had an HDC when compared with those that had an LDC. Moreover, the gap junctional intercellular coupling rate was significantly higher in cumulus from COCs deemed to have an HDC. Significantly higher expression of the cumulus health markers luteinizing hormone receptor and cytochrome p450 19A1 was found in the cumulus originating from oocytes with HDC, suggesting that this system may provide a mechanism for noninvasively testing for oocyte health in preselected bovine follicles.     

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes.Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall.For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only.Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.     

Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro

Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower (P less than .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.     

Relationship between the morphological changes of somatic compartment and the kinetics of nuclear and cytoplasmic maturation of oocytes during in vitro maturation of porcine follicular oocytes

Based on the morphology and expansion of the cumulus cells, several different classes of porcine cumulus-oocyte complexes (COCs) can be distinguished, during their maturation in vitro. The goal of the present study was to find out the rate of each morphologic category in case of COCs and granulosa-cumulus-oocyte complexes (GCOCs), the characteristics of their nuclear progression, cytoplasmic maturation, and the frequency of monospermy after IVF. It was found that the frequency of cumulus expansion is higher in case of GCOCs than that of COCs. Nuclear progression of COCs was more accelerated than that of GCOCs. Oocytes attached to the bottom of culture dish with dark, compact cumulus underwent nuclear and acquired their ability to be activated earlier than that of oocytes showing normal cumulus expansion. The rate of monospermic fertilization after IVF of normal COCs showing normal cumulus expansion was higher than that of COCs attached to the dish. These results suggest that diverse behavior of cumulus cells during in vitro culture affects nuclear and cytoplasmic maturation of porcine oocytes, which also affects IVF results. It can be concluded that granulosa cells promote normal cumulus expansion thus decrease heterogeneity in nuclear and cytoplasmic maturation amongst oocytes.     

Stereological characterization of bovine (Bos taurus) cumulus-oocyte complexes aspirated from small antral follicles during the metestrous and proestrous phases

Bovine cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries are used for in vitro maturation and fertilization after selection on the basis of morphological appearance of the cumulus and ooplasm. In this context, a quantitative characterization of COCs could provide additional criteria for selecting the most competent complexes. Bovine COCs from small (1-4mm) antral follicles were aspirated from metestrous and proestrous stage ovaries of Holstein-Friesian cows, fixed in glutaraldehyde, randomly embedded in glycol-methacrylate, and sectioned at 20 microm. The unbiased nucleator principle of stereology was used for estimating the mean volumes of complexes, oocytes, cumulus cells, and nuclei of oocytes and cumulus cells. The thickness of the zona pellucida and the relative numerical percentages of the several morphological types (C1, C2 and C3) of cumulus cells were also evaluated. The optical dissector procedure was used for cumulus cell sampling. Quantitative data show that the variability among complexes is generally high, especially for the volume of COCs. There were no linear correlations between the studied parameters, except between the volume of the oocyte and nucleus at metestrus. At proestrus, the volumes of COCs, oocytes and nuclei of oocytes, the volume of follicular cells and the thickness of the inner zona pellucida, were significantly higher than at metestrus. The relative numerical frequency of follicular type C1 cells was lower whereas that of type C3 cells was higher at proestrus than at metestrus. In conclusion, small antral follicles had larger COCs and oocytes at proestrus compared to metestrus and the COCs also had a higher percentage of follicular type C3 cells. Results suggest that for the same type of follicle size there may exist different functional populations of COCs at distinct stages of the bovine ovarian cycle.     

The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.     

Effects of maturational stage, cumulus cells and coincubation of mature and immature cumulus-oocyte complexes on in vitro penetrability of porcine oocytes

The in vitro penetrability of porcine oocytes is conditioned by several factors, some of which remain unclear. Knowledge of the different effects of the cellular components involved in penetrability would no doubt serve to simplify laboratory IVF methods. This study was designed to evaluate the effects of the following factors on penetrability: oocyte maturational stage, the presence of isolated or oocyte-attached cumulus cells, and coincubation of in vitro-matured and immature oocytes. Immature oocytes and oocytes matured in Waymouth medium were obtained from non atretic follicles and fertilized in TCM 199 medium. Sperm-rich fractions were collected by the gloved hand method and semen was used for IVF at a final concentration of 1 x 10(6) cells/mL in all experiments. Under the same conditions of IVF, the penetrability of the immature cumulus-oocyte complexes (COCs) was significantly lower than that of mature COCs, in terms of penetration rate and mean number of sperm per penetrated oocyte. This difference was abolished when the oocytes were denuded, leading to similar penetration rates. Coincubation of mature and immature COCs reduced the penetrability of immature COCs compared with that observed when these were incubated in isolation. However, neither the addition of isolated cumulus cells from decumulated mature oocytes nor the addition of denuded mature oocytes to immature COCs modified the penetration rate. These findings suggest that the presence of surrounding cumulus cells is mainly responsible for the differences observed in penetrability, regardless of the maturational stage of the oocyte. Moreover, when mature and immature COCs are coincubated, penetrability of immature COCs is diminished by the effects of the mature COC and not by the independent actions of the cellular components.     

Recovery, morphological quality, and in vitro maturation of follicular oocytes from bitches with pyometra

The objective of this study was to collect oocytes from ovaries of bitches with pyometra and to characterize the quality of the oocytes recovered. In 10 of 12 cases of pyometra, follicles with a diameter of 500 microm to 1mm were observed in the ovaries. A total of 710 oocytes were collected from 10 bitches by puncturing individual follicles after slicing the ovarian tissues. Oocyte recovery was successful from a bitch with severe clinical signs of pyometra. Of the oocytes collected, 53.5% were surrounded by > or =2 layers of cumulus cells, and 55.0% of these cumulus-oocyte complexes (COCs) had a darkly pigmented ooplasm >110 microm in diameter (large-dark COCs). The number of large-dark COCs per bitch varied from 1 to 72. A germinal vesicle with fine filaments of chromatin (Type A) was observed in 51.8% (range 21.1-100%) of the oocytes of large-dark COCs. Out of 50 oocytes cultured for 72 h, 6.0% developed to Metaphase II. In conclusion, there were many follicles with a diameter of 500 microm to 1mm in ovaries of bitches with pyometra, and many oocytes recovered from these follicles underwent meiotic maturation in vitro. The number of oocytes and COCs, and the morphological quality of the germinal vesicles varied among individual bitches.     

Lanosterol 14alpha-demethylase expression in the mouse ovary and its participation in cumulus-enclosed oocyte spontaneous meiotic maturation in vitro

The expression of lanosterol 14alpha-demethylase (LDM) in the mouse ovary after gonadotrophin administration was examined and the action of follicle fluid meiosis activating sterol (FF-MAS), derived from lanosterol by the action of LDM, on oocyte spontaneous maturation was also evaluated in cumulus cell enclosed oocytes (CEOs). Expression of LDM was primarily in oocytes in primordial and secondary follicles prior to administration of gonadotrophins, but obvious LDM expression was apparent in ovarian somatic cells 48 h after administration of equine chorionic gonadotrophin (eCG), especially in luteal and cumulus cells 54 h after eCG or 48 h after eCG plus 6 h after human chorionic gonadotrophin (hCG). The LDM expression in oocytes was only slightly elevated in larger growing follicles after eCG treatment. On the contrary, 48 h after hCG treatment, the elevated expression of LDM was only detected in interstitial cells. Therefore, eCG may be the primary gonadotrophin for LDM expression, and furthermore for production of FF-MAS in mouse cumulus cells (which are indispensable for oocyte maturation in vivo). Conversely, inhibitors of LDM, either 40 microM azalanstat or 50 microM RS-21745, significantly inhibited oocyte germinal vesicle breakdown (GVB) after 4h of in vitro culture; GVB rates decreased to 14 or 20%, compared to 90% in spontaneous maturation, respectively. There was no significant increase in GVB in CEOs following specific inhibitor of sterol Delta14-reductase and Delta7-reductase, AY9944-A-7 (5-100 microM), until marked oocytes degeneration appeared (50 microM). The phenomena may be ascribed to slow, passive accumulation of FF-MAS by AY9944-A-7, which cannot be associated with fast spontaneous progression. Furthermore, in spontaneous-matured CEOs, LDM was expressed preferentially in cumulus cells instead of oocytes. Therefore, FF-MAS may have a positive role in the spontaneous maturation of CEOs. In conclusion, there was an eCG-dependent dual LDM expression pattern on both oocytes and somatic cells in growing follicles in vivo, which may increase LDM expression and FF-MAS production in cumulus cells for oocyte maturation. For the first time, the inhibitory effect of LDM inhibitors on spontaneous maturation, together with the strong LDM expression in spontaneous matured CEOs, indicated that FF-MAS produced by cumulus cells might participate in spontaneous maturation of mouse CEOs.     

Estrogen regulation of nitric oxide synthesis in the porcine oocyte

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 microM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17beta-estradiol (E2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E2 action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E2 did not increase NOS-3 expression. In addition, estrogen receptor beta was detected in oocytes and cumulus cells, and estrogen receptor alpha was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E2 without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.