Relationship of morphology and follicular fluid environment of bovine oocytes to their developmental potential in vitro

Morphology of bovine cumulus oocyte complexes (BCOC) and follicular fluid parameters were examined as potential criteria for selecting in vitro developmentally competent oocytes. Immature BCOC, from antral follicles, exhibiting similar morphological characteristics were grouped and the blastocyst development for oocytes in each group was examined. In a second experiment, follicles were individually aspirated to recover BCOC with their associated follicular fluid. Each oocyte was categorized and individually matured, fertilized and cultured. Radioimmunoassays for follicular progesterone, estradiol-17beta and insulin-like growth factor-I (IGF-1) concentrations were performed. The BCOC were categorized into 9 groups based on the homogeneity of the ooplasm, compactness of the cumulus investment, color and size. Oocytes classified into Groups 1, 2 and 3 demonstrated the highest rates of development to the blastocyst stage at 13, 16 and 20%, respectively. Therefore, Groups 1, 2 and 3 were pooled and designated as the enhanced developmental potential (EDP) group, and the remaining groups were designated as the reduced developmental potential (RDP) group. The progesterone concentration (+/- SEM) in follicles containing EDP oocytes (27.54 +/- 5.08 ng/ml) was significantly lower than the RDP group (72.72 +/- ng/ml; P < 0.001). Follicles containing oocytes that developed to the blastocyst stage (28.65 +/- 7.18 ng/ml) possessed significantly lower progesterone levels than all remaining follicles (52.9 +/- 7.51 ng/ml; P < 0.05). No significant differences were found for estradiol-17beta or IGF-I concentrations between the EDP and RDP groups or developmental stages. These results indicate that morphological criteria and follicular fluid progesterone concentration may be used to select BCOC for high potential of blastocyst development.     

Relationship between growth hormone concentrations in bovine oocytes and follicular fluid and oocyte developmental competence

In the last few years, several works suggest that Growth Hormone (GH) is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH. We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs) and the concentration of GH in the oocytes and in the follicular fluids (FF) from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi) show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo). At the same time we measured Estrogen (E2) and Progesterone (P4) concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA). We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrine-paracrine manner. Moreover, E2, and P4 levels in FF suggest that, in our model, atresia processes are also involved in oocyte developmental capability and that the highest level of GH may represent a local reaction to these phenomena.     

Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro

Described in the present paper is a culture system that preserves oocyte and granulosa cell morphology in bovine preantral follicles during 5 d in vitro. The effects of additional hypoxanthine and energy substrata (i.e., pyruvate and glutamine) on the morphology of cultured preantral follicles were investigated. It was shown that addition of a mixture of pyruvate, glutamine and hypoxantine to the culture medium increased the percentage of follicles with an intact oocyte from 29.4 to 78.6%. Morphological criteria are described to discriminate between normal and degenerated preantral follicles during culture by inverted microscopy. In addition, the importance of histological evaluation to judge the quality of oocyte and granulosa cells is demonstrated.     

Toxic effects of in vitro exposure to p-tert-octylphenol on bovine oocyte maturation and developmental competence

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors alpha and beta (ERalpha and ERbeta) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1-0.0001 microg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17 beta-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 microg/ml. Groups treated with > or =0.001 microg/ml OP showed impaired embryo development. No adverse effects of E2 were observed. In the E2-treated COCs, ERalpha mRNA was decreased but PR mRNA was upregulated compared with controls. Treatment with 0.001 and 0.0001 microg/ml OP induced a decrease in ERalpha mRNA, but ERbeta and PR mRNA were not affected. Treatment with 0.01 microg/ml OP did not produce changes in the expression of any of the mRNAs studied. OP impairs meiotic progression and developmental competence of bovine oocytes without demonstrating clear estrogen-mimic activity.     

Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro

The present study was carried out to evaluate if the addition of cysteamine to the culture medium during in vitro maturation of bovine oocytes increased the glutathione (GSH) levels in the mature oocytes, and if these changes may promote an improvement on in vitro development to the blastocyst stage. Follicular oocytes from slaughterhouse ovaries were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 (control), 25, 50, or 100 μM of cysteamine for 24 hr. After in vitro maturation the oocytes were fertilized and cultured for 8 days. The percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.01) for oocytes matured in medium containing 100 μM of cysteamine than for those matured in control medium. Moreover, the intracellular GSH levels were increased (P < 0.05) in oocytes matured with 100 μM of cysteamine with respect to control. No differences were observed in maturation and cleavage rates, and in the mean cell numbers per blastocyst among treatments (P > 0.05). These results indicate that the addition of thiol compounds such as cysteamine to maturation medium increases the efficiency of in vitro blastocyst production from immature bovine oocytes. The higher levels of GSH in oocytes matured in the presence of cysteamine suggest that the beneficial effects of cysteamine on in vitro maturation and subsequent development after in vitro fertilization are mediated by GSH. © 1995 wiley-Liss, Inc.     

Amino acid turnover by bovine oocytes provides an index of oocyte developmental competence in vitro

Amino acid profiling has been used to distinguish between human embryos of differing developmental competence. We sought to determine whether amino acid profiling could be used to distinguish between metaphase II (MII) bovine oocytes with different developmental capabilities in vitro. Amino acid turnover was assayed during the final 6 h of in vitro maturation prior to oocytes undergoing individual fertilization in vitro. Following insemination, zygotes were immobilized in groups of 16 on the base of a Petri dish using Cell-Tak tissue adhesive to enable the developmental progress of each to be tracked to the blastocyst stage. Spent droplets of in vitro maturation medium were analyzed by high performance liquid chromatography, which revealed glutamine, arginine, and asparagine were depleted in the greatest quantities. Incompetent MII oocytes that failed to cleave by 72 h postfertilization depleted significantly more glutamine from (P?=?0.0006) and released more alanine (P?=?0.0001) into the medium than oocytes that cleaved. When cutoff values were selected for the turnover of alanine, arginine, glutamine, leucine, and tryptophan and modeled to predict fertilization and cleavage potential, oocytes that did not exceed the cutoff values for ≥2 of these key amino acids were more likely to cleave. The sensitivity, specificity, accuracy, and positive predictive value of this model were 60.5%, 76.8%, 63.5%, and 92.0%, respectively. Significant differences (P ≤ 0.015) in the consumption/production of alanine and glutamine were also observed when comparing uncleaved oocytes with those that produced blastocysts. The data show that noninvasive amino acid profiling can be used to measure oocyte developmental competence.     

Prepubertal bovine oocyte: A negative model for studying oocyte developmental competence

To identify potential markers of maturation quality, differences in developmental capacity between cow and calf oocytes were compared in parallel with their constitutive and neosynthetic protein profiles before and after in vitro maturation (IVM). A comparison was also made between the protein profiles of follicular fluid (FF) from calf and cow ovaries. The effect of epidermal growth factor (EGF) during IVM on the subsequent development of prepubertal calf oocytes was examined. The effect of the presence of fetal calf serum (FCS) during development of embryos originating from calf oocytes was also examined. No differences were noted between the constitutive proteins of cow and calf oocytes and only a minor modification was observed before IVM in the pattern of neosynthesized proteins (presence of a band of 37 kD and a slight increase in the intensity of band of 78 kD in cow as compared to calf oocytes). However, the comparison of constitutive protein profiles from calf and cow FF demonstrated quantitative (the bands of 34 and 45 kD were more intense for cow than for calf) differences. EGF receptors (EGF-R) were demonstrated on cumulus—oocytes complexes (COCs) by immunofluorescence. There was no difference in intensity between cow and calf COCs. Furthermore, the addition of EGF during IVM of calf oocytes dramatically stimulated cumulus expansion and significantly increased the cleavage rate at 72 h post-insemination (82% vs 67%), as well as the proportion of embryos at the 5- to 8-cell stage at this time (54% vs 43%). Also, blastocyst yields at day 6 (11% vs 5%) and at day 8 (17% vs 10%) were significantly higher in the presence of EGF P < 0.05). The addition of FCS to synthetic oviduct fluid droplets at day 2 of culture (48 hpi) had no effect on cleavage, blastocyst yield, or blastocyst cell number. In conclusion, differences in developmental ability between calf and cow oocytes would appear to be not solely linked to differences in oocyte protein patterns. It is likely that the FF, which constitutes the microenvironment in which the oocyte develops, plays a major modulating role in determining the fate of the oocyte/follicle. © 1996 Wiley-Liss, Inc.     

Apoptosis in porcine cumulus‐oocyte complexes: Relationship with their morphology and the developmental competence

The aim of this study was to assess the presence and distribution of apoptosis in porcine cumulus‐oocyte complexes (COCs) and its relations with COC morphology and developmental competence. The COCs were obtained from slaughterhouse ovaries, classified into A1 (top category), A2, B1, B2, C, and D based on their morphology. A1, A2, and B1 were matured and fertilized in vitro, and blastocyst rate was compared among them. Before and after in vitro maturation (IVM), annexin‐V staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to assess early and late apoptosis, respectively. There was a significant increase in both annexin‐V (+) oocytes and TUNEL (+) cumulus cells as morphology further deteriorated. There were no statistical differences regarding annexin‐V (+) oocytes within immature and post‐IVM COCs, but TUNEL (+) oocytes were only observed in post‐IVM COCs. Early and late apoptosis was detected in cumulus cells of all categories of immature and post‐IVM COCs. However, the difference was only significant for annexin‐V (+). There were no significant differences in embryo development. Therefore, apoptosis increases as the morphological features of the immature COCs decrease. In conclusion, the selection of COCs from Categories A1, A2, and B1 may be used as a selection criterion for in vitro development.     

Effects of aspiration vacuum and needle diameter on cumulus oocyte complex morphology and developmental capacity of bovine oocytes

The effects of aspiration vacuum and needle diameter on the morphology of the cumulusoocyte-complex (COC) and developmental capacity of the oocyte after IVF was studied in 2 experiments using a disposable ovum pick-up needle guidance system whose construction permits its use in vitro. In Experiment 1, the relationship was determined between the aspiration vacuum, expressed in millimetre of mercury, and the actual amount of water aspirated by the system, expressed in millilitre per minute. In Experiment 2, five different levels of aspiration vacuum for 3 different needle diameters (18g, 19g and 21g) were tested in slaughterhouse ovaries. The cumulus-oocyte complexes (COCs) were divided into 3 categories: 1) oocytes with a compact cumulus, 2) oocytes with an expanded cumulus and 3) naked oocytes. The results show that a change of needle diameter can triple the amount of fluid actually aspirated. The highest oocyte recovery rates are obtained when using the thickest needle (18-g), regardless of the aspiration vacuum. On the average, for all needle types, more oocytes are recovered at the highest aspiration vacuum. For all needle diameters, the proportion of oocytes surrounded by a compact cumulus decreases progressively as the vacuum increases. Regardless of the vacuum applied, thinner needles result in a higher proportion of recovered COCs with a compact cumulus. At a high aspiration vacuum, naked oocytes become predominant regardless of the needle diameter. The prevalence of blastocysts, expressed in proportion to the recovered COCs, decreases as the aspiration vacuum increases, being especially noticeable between 70 and 130 mm Hg.     

Relationship between streaming potential and compressive stress in bovine intervertebral tissue

The intervertebral disc is formed by the nucleus pulposus (NP) and annulus fibrosus (AF), and intervertebral tissue contains a large amount of negatively charged proteoglycan. When this tissue becomes deformed, a streaming potential is induced by liquid flow with positive ions. The anisotropic property of the AF tissue is caused by the structural anisotropy of the solid phase and the liquid phase flowing into the tissue with the streaming potential. This study investigated the relationship between the streaming potential and applied stress in bovine intervertebral tissue while focusing on the anisotropy and loading location. Column-shaped specimens, 5.5 mm in diameter and 3 mm thick, were prepared from the tissue of the AF, NP and the annulus–nucleus transition region (AN). The loading direction of each specimen was oriented in the spinal axial direction, as well as in the circumferential and radial directions of the spine considering the anisotropic properties of the AF tissue. The streaming potential changed linearly with stress in all specimens. The linear coefficients k_e of the relationship between stress and streaming potential depended on the extracted positions. These coefficients were not affected by the anisotropy of the AF tissue. In addition, these coefficients were lower in AF than in NP specimens. Except in the NP specimen, the k_e values were higher under faster compression rate conditions. In cyclic compression loading the streaming potential changed linearly with compressive stress, regardless of differences in the tissue and load frequency.     

Effects of needle tip bevel and aspiration procedure on the morphology and developmental capacity of bovine compact cumulus oocyte complexes

Effects of the needle tip bevel and the aspiration procedure on the morphology of cumulusoocyte-complexes (COCs) and the developmental capacity of the oocytes after IVF were studied in 2 in vitro oocyte pick-up (OPU) simulations using a disposable ovum pick-up needle guidance system. In Experiment 1, the influence of the length of the needle bevel was investigated using a short and a long bevelled 20-g disposable needle. After being aspirated from slaughterhouse ovaries, the retrieved COCs were divided into 3 categories: 1) oocytes surrounded by a compact cumulus, 2) oocytes with an expanded cumulus, 3) partially naked oocytes. In Experiment 2, the influence of 5 different levels of aspiration vacuum for 3 different needle diameters (18-g, 19-g, 20-g) and 2 different needle bevels (long, short) was tested on the recovery and on the morphology of the cumulus investment of a fixed number of previously scored compact cumulus oocytes complexes (CCOCs), retrieved after slicing slaughterhouse ovaries. The re-retrieved COCs were allocated to Categories 1 and 3. The results show that the length of the needle bevel has a significant effect on oocyte recovery, in favor of the long-bevelled needle. As soon as higher aspiration vacua are used, a decrease of the number of CCOCs can be observed, which is less prominent for the short-bevelled needle compared to the long-bevelled one. The final number of blastocysts is similar for both needle types. In Experiment 2, the disposable needle system proved to be highly effective since nearly 80% of the CCOCs were retrieved. At low aspiration vacuum, up to 90% of the CCOCs withstand the aspiration procedure undamaged. Increasing the aspiration vacuum results in a decrease of the number of CCOCs, which is less pronounced using thinner needles. Averaged over all needle types, the prevalence of blastocysts expressed relative to the number of recovered oocytes decreases with higher aspiration vacuum.     

Effect of follicle size on bovine oocyte quality and developmental competence following maturation,fertilization, and culture in vitro

The aim of the present series of experiments was to investigate the effect of the size of follicle from which the oocytes originate on their subsequent in vitro developmental ability. Ovarian follicles were isolated and grouped according to size (2–6 mm, >6 mm). Primary oocytes were carefully liberated and grouped according to morphology into one of five categories: denuded; expanded; with two or three layers of cumulus; with four or five layers; and with many (six or more) layers. Following in vitro maturation (IVM), fertilization (IVF), and culture (IVC), more oocytes with many layers of cumulus (P < 0.01, 70.2%, 73/104 vs. 46.8%, 87/186, respectively) and a higher proportion of blastocysts were obtained from follicles > 6 mm compared to 2–6 mm follicles (P < 0.01, 65.9%, 60/91 from >6 mm follicles vs. 34.3%, 34/99 from 2–6 mm follicles, respectively). Use of follicular fluid (BFF) from follicles of different sizes in the IVM medium did not significantly increase the cleavage rate or blastocyst yield compared to controls. Administration of procine folliclestimulating hormone (pFSH) to donors prior to slaughter was investigated as a possible means of increasing the number of larger sized follicles in the ovaries and, thereby, the quality of the recovered oocytes. It was found that administration of six injections of pFSH beginning 3 days prior to slaughter resulted in a significant increase (P < 0.001) in the proportion of follicles >6 mm in diameter (31.6%) compared to that in nontreated controls (6.6%) and to animals that received only four injection groups (9.4%). The blastocyst yield from oocytes originating from >6 mm follicles, whether from unstimulated or from pFSH-treated animals, was approximately double that of oocytes from 2–6 mm follicles (P < 0.01; 42.9%, 24/56 for >6 mm follicles vs. 22.8%, 21/92 for 2–6 mm follicles, respectively, for the 6 pFSH group; P > 0.05; 62.5%, 5/8 for >6 mm follicles vs. 32.8%, 22/67 for 2–6 mm follicles, respectively, for the control). © 1994 Wiley-Liss, Inc.     

Structural aspects of bovine oocyte maturation in vitro.

Bovine cumulus-oocyte complexes (COCs) were collected from 4-8 mm follicles and graded into four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of oocytes that had matured in vivo and were collected 20-23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphological characteristics. After in vitro maturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: 1) vacuolization, 2) flattening of cumulus cells, and 3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: 1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, 2) retardation of some aspects of the cytoplasmic maturation, and 3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.     

Effects of non-esterified fatty acids on bovine granulosa cells and developmental potential of oocytes in vitro

Cryopreservation of bull semen is sub-optimal, causing cell death of a majority of spermatozoa. Even the surviving cells are affected post-thaw, either structurally or functionally. The aim of this study was to investigate the sequence of events that take place when sperm plasma membrane and acrosome deteriorate during a 4 h incubation period post-thaw, with special attention paid to the acrosome status of dying cells. Frozen-thawed semen of six AI dairy bulls was used. Three straws per batch were pooled and incubated at 37 °C. Sub-samples were taken at 30 min intervals and stained with SYBR 14, propidium iodide (PI) and phycoerythrin-conjugated peanut agglutinin (PE-PNA). Plasma membrane and acrosome integrity were measured by flow cytometry. The experiment was repeated three times. Immediately after thawing, only 3.45% of the dying cells showed acrosomal exocytosis. This number increased dramatically during incubation, reaching 67% after 4 h. Within the intact cell population, the overall decrease in viability and acrosome integrity was kept at five percentage points. Flow cytometry and the triple fluorochrome combination presented a detailed picture of the time course in plasma membrane and acrosome deterioration of frozen-thawed bull semen. The results are expected to be useful for monitoring new cryopreservation protocols.     

In vitro inhibition of oocyte nuclear maturation in the bovine

Bovine follicular oocytes (N = 5991) were exposed to an analog of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cyclic AMP (db-cAMP) (2.5, 5, and 10 mM), the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX, 0.2 mM), or the purine, hypoxanthine (0.5, 1.0, 2.0 mM), and the nucleoside, adenosine (0.05, 0.1, 0.2 mM), for 6 or 21 h to assess their effects on oocyte nuclear maturation. Potential effects of bovine follicular fluid (BFF) were also evaluated after different preculture washing procedures. In a separate experiment, denuded oocytes were used to study the effect of cumulus removal on meiotic inhibition. Db-cAMP decreased the frequency of germinal vesicle breakdown (GVBD) at 6 h (88% for control and 51%, 45%, and 32% for 2.5, 5, and 10 mM concentrations, respectively). IBMX had a comparable effect with only 41% of the oocytes resuming meiosis. Hypoxanthine and adenosine alone or in combinations also decreased the number of oocytes undergoing GVBD at 6 h. Only 22% GVBD occurred when the combined highest concentration of both substances was used compared to 88% in controls. If oocytes were incubated in 50% BFF after a wash in control medium during processing, 56% would resume meiosis at 6 h vs. 35% if the washing procedure included inhibitors (db-cAMP + IBMX). Total BFF (100%) during washing and maturation prevented 72% of the oocytes from resuming meiosis. Db-cAMP and IMBX combined or BFF also inhibited meiotic resumption of denuded oocytes. At 21 h, the inhibitory effects were less pronounced, with most oocytes only delayed in completing the first reduction division.(ABSTRACT TRUNCATED AT 250 WORDS)     

The in vitro developmental competence of bovine oocytes can be related to the morphology of the ovary

This study was designed to assess whether the developmental potential of bovine cumulus-oocyte complexes (COCs) could be related to the morphology of their originating ovary, providing a simple, noninvasive and objective selection criterion. Ovaries were divided into 3 categories on the basis of: A) presence of a follicle > 10 mm in diameter, B) presence of more than 10 follicles of 2 to 5 mm in diameter and no follicles > 10 mm, and C) presence of less than 10 follicles of 2 to 5 mm in diameter and no follicles > 10 mm. The COCs, isolated from ovaries of Category C, showed lower rates of maturation and blastocyst formation than those from Categories A and B. Moreover, blastocysts derived from Category C ovaries had fewer cells than those derived from the other 2 categories. It is concluded that ovarian morphology is a simple and noninvasive parameter for an effective selection of oocytes with better developmental competence.