Relationship of macroscopic appearance of the surface of bovine ovarian follicles concentrations of steroids in follicular fluid, and maturation of oocytes in vitro

The relationship among opaqueness of the surface of bovine ovarian follicles, concentrations of follicular steroids, and capacity of oocytes to achieve nuclear maturation in vitro was examined in this study. Follicles greater than or equal to 5 mm in diameter were classified as clear (n=68) or opaque (n=72) based on their surface appearance. An oocyte and follicular fluid (FF) were removed from each follicle. Each oocyte was cultured, and the concentration of estradiol (E), progesterone (P), and testosterone (T) was determined for each sample of FF. Oocytes that extruded the first polar body by 30 h in culture were considered mature. All other oocytes were immature. More (p less than 0.05) mature oocytes came from clear (56%) than opaque follicles (29%). Clear follicles had lower concentrations of E (p less than 0.05) and P (p less than 0.10) in FF than opaque follicles. Follicles with mature oocytes had greater (p less than 0.05) concentrations of P than follicles with immature oocytes. Follicles were separated into three categories based on ratio of P:E in FF: high = P:E greater than or equal to 10, medium = P:E greater than or equal to 1 less than 10, and low = P:E less than 1. The percentage of mature oocytes from clear follicles was similar for high (64%), medium (48%), and low (57%) P:E groups; however, the percentage of mature oocytes from opaque follicles was greater (p less than 0.05) for the high (59%) than for the medium (21%) or low (19%) P:E groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronological Changes of Bovine Follicular Oocyte Maturation in Vitro

Chronological changes of bovine follicular cumulus-oocyte-complexesi were studied after in vitro maturation over a period of 48 h. According to their thickness and compactness of cumulus investments they were classified into 4 groups and cultured in enriched Ham’s F-10 medium with or without human chorionic gonadotrophin (hCG) and estradiolbenzoate (EB) for 0, 6, 12, 18, 21, 24, 27, 30 and 48 h. Representative samples were taken at each time interval for evaluation of nuclear maturation stages, ooplasm quality and size of the peri vitelline space (PVS). The results showed that oocyte nuclear breakdown (ONBD) required 6 to 12 h culture, and the peak of the first polar abstriction occurred at 24 h. The culture period required for ONBD and abstraction of the first polar body were related to the thickness and compactness of cumulus investments with and approximately 6 h delay in heavily compacted complexes. Ooplasm quality evaluation failed to show a clear trend, but the PVS increased in size from 0 h to 30 h and then, retracted again from 30 to 48 h. The overall maturation rate in the presence of hCG and EB was 79.1 %, and a substantial proportion (68.8 %) of nude or partially covered oocytes reached metaphase II stage. In the presence of hCG and EB no block at either metaphase I or at anaphase-telophase I was observed. In the absence of hCG and EB the percentage of oocytes reaching metaphase II was much lower (48.6%) in comparison with oocytes matured in the presence of these hormones (79.1 %). It was concluded a very high proportion of slaughterhouse oocytes could be matured in vitro and that the cumulus investments and addition of certain hormones affected the maturation rate.     

Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment

The objective of this study was to investigate the relationship between oocyte morphology and follicular fluid steroid concentrations in patients being treated with intracytoplasmic sperm injection. A total of 82 IVF cycles were evaluated in patients aged 24-40 years. Oocytes at metaphase II were graded into four groups according to the status of the first polar body and the size of the perivitelline space. The proportion of oocytes at the germinal vesicle and germinal vesicle breakdown stages, and the proportion of degenerated oocytes and oocytes with a large polar body were compared with different concentrations of oestradiol, progesterone and testosterone in the follicular fluid. The association between these oocyte characteristics and the ratio of oestradiol:testosterone and oestradiol:progesterone was also analysed. The results showed that oocyte morphology, as assessed by the status of the first polar body and the size of the perivitelline space, is associated with the ratio of oestradiol:testosterone and oestradiol:progesterone but not with the absolute concentrations of oestradiol, progesterone and testosterone in the follicular fluid. A ratio of oestradiol:testosterone > 200 is the best indicator for a small proportion of grade 1 and 2 oocytes (poor quality), a large proportion of grade 3 and 4 oocytes (good quality), and a small proportion of oocytes with cytoplasmic inclusions. These results will be of clinical use in evaluating oocyte quality.     

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.     

Ultrastructure of in-vitro oocyte maturation in cattle

Cumulus-oocyte complexes were collected from cows at an abattoir by aspiration from small (1-6 mm) antral follicles. After different periods of culture the complexes were processed for electron microscopy. Cumulus expansion occurred at 12-18 h of culture and concomitantly enlargement of cumulus cell projections in the perivitelline space was seen. At 48 h the innermost cumulus cells flattened and adhered tightly to the zona pellucida. In the oocyte the following changes occurred: at 0-3 h of culture the perivitelline space developed; at 3-12 h disconnection of the junctions between cumulus cell projections and the oolemma, and the concomitant breakdown of the nucleus was observed; at 12-18 h the mitochondria moved from a peripheral location to a more even spatial distribution and the Golgi complexes decreased in size; at approximately 18 h the smooth endoplasmic reticulum formed large aggregates surrounded by mitochondria; at 18-21 h the first polar body was abstricted; at 24-40 h the cortical granules spread; at 30-40 h the polar body degenerated in some specimens; at 40-48 h the perivitelline space decreased in size; and at 48 h one oocyte was in the process of fragmentation. It is concluded that nuclear and cytoplasmic oocyte maturation is simulated in vitro. However, certain deviations were noticed compared to in-vivo maturation.     

Ultrastructural features of preovulatory oocyte maturation in superovulated cattle

Cows and heifers were induced to superovulate by treatment with PMSG or FSH. The ultrastructural features of the oocytes were related to the time of the LH peak and the progesterone/oestradiol-17 beta ratios in the follicular fluid. At 0-2 h after the LH peak the perivitelline space developed; at 9-12 h there was disconnection of the junctions between cumulus cell projections and oolemma, and the concomitant breakdown of the oocyte nucleus; at approximately 15 h there were spatial rearrangements in the ooplasm of (a) mitochondrial clusters from a peripheral to an even distribution and (b) vesicles from an even distribution to a more central location; at approximately 19 h there was abstriction of the first polar body with dislocation of mitochondrial clusters and vesicles towards the site of polar body formation; at 21-22 h there was migration of cortical granules to solitary positions along the oolemma and decrease in the sizes of Golgi complexes and, on some occasions, the smooth endoplasmic reticulum. These ultrastructural changes were accompanied by an increase in progesterone/oestradiol ratios in the follicular fluids. It is concluded that preovulatory oocyte maturation in gonadotrophin-stimulated cattle comprises nuclear as well as cytoplasmic changes accompanied by steroidogenic changes in the follicle, each of which are closely related to the time of the LH peak. However, some variation existed between animals, between follicular and oocyte maturation and even within oocytes between nuclear and cytoplasmic maturation.     

Proteolytic activity of the 26S proteasome is required for the meiotic resumption, germinal vesicle breakdown, and cumulus expansion of porcine cumulus-oocyte complexes matured in vitro

The resumption of oocyte meiosis in mammals encompasses the landmark event of oocyte germinal vesicle (GV) breakdown (GVBD), accompanied by the modification of cell-to-cell communication and adhesion between the oocyte and surrounding cumulus cells. The concomitant cumulus expansion relies on microfilament-cytoskeletal remodeling and extracellular matrix (ECM) deposition. We hypothesized that this multifaceted remodeling event requires substrate-specific proteolysis by the ubiquitin-proteasome pathway (UPP). We evaluated meiotic progression, cytoskeletal dynamics, and the production of cumulus ECM in porcine cumulus-oocyte complexes (COCs) cultured with or without 10-200 microM MG132, a specific proteasomal inhibitor, for the first 22 h of in vitro maturation, followed by 22 h of culture with or without MG132. Treatment with 10 microM MG132 arrested 28.4% of oocytes in GV stage (vs. 1.3% in control), 43.1% in prometaphase I, and 16.2% in metaphase I, whereas 83.7% of control ova reached metaphase II (0% of MG132 reached metaphase II). The proportion of GV-stage ova increased progressively to >90% with increased concentration of MG132 (20-200 microM). Furthermore, MG132 blocked the extrusion of the first polar body and degradation of F-actin-rich transzonal projections (TZP) interconnecting cumulus cells with the oocyte. The microfilament disruptor cytochalasin E (CE) prevented cumulus expansion but accelerated the breakdown of TZPs. Ova treated with a combination of 10 microM MG132 and 10 microM CE underwent GVBD, despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10 microM MG132 remained in GV stage, compared with 16.7% GV ova in control. Cumulus expansion, retention of hyaluronic acid, and the deposition of cumulus ECM relying on the covalent transfer of heavy chains of inter-alpha trypsin inhibitor (IalphaI) were also inhibited by MG132. Cumulus expansion in control COCs was accompanied by the degradation of ubiquitin-C-terminal hydrolase L3, an important regulator of UPP. RAC1, a UPP-controlled regulator of actin polymerization was maintained at steady levels throughout cumulus expansion. We conclude that proteasomal proteolysis has multiple functions in the progression of oocyte meiosis beyond GV and metaphase I stage, polar body extrusion, and cumulus expansion.     

Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes

Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 microl droplets of three different culture systems: (1) TCM-199 + steer serum (10%): (2) TCM-199 + steer serum (10%) + PMSG (40 IU/ml); and (3) TCM-199 + steer serum (10%) + PMSG (40 IU/ml) + estradiol 17beta (1 microg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 degrees C) mineral oil and incubated in a CO2 incubator (39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 16-18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9-10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22-24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.     

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.     

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.     

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.     

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Factors affecting follicular population, oocyte yield and quality in camels (Camelus dromedarius) ovary with special reference to maturation time in vitro

Three experiments were conducted to study a series of factors affecting in vitro reproductive parameters in camels. In Experiment 1, the effect of season and presence of a corpus luteum (CL) on ovarian follicular populations, oocyte yield and quality was studied using a total of 252 and 208 ovaries collected during the breeding and non-breeding season, respectively. Small, medium, large and the total number of ovarian follicles, oocyte yield and quality were measured. In Experiment 2, the effect of methods of oocyte retrieval and needle gauge on oocyte yield and quality was evaluated with oocytes recovered using slicing and aspiration with 18-, 19- or 20-gauge needle. Oocytes were evaluated microscopically and classified into three categories. The objective of Experiment 3 was to identify the optimum time for oocyte maturation in the dromedary camel. Oocytes were cultured in CR1aa medium at 38.5 degrees C under 5% CO(2) for 24, 32, 36, 48 and 72h. Maturation was calculated as the percentage of cumulus expansion and oocytes reaching metaphase II (MII). The number of small, medium, large and the total number of ovarian follicles were higher (P<0.01) during the breeding than non-breeding season. The recovery of total number of oocytes and Category I oocytes were also greater (P<0.01) during the breeding season. Ovaries without a CL possessed significantly (P<0.01) more ovarian follicles and more (P<0.05) small and large follicles. The total number of oocytes and Category I oocytes were also greater (P<0.01) in ovaries without CL. Slicing of camel ovaries increased (P<0.01) the yield of oocytes as compared to aspiration. The aspiration of follicles using a 20-gauge needle had greater yields of the total number of oocytes and Category I oocytes than when using 19- (P<0.05) and 18-gauge needle (P<0.01). The culture of camel oocytes for 36h produced higher (P<0.01) percentages of cumulus expansion and oocytes at MII. Increasing culture times up to 48 or 72h increased (P<0.01) the percentage of degenerated oocytes.In conclusion, the growth and development of ovarian follicles in the camel as well as yields of Category I oocyte were greater during the breeding season. Slicing or aspirations using a 20-gauge needle yielded greater numbers of total and Category I oocytes. Finally, maturation of oocytes in CR1aa medium for 36h produced higher percentages of cumulus expansion and oocytes at MII stage.     

Ovine cumulus cells estradiol-17Beta production in the presence or absence of oocyte

The objective of the present study was to compare the in vitro production of estradiol-17Beta (E(2)) by cumulus cells in the presence or absence of ovine oocyte. Moreover, the relationship between the concentration of produced estradiol-17Beta and oocyte nuclear maturation was assessed. Ovaries collected from the local abattoir were transported to the laboratory in saline at 30-35 degrees C within 1-3 h after collection. The oocytes of follicles, 2-6 mm in diameter, were recovered by aspiration. The oocytes with evenly granulated cytoplasm and which were surrounded with at least three layers of cumulus cells were selected and subjected to culture in pre-incubated oocyte culture medium (OCM). Before culturing, the selected oocytes were randomly divided into five treatment groups: Group 1, cumulus enclosed oocytes cultured in OCM (Group COCs); Group 2, denuded oocytes cultured in OCM (Group D); Group 3, denuded oocytes co-cultured with a cumulus cell-monolayer in OCM (Group D+M); Group 4, denuded oocytes co-cultured with previously cultured (for 26 h) cumulus cell-monolayer (10(5) cells/ml) in refreshed OCM (Group D+M(26)); Group 5, cumulus cell-monolayer (10(5) cells/ml) cultured in OCM (Group M). After an incubation period (26 h at 38.6 degrees C, 5% CO(2) and 100% humidity), the media were collected and kept at -20 degrees C until hormonal assay. The concentration of E(2) was determined by RIA method. For assessment of nuclear status, the completely denuded oocytes were subjected to DAPI staining. The highest percentage of metaphase II (MII) stage oocytes was observed in Group N (91%) and the lowest percentage was observed in Group D (6%) and Group D+M(26) (6%). The mean production of E(2) was highest and lowest in Group D+M (378.69+/-54.34 pg/ml) and Group D+M(26) (109.15+/-8.24 pg/ml), respectively. The production of E(2) was significantly (P<0.01) higher in Group D+/-M when compared with Groups M and D+/-M(26). Regarding the nuclear maturation, the percentage of MII stage oocytes was significantly (P<0.001) higher in Group COCs compared to the other groups. The results suggest that steroidogenic activity of cumulus cells in in vitro condition can be influenced by the pattern of connection between cumulus cells and the oocyte. Moreover, the nuclear maturation of oocytes is not influenced by the different production levels of E(2).     

Meiotic competence of equine oocytes and pronucleus formation after intracytoplasmic sperm injection (ICSI) as related to granulosa cell apoptosis

Follicle atresia and granulosa cell apoptosis may be related to oocyte meiotic and developmental competence. We analyzed the relationships among granulosa cell apoptosis, initial cumulus morphology, oocyte nuclear maturation in vitro, and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the horse. For each follicle, the size was measured and granulosa cells were used for DNA laddering analysis. Oocytes were evaluated for cumulus morphology, cultured for in vitro maturation, and submitted to ICSI. Apoptosis was categorized as absent, intermediate, or advanced according to the relative concentrations of two DNA fragments at 900 and 360 base pairs (bp). In 98 oocyte-follicle pairs, 52 oocytes were classified as expanded (Exp), 39 as compact (Cp), and 7 as having a partial (P) cumulus. Advanced apoptosis was detected in 55% (54/98) of follicles; 37% (36/98) of follicles showed an intermediate level of apoptosis; and 8 follicles (8%) were nonapoptotic. Follicle size was not significantly correlated with granulosa cell apoptosis (P > 0.05). Significantly more Exp than Cp oocytes originated from follicles with advanced apoptosis (P < 0.001). The proportion of oocytes maturing in vitro was significantly higher in oocytes issuing from apoptotic follicles than in oocytes issuing from healthy follicles (P < 0.05). The proportion of normally (two pronuclei) or abnormally fertilized oocytes (one or greater than two pronuclei, or partially decondensed sperm) did not differ in relation to granulosa cell apoptosis. We conclude that, in the mare, granulosa cell apoptosis is related to cumulus expansion and an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. These results provide selection criteria for horse oocytes used in assisted reproductive techniques so that embryo production may be maximized.     

Effect of follicle cells on in vitro fertilization of pig follicular oocytes

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.     

Oocyte size and intrafollicular position in polyovular follicles in rabbits

Healthy follicles with 2-24 oocytes were observed in adult rabbit ovaries during all phases of folliculogenesis from primary to preovulatory follicles. Most follicles contained 2-3 oocytes which developed according to their topographical situation in the follicle. The central oocyte in a normal topographical situation has an almost normal growth and development up to metaphase II and cumulus expansion. The peripheral oocytes grow more slowly: most do not attain the normal size or resume meiosis and remain surrounded by ordinary granulosa cells. When the number of oocytes is higher than 3, the peripheral oocytes develop even more slowly, as do the central ones. It demonstrates the necessity for the oocyte to occupy a certain position inside the follicle and to reach a size which allows resumption of meiosis; the cumulus responds only to oocytes of normal size and position. We suggest that, despite the relative frequency of binovular follicles, fertilization of two oocytes originating from one follicle is unlikely.     

Structural and endocrine aspects of equine oocyte maturation in vivo

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring ≤30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol-17β (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON-I) stage, 3) the peripheral flattened oocyte nucleus (PON-II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M-I) stage, and 6) the metaphase II (M-II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10-fold increase during follicle maturation, particularly associated with the progression from M-I-to M-II-stage oocytes. The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division. The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane-bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well-defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high. © 1995 wiley-Liss, Inc.     

Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles

The aim of the present study was to examine the growth and survival in culture, and the subsequent meiotic competence, of bovine oocytes recovered from early antral ovarian follicles. Follicles isolated by microdissection of the ovarian slices were sorted into two size groups: (I) 0.2-0.5 mm diameter; and (II) 0.4-0.7 mm diameter. Group I follicles were cultured intact while in Group II, cumulus-oocyte complexes with pieces of parietal granulosa were dissected from the follicles and cultured. Follicles or cumulus-oocyte complexes with parietal granulose were embedded in collagen gel and cultured in TCM 199 supplemented with 3% BSA and 4 mM hypoxanthine for 14 days (Group I) or 7-10 days (Group II). After this, cumulus-oocyte complexes were recovered from the gel. Oocytes that had lost the majority of the cumulus were fixed immediately after recovery. Cumulus-oocyte complexes showing normal morphology were either fixed immediately or were subjected to IVM for an additional 24h, and then were fixed. At the end of the growth culture, 57.6% of the compact COCs in Group I follicles were preserved in the GV configuration, 16.7% had resumed meiosis, and 25.8% were degenerated or did not show detectable chromatin. After IVM, the proportion of oocytes resuming meiosis increased significantly (from 16.7% versus 42.7%; P < 0.05), and 9.1% of all oocytes had reached TI or MII. The isolated cumulus-oocyte complexes in Group II began creating follicle-like structures following 24 h of growth culture (7.1%). The proportion of these structures reached 50.8% on days 2-3, and then gradually decreased due to degeneration. On day 10 only 5.8% of cumulus-oocyte complexes were classified as intact. Of the cumulus intact oocytes recovered from the newly created follicle-like structures at 7-10 days, 54.7% were in the germinal vesicle stage, 31.0% underwent germinal vesicle breakdown, 14.3% were degenerated or the chromatin configuration was not detectable. After 24 h of IVM, 67.6% of oocytes had resumed meiosis, and 21.6% of all oocytes had reached TI and MII. These results show that isolated early follicles and cumulus-oocyte complexes from intact early antral follicles can grow in culture and can develop meiotic competence.