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.     

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.     

Effects of resazurin on bovine oocyte fertilization and embryo development in vitro

Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n=2767) were fertilized with spermatozoa exposed to resazurin (17.6 μg/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P>0.05) difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes (n=1671) were treated with resazurin (1.8 μg/ml) during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant (P>0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P<0.05) less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with resazurin may be detrimental to embryonic development.     

Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development

The objective was to evaluate the effects of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin on the nuclear and cytoplasmic maturation of bovine oocytes in the presence of follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22h in the presence of follicular cells (control with cells) and Ang II, IGF-I or insulin (treatments), or in the absence of follicular cells (control without cells). Using these five groups, Experiment 1 was conducted with and without the addition of gonadotrophins. Only oocytes in the Ang II group resumed meiosis at rates (88.2+/-1.8% and 90.7+/-4.3% for oocytes cultured in the absence or presence of LH/FSH, respectively) similar to those observed for oocytes cultured in the absence of follicular cells (89.7+/-0.3% and 92.6+/-2.6%; P<0.01). In Experiment 2, the effect of Ang II alone and in combination with IGF-I or insulin on oocyte maturation for 7h (germinal vesicle breakdown), 12h (metaphase I) and 22h (metaphase II) was evaluated in a design similar to that of the first experiment. Ang II plus IGF-I or insulin induced the resumption of meiosis, irrespective of the presence of gonadotrophins (P<0.01). Experiment 3 used groups similar Experiment 2 to determine the rate of subsequent embryo development, using fetal calf serum (FCS) in the culture medium. The COCs were cultured in maturation medium for 1h (1+23h), 12h (12+12h) or 24h in the presence of follicular cells and the respective treatments and for the remaining period in the absence of follicular cells to complete 24h. In Experiment 4, BSA was used in lieu of serum in the maturation medium in a 12+12h maturation system. Oocytes matured using the 12+12h system with BSA or FCS in the presence of Ang II+IGF-I had higher rates of blastocyst formation than the other treatments (P<0.05). In conclusion, Ang II reversed the inhibitory effect of follicular cells on nuclear maturation of bovine oocytes, irrespective of the presence of gonadotrophins, IGF-I and insulin. However, oocyte cytoplasmatic maturation (i.e., subsequent embryo development), was higher when Ang II and IGF-I were present in the maturation medium containing follicular cells cultured for 12+12h.     

Influence of progesterone on oocyte quality and embryo development in cows

In cattle, the majority of embryo loss occurs very early during pregnancy (approximately Day 16), around or prior to maternal recognition of pregnancy. The actions of P4 in controlling LH pulsatility and ovarian follicular development may impinge negatively on oocyte quality. A considerable proportion of embryo loss may be attributable to inadequate circulating progesterone (P4) concentrations and the subsequent downstream consequences on endometrial gene expression and histotroph secretion into the uterine lumen. Conceptus growth and development require the action of P4 on the uterus to regulate endometrial function, including conceptus–maternal interactions, pregnancy recognition, and uterine receptivity for implantation. This review summarizes recent data highlighting the role of progesterone in determining oocyte quality and embryo development in cattle.     

Influence of progesterone on oocyte quality and embryo development in cows

In cattle, the majority of embryo loss occurs very early during pregnancy (approximately Day 16), around or prior to maternal recognition of pregnancy. The actions of P4 in controlling LH pulsatility and ovarian follicular development may impinge negatively on oocyte quality. A considerable proportion of embryo loss may be attributable to inadequate circulating progesterone (P4) concentrations and the subsequent downstream consequences on endometrial gene expression and histotroph secretion into the uterine lumen. Conceptus growth and development require the action of P4 on the uterus to regulate endometrial function, including conceptus-maternal interactions, pregnancy recognition, and uterine receptivity for implantation. This review summarizes recent data highlighting the role of progesterone in determining oocyte quality and embryo development in cattle.     

Effect of follicle diameter on oocyte apoptosis, embryo development and chromosomal ploidy in prepubertal goats

The aim of this study was to assess the following parameters in prepubertal goat oocytes of different follicle diameter (≥3 mm, <3 mm, control): oocyte diameter, early (Annexin-V) and late (TUNEL) apoptosis, embryo development and chromosomal ploidy of these blastocysts using Fluorescence In Situ Hybridization (FISH). Before in vitro maturation, oocytes were measured and stained with Annexin-V or TUNEL. The rest of the oocytes were matured, fertilized, and cultured in vitro for 8 days. Oocytes from follicles of ≥3 mm showed greater mean oocyte diameter (128.27 ± 7.20 μm vs. 125.35 ± 7.59 μm), higher percentages of TUNEL positive (42.86 vs. 24.23%), higher cleavage (47.85 ± 3.98 vs. 23.07 ± 2.44 %) and blastocyst rates (19.77 ± 3.04 vs. 4.11 ± 1.10 %) than oocytes from follicles of <3 mm.. Blastocyst mean cell numbers did not show differences between follicular groups (123.83 ± 49.62 vs. 104.29 ± 36.09 for follicles of ≥3 mm and <3 mm, respectively). A total of 54 blastocysts with 7084 nuclei were hybridized with specific probes to chromosomes X and Y. Ninety-eight percent (98%) of the embryos presented at least one cell carrying an abnormal number of chromosomes, but 78% of them presented less than 25% of chromosomal abnormal cells. No differences in the percentage of blastocysts with abnormal ploidy were found in embryos produced from oocytes of different follicle 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.     

The relationship of follicle atresia to follicle size, oocyte recovery rate on aspiration, and oocyte morphology in the mare

Oocytes were collected by aspiration of follicles from horse ovaries obtained at surgery or post-mortem. The oocytes were classified according to morphology of the ooplasm and cumulus. The size of the corresponding follicles was measured, and sections of the follicles were fixed and examined histologically to determine the stage of viability or atresia. In Part 1, 11 pairs of ovaries were examined and all follicles were sectioned; in Part 2, 9 pairs of ovaries were examined and only those follicles from which oocytes were recovered were sectioned. The number of follicles examined per pair of ovaries in Part 1 (average +/- SD) was 12.9 +/- 4.1. The proportion of follicles that were viable increased with increasing follicular size (P < 0.01); the percentage of viable follicles was 21, 42 and 83% for follicles < 10 mm, 10 to 19 mm, and >/= 20 mm in diameter, respectively. The overall oocyte recovery rate on aspiration of follicles was 46%. There was no significant difference in the oocyte recovery rate between viable and atretic follicles. A significantly higher proportion of oocytes recovered from viable follicles had granular ooplasm (64 vs 39%; (P < 0.05); whereas significantly more oocytes from atretic follicles had a misshapen or dense ooplasm (23 vs 6%; P < 0.05), or an expanded or pyknotic cumulus (24 vs 6%; P < 0.05). The most common cumulus morphology (63% of oocytes from viable follicles and 48% of oocytes from atretic follicles) was presence of only the corona radiata. Only 11% of oocytes from viable follicles and 9% of oocytes from atretic follicles had a complete cumulus present.     

Effect of adiponectin on bovine granulosa cell steroidogenesis,oocyte maturation and embryo development

Background  

Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor) in bovine ovary and its role on ovarian cells and embryo, remain however to be determined.     

Influence of somatotropin and nutrition on bovine oocyte retrieval and in vitro development

The objective of this study was to determine the effects of supplemental bovine somatotropin (bST) and limit feeding on follicular growth and oocyte competence in yearling beef heifers. Sixteen growing heifers (424+/-4 kg) were randomly assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement, with main effects of bST (0 or 33 microg/kg BW/d) and feeding regimen (ad libitum or 0.75 ad libitum intake). Animals were treated for 100 d prior to follicular aspiration, and treatments continued for the 42-d period that follicles were aspirated. Follicles were observed ultrasonically then aspirated, and recovered oocytes were matured, fertilized and developed in vitro. The number of follicles observed ultrasonically was greater with bST treatment (P<0.01) but was unchanged by plane of nutrition. The number and quality of recovered oocytes were similar among treatments, as was the number of oocytes resulting in blastocyst formation.     

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.     

The effect of the composition of the culture media on bovine oocyte maturation and embryo development in vitro

We studied the capacity of the cattle oocyte for the resumption of meiosis and achievement of metaphase II in various protein-free culture media (DMEM, TCM-199, Ham's F-10, and Ham's F-12) and the pattern of influence of the estrous serum on in vitro development of fertilized cattle oocytes, with special reference to the time of its addition to the synthetic oviduct fluid containing BSA. In the first experimental series, it was shown that the highest number of oocytes (76.1%) resumed meiosis in DMEM medium. Meiosis was not resumed in Ham's F-12. Intermediate results were obtained for TCM-199 (55.1%), which is commonly used for maturation of cattle oocytes in vitro, and for Ham's F-10 (51.7%). The oocytes reached metaphase II in DMEM at a higher rate (45.3%) than in TCM-199 or Ham's F-10 (29.4 and 8.6%, respectively). In the second experimental series, the estrous serum was added to the culture medium within 20 h (control) or 42 h (experiment) after the beginning of fertilization. The estrous serum did not inhibit the first cleavage division (the percentage of cleaving embryos did not differ reliably: 32.7 and 37.9%, respectively). However, a later serum addition to the culture medium (within 42 h after the beginning of fertilization) reliably increased the percentage of embryos that reached the blastocyst stage (6.5% in the control and 17.8% in the experiment) and the hatched blastocyst stage (2 and 9.2%, respectively).     

Effects of once- versus twice-weekly transvaginal follicular aspiration on bovine oocyte recovery and embryo development

Ultrasound-guided transvaginal follicular aspiration combined with in vitro maturation/in vitro fertilization (IVM/IVF) and culture was used to obtain bovine preimplantation stage embryos. Evaluated were the effects of aspiration frequency on oocyte recovery and embryo development following IVM/IVF. In Experiment 1, transvaginal follicular aspiration was performed once (n=5) or twice (n=5) weekly in multiparous Angus cows with the aid of a transvaginal sector transducer (5-MHz). In Experiment 2, aspiration was performed on Angus cows once weekly (n=6), twice weekly (n=4), or twice weekly after treatment with FSH (15 mg; n=4). Follicles (>2 mm) were punctured using a 55-cm needle (17g), and oocytes were aspirated through the needle and silastic tubing (2 m) by vacuum suction (75 mmHg). The oocytes were examined for morphology and were in vitro matured and fertilized. Following IVF, all ova were co-cultured in vitro for 7 d on Buffalo Rat liver cells. Oocyte recovery rates per asp?ration session in Experiment 1 were not different between groups aspirated once or twice weekly (6.8+/-2.0 vs 6.3+/-1.1 oocytes/session; x+/-SEM) or in Experiment 2 between groups aspirated once, twice, or twice plus FSH treatment (7.7+/-1.8 vs 9.5+/-1.1 vs 6.2+/-1.1; P>0.10). In vitro development to the blastocyst stage was not different between the once, twice or twice-weekly aspiration plus FSH treatments or control oocytes obtained from cows at slaughter (23.1 vs 26.1 vs 18.0 vs 27.9%; P>0.10). Oocytes from the twice-weekly and twice-weekly plus FSH aspiration groups generated a higher percentage of Grade-1 quality embryos than the once-weekly group (P<0.05). In commercial bovine oocyte aspiration, more transferable embryos can be generated from twice-weekly aspirations than from once-weekly aspiration.     

Endocrine regulation of ovarian antral follicle development in cattle

Antral follicle growth in cattle occurs in two distinct phases; the first 'slow' growth phase spans the time from antrum acquisition to a size of approximately 3 mm detectable by transrectal ultrasound, and the second 'fast' phase is gondadotrophin-dependent and includes cohort growth, dominant follicle (DF) selection, and DF growth. This review summarises current concepts of the relative roles FSH and LH, ovarian and metabolic hormones play mainly in the second phase of antral follicle growth in animals of different reproductive and nutritional states. It is proposed that differential FSH response may enable one cohort follicle to become selected, and that follicular secretions, particularly inhibin, suppress FSH and thus are responsible for DF selection and dominance. Acute dependence of the DF on LH pulses will determine DF lifespan, and the LH pulse profile can be influenced by metabolic hormones such as leptin, providing one possible link for nutritional state and reproduction. Direct ovarian effects of acute and chronic changes in growth hormone, insulin and insulin-like growth factor (IGF)-I have been described on cohort follicles, DF oestrogen activity and on DF growth. Influences of metabolic hormones on early antral follicles undergoing their first 'slow' growth phase are less well described, yet metabolic hormones appear to enhance growth into the cohort available for FSH-induced emergence, and may influence subsequent developmental competence of oocytes.