Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.     

Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes

Success of in vitro maturation (IVM) and production of bovine embryos as related to aspects of follicle source and oocyte size were evaluated. First, it was determined that bovine oocytes continue growing in all follicular sizes studied, including >1- to 15-mm follicles. Populations of oocytes were collected from surface visible (peripheral) and cortical follicles from the same ovaries. When the number of oocytes from both peripheral and cortical follicles was combined, the yield of oocytes was approximately double that collected from 1 ovarian site alone. Oocytes from cortical follicles were smaller than those from the surface population, and the smaller cortical oocytes had a lower potential for both meiotic maturation and embryo development Only cortical oocytes with the largest diameters underwent IVM and subsequently developed to blastocysts at rates comparable to oocytes from peripheral follicles. As the diameter of the oocytes recovered from peripheral follicles increased, so did their developmental potential. When the stage of the estrous cycle was observed, it was found to have no effect on developmental potential. Finally, oocytes which extruded polar bodies at an earlier time during maturation were, on average, larger than those which extruded polar bodies later. The results serve a practical purpose in assisting selection of oocytes capable of developing into blastocysts and they give useful correlates of oocyte competencies based on knowledge of follicle source and oocyte stage.     

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.     

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.     

In vitro fertilization and development of cumulus oocytes complexes collected by ultrasound-guided follicle aspiration in superstimulated llamas

The objective was to evaluate the developmental competence of cumulus-oocyte complexes (COC) collected by follicular aspiration in llamas treated with FSH or eCG. Llamas were assigned randomly to two groups (n = 16 per group) and treated, at the time of ovarian follicular wave emergence, with either: 1) 25 mg of FSH im, twice daily for 4 d; or 2) 1000 IU of eCG as a single i.m. dose. The start of gonadotropin treatment was considered Day 0. Both groups were given 5 mg of Armour Standard LH im on Day 6, and COC were collected by follicle aspiration on Day 7. Expanded COC collected from FSH- (n = 157) and eCG-treated llamas (n = 151) were fertilized in vitro using epididymal sperm, and presumptive zygotes were in vitro cultured in SOF medium for 8 d. The FSH and eCG treatment groups did not differ with respect to: the number of follicles ≥7 mm (16.0 ± 2.7 vs 14.0 ± 1.9, respectively; P = 0.5); the number of COC collected (11.5 ± 1.9 vs 9.7 ± 1.2; P = 0.4); the number of expanded COC (9.8 ± 1.4 vs 9.4 ± 1.2; P = 0.8); or the percentage of presumptive zygotes which developed into 2 to 8 cell stage embryos (65.3 vs 63.1), morulas (46.2 vs 42.5), or blastocysts (23.1 vs 20.5; P > 0.05). In conclusion, FSH and eCG treatments were equally effective for recovery of a high number of expanded COC which were used directly for in vitro fertilization. Furthermore, rate of embryo development was not significantly affected by the gonadotropin treatment used.     

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology,fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.     

Relationship between antral follicle size,oocyte diameters and nuclear maturation of immature oocytes in pigs

We designed the present study to examine the possible relationship between oocyte, antral follicle size and the nuclear heterogeneity of immature pig oocytes, in order to study the heterogeneity of oocyte populations in ovaries obtained from slaughterhouses. Previously, we carried out an initial experiment to determine, by histological analysis, the effectiveness of the macroscopic criteria (MC) used to screen atretic and nonatretic antral follicles. We recovered 239 follicles by mechanical dissection, measured them with a computerized image analysis system, and classified them into five size categories according to their diameter (FD): Group 1 (0.40-0.99 mm), Group 2 (1.00-2.19 mm), Group 3 (2.20-2.79 mm), Group 4 (2.80-3.59 mm) and Group 5 (3.60-6.50 mm). In relation to histological analysis, the results showed that MC is an effective method to select atretic and nonatretic antral follicles from 0.40 to 6.50 mm in diameter (overall accuracy was 80.75%, with sensitivity and specificity rates of 79.33 and 82.20%, respectively). In a second experiment, we recovered 454 nonatretic follicles, then measured and classified them as mentioned above. We removed oocytes individually from follicles and measured their size (oocyte diameter without and with zona pellucida, OD and TOD, respectively). Finally, we evaluated the relationship between OD, FD and nuclear maturation of immature oocytes (germinal vesicles (GV) Stages 0, I, II, III and IV; diakinesis, prophase I, and metaphase I). Overall OD was 101.77 +/- 0.65, 109.19 +/- 0.45, 113.55 +/- 0.50, 116.92 +/- 0.46 and 117.13 +/- 0.47 microm (Groups 1, 2, 3, 4, and 5, respectively). Differences in OD between groups were significant (P < 0.01), although from 2.80 to 6.50 mm follicles, the oocytes were not different in size. There was a certain heterogeneity in OD within each follicular group. Although we observed a certain degree of nuclear variability, regardless of FD or OD, the present study showed a clear progression in GV when FD increased from 0.40 to 6.50 mm. A positive correlation (r2 = 0.4248; P > 0.05) was established mainly between the nuclear stage and oocyte diameter.     

A light- and electron-microscope study of the oocyte nucleus during development of the antral follicle in the prepubertal mouse.

The ordered changes which occur in the structural organization of the mouse oocyte nucelus during the preparatory, the maturative and the preovulatory stages of antral follicle development, have been studied under both light and electron microscopy. All observations have been made on those antral follicles whose development is initiated on postnatal day 14 and completed by postnatal day 28 in prepubertal animals of the ICR albino mouse strain. The formed entities that can be recognized within the oocyte nucleus during that period are the condensing bivalents, the heterochromatic knobs, the nucleolus and the extranucleolar bodies. At the onset of antral follicle development, the highly unravelled dictyate bivalents are seen to take on a lampbrush-type configuration. Subsequent condensation of these lampbursh bivalents appears to be a very gradual and lengthy process that extends over almost the entire period of antral follicle development. The shortening and thickening of the lampbrush bivalents are best interpreted as resulting from the withdrawal of their lateral loop-like projections into the chromosome axes and from the focal aggregation of these axes into compact chromatin masses. Electron-opaque granules, which appear within the oocyte nucleus during the preparatory and maturative follicle stages, are seen to be intimately associated with these condensing bivalents. A number of Feulgen-positive heterochromatic knobs make their appearance in contact with certain bivalents during the preparatory follicle stage. These knobs are not reincorporated as such into the condensing chromatin masses and undergo disintegration and dissolution during the preovulatory follicle stage. The size, shape and ultrastructural features of the nucleolus remain unchanged thoughout the period of antral follicle development. Breakdown and dissolution of the nucleolar mass is a swift process that takes place only in the fully mature preovulatory follicle and more or less concomitantly with the dismantling of the nuclear envelope. The extranucleolar bodies increase noticeably in size during the preparatory and the maturative follicle stages; they shrink in size and undergo dissolution during the preovulatory stage of antral follicle development. An attempt is made to interpret these morphological changes in the light of current knowledge concerning the architectural and functional organization of the oocyte nucleus in general during meiotic prophase. The relevant observational evidence would be consistent with the view that, during antral follicle deveopment, the mouse oocyte nucleus is not, as too often assumed, in a period of arrested evolution; its formed components undergo structural, maturational and functional changes which are of significance not only for the resumption of the first meiotic prophase but also for the early development of the embryo.     

CPEB controls oocyte growth and follicle development in the mouse

CPEB is a sequence-specific RNA-binding protein that regulates polyadenylation-induced translation. In Cpeb knockout mice, meiotic progression is disrupted at pachytene due to inhibited translation of synaptonemal complex protein mRNAs. To assess the function of CPEB after pachytene, we used the zona pellucida 3 (Zp3) promoter to generate transgenic mice expressing siRNA that induce the destruction of Cpeb mRNA. Oocytes from these animals do not develop normally; they undergo parthenogenetic cell division in the ovary, exhibit abnormal polar bodies, are detached from the cumulus granulosa cell layer, and display spindle and nuclear anomalies. In addition, many follicles contain apoptotic granulosa cells. CPEB binds several oocyte mRNAs, including Smad1, Smad5, spindlin, Bub1b, Mos, H1foo, Obox1, Dnmt1o, TiParp, Trim61 and Gdf9, a well described oocyte-expressed growth factor that is necessary for follicle development. In Cpeb knockdown oocytes, Gdf9 RNA has a shortened poly(A) tail and reduced expression. These data indicate that CPEB controls the expression of Gdf9 mRNA, which in turn is necessary for oocyte-follicle development. Finally, several phenotypes, i.e. progressive oocyte loss and infertility, elicited by the knockdown of CPEB in oocytes resemble those of the human premature ovarian failure syndrome.     

Influence of sperm:oocyte ratio during in vitro fertilization of in vitro matured cumulus-intact pig oocytes on fertilization parameters and embryo development

The present study was conducted to evaluate the influence of sperm:oocyte ratio during in vitro fertilization (IVF) of in vitro matured cumulus-intact oocytes on fertilization parameters and embryo development in pigs. In vitro matured oocytes surrounded by intact cumulus cells (COC) were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000:1, 3000:1, 4000:1, 6000:1, and 8000:1). Denuded oocytes inseminated with 2000 frozen-thawed spermatozoa:oocyte were the control group. A total of 2546 oocytes in five replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture (EC) medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The penetration rate increased in the COC groups with the sperm:oocyte ratio, reaching the highest rates with 8000:1 spermatozoa:oocyte (72.1 +/- 6.5%), similar to the control (73.5 +/- 3.5%). However, the monospermy was highest with the lower spermatozoa:oocyte rates (82.6-94.8%) and decreased drastically (P<0.05) in the COC group fertilized with 8000 sperm:oocyte (36%). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) showed no difference among the COC groups (20-30%) but they were significantly lower (P<0.007) than those obtained by the control group (43.7 +/- 2%). Embryo development was highest in the control group (58% for cleavage and 23% for BF) but not significantly different with the 6000 and 8000 sperm:oocyte COC groups (47 and 50% for cleavage and 19 and 17% for BF, respectively). These results indicate that the use of COC for IVF involves a drop in the efficiency of the fertilization and the necessity to increase the frozen-thawed sperm:oocyte ratio three to four times more to obtain similar embryo development to denuded oocytes.     

Effect of growth factors on in vitro development of caprine preantral follicle oocytes

The objective of this work was to examine the effect of various growth factors including epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), either individually or in association, in the presence of follicle stimulating hormone (FSH) on the in vitro growth and viability of caprine preantral follicle oocytes. Preantral follicles were disassociated enzymatically and mechanically from prepuberal caprine ovaries after the animals were anesthetically ovariectomized. In experiment, caprine preantral follicles in groups 1–4 were cultured in growth culture medium, growth culture medium + EGF, growth culture medium + IGF-I and growth culture medium + IGF-I + EGF, respectively, for 9 days. The results indicated that EGF (50 mg/l) increased the survival rate of oocytes, but decreased the growth rate of oocytes; IGF-I (100 mg/l) effectively maintained the survival of oocytes and stimulated their growth; IGF-I (100 mg/l) and EGF (50 mg/l) in combination produced a higher effect on both of the survival and the growth rate of oocytes than IGF-I or EGF alone. Conclusively, the growth factors can effectively maintain the survival of caprine preantral follicle oocytes and regulated their growth in culture. EGF and IGF-I in association could synergically meliorate the culture system of caprine preantral follicle oocytes.     

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 competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.     

Regulation of mouse oocyte growth: probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth

Intercellular communication, as determined by two different assay procedures, was established in vitro between mouse oocytes free of adhering follicle cells and monolayers of either follicle or 3T3 cells. Both of these cell types are known to be able to form homologous gap junctions, and follicle cells naturally form heterologous gap junctions with oocytes in vivo. Monolayers of L cells that are communication deficient did not establish intercellular communication with oocytes as determined by the two different assays for intercellular communication. The diameter of oocytes cultured for 4 days in medium or on monolayers of L cells decreased markedly, 9.7 and 13.1 micron, respectively. In contrast, oocytes cultured for 4 days on follicle cell monolayers increased on the average about 4.7 micron in diameter. Oocytes cultured for 4 days on monolayers of 3T3 cells decreased slightly in diameter, i.e., 2.1 micron. Results from these experiments support a nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.     

Coculturing cumulus oocyte complexes with denuded oocytes alters zona pellucida ultrastructure in in vitro matured bovine oocytes

Oocyte quality is a key factor affecting success of in vitro embryo production in cattle. Improving the microenvironment of oocytes during in vitro maturation (IVM) can increase developmental rate and embryo quality. Therefore, the objective was to determine whether denuded oocytes (DO) affect embryo development and ultrastructure of the zona pellucida (ZP) in in vitro matured bovine oocytes. Intact immature cumulus-oocytes complexes (COC) obtained from a local abattoir or by ovum pick-up (OPU) were cocultured with and without abattoir-obtained DO at a COC:DO ratio of 1:5. After IVM, DO were removed and intact DO were either fertilized or observed by scanning electron microscopy. Blastocyst quality was evaluated using a TUNEL assay. The ZP pore size decreased after IVM in COC + DO coculture, regardless of their origin (OPU, 310.5 ± 92.5 vs. 428.9 ± 148.5 nm; abattoir, 317.5 ± 68.5 vs. 358.9 ± 128.5 nm; P < 0.05; mean values ± standard deviation). Moreover, the number of ZP pores in OPU COC + DO and COC + DO was greater than those in OPU COC and COC (control) groups (56 ± 4 and 55 ± 7 vs. 50 ± 6 and 42 ± 4; P < 0.05). The rate of blastocyst development in COC + DO and OPU COC + DO groups was greater those in control and OPU COC groups (36.6% and 55.5% vs. 28.1% and 40.0%; P < 0.05). Moreover, the total cell numbers of blastocysts in COC + DO group exceeded that of control (132.91 ± 30.90 vs. 115.44 ± 24.95; P < 0.05), with no significant between OPU COC + DO and OPU COC groups (139.31 ± 42.51 vs. 137.00 ± 61.34). In conclusion, in vitro embryo development competence and quality improved when oocytes were cocultured with DO. Furthermore, there more, but smaller, ZP pores.     

Effects of oviductal and cumulus cells on in vitro fertilization and embryo development of porcine oocytes fertilized with epididymal spermatozoa

This study was designed to evaluate the effects of adding porcine oviductal epithelial cell (POEC) monolayers before or during the fertilization of denuded or cumulus-enclosed oocytes, in terms of fertilization results and subsequent embryo development. The variables determined were: penetration rate, mean number of spermatozoa per oocyte, male pronucleus formation rate, monospermy rate, cleavage rate after 48 h of fertilization, blastocyst rate, and mean number of nuclei per blastocyst. We used cumulus-free and cumulus-enclosed oocytes preincubated or fertilized in the presence of POEC, once the purity in epithelial cells of these cultures had been assessed. All the experiments involved the use of frozen-thawed epididymal spermatozoa to avoid replicate variability. The POEC cultures prepared showed a high proportion of epithelial cells (over 95%). Preincubation of oocytes with POEC before fertilization showed no effects on the fertilization variables determined. In contrast, during IVF under our experimental conditions, these cells attached to the cumulus cells and their interaction had a significant effect on some of the fertilization variables analyzed. The presence of POEC and cumulus cells during IVF increased oocyte penetrability. Moreover, in the absence of POEC, cumulus cells resulted in a reduced monospermy rate. On subsequent embryo culture, a lower cleavage and blastocyst formation rate were recorded when the oocytes had been preincubated with POEC before IVF.     

Effects of gonadotropin treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of oocytes in prepubertal gilts

This study was undertaken to compare the effects of FSH-pituitary (FSH-P), eCG, and a combination of gonadotropins containing 400 IU eCG and 200 IU hCG (PG 600) on the growth of large follicles, oocyte quality and in vitro fertilization (IVF) rate of in vitro matured (IVM) oocytes in prepubertal gilts. The ovaries were removed via midventral laparotomy 48 h (Experiment 1) or 72 h (Experiment 2) after the first injection. In Experiment 1, 30 gilts received 1 of 5 treatments: 1) saline (3 ml i.m., once, n = 6); 2) FSH-P8 (8 mg i.m., twice, with a 24-h interval, n = 6); 3) FSH-P16 (16 mg i.m., twice, with a 24-h interval, n = 6; 4) eCG (1000 IU i.m., once, n = 6); or 5) PG 600 (5 ml i.m., once, n = 6). Compared with saline, treatment with PG 600 or eCG induced significant (P < 0.05) growth of large follicles (> or = 6 mm). In Experiment 2, 16 gilts received 1 of 5 treatments: 1) saline (n = 4); 2) FSH-P8 (n = 4); 3) FSH-P16 (n = 4); 4) eCG (n = 4), or 5) PG 600 (n = 4). The same injection protocol as in Experiment 1 was used. Compared with treatment with FSH-P8 or FSH-P16, eCG increased (P<0.05) the number of large follicles. The proportion of good oocytes was increased (P<0.05) with FSH-P8 or FSH-P16 compared with treatment with eCG or PG 600. Moreover, oocytes from eCG-treated gilts had a greater (P<0.05) rate of male and female pronuclei than FSH-P or saline-treated gilts. In conclusion, treatment with FSH-P resulted in a higher proportion of oocytes with multilayer cumulus cells, whereas treatment with eCG resulted in higher pronuclear rates following in vitro fertilization in prepubertal gilts.     

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.