Nucleoli from growing oocytes support the development of enucleolated full‐grown oocytes in the pig

Recent research has shown that the maternal nucleolus is essential for embryonic development. The morphology of the nucleolus in growing oocytes differs from that in full‐grown oocytes. We determined the ability of nucleoli from growing oocytes to substitute for nucleoli of full‐grown oocytes in terms of supporting embryonic development in this study. Growing (around 100 µm in diameter) and full‐grown porcine oocytes (120 µm) were collected from small (0.6–1.0 mm) and large antral follicles (4–5 mm), respectively. The nucleolus was aspirated from full‐grown oocytes by micromanipulation, and the resulting enucleolated oocytes were matured to metaphase II; the nucleoli originating from full‐grown and growing oocytes were then injected into the oocytes. The Chromatin of growing oocytes was aspirated with the nucleolus during the enucleolation process. Growing oocytes were thus treated with actinomycin D to release the chromatin from their nucleoli, and the nucleoli were collected and transferred to the enucleolated and matured full‐grown oocytes. After activation by electro‐stimulation, nucleoli were formed in pronuclei of sham‐operated oocytes. Enucleolated oocytes that had been injected with nucleoli from either full‐grown or growing, however, did not form any nucleoli in the pronuclei. No enucleolated oocytes developed to blastocysts, whereas enucleolated oocytes injected with nucleoli from full‐grown oocytes (15%) or growing oocytes (18%) developed to blastocysts. These results indicate that the nucleoli from growing oocytes can substitute for nucleoli from full‐grown oocytes during early embryonic development. Mol. Reprod. Dev. 77: 167–173, 2010. © 2009 Wiley‐Liss, Inc.     

Comparative analysis of calf and cow oocytes during in vitro maturation

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3–5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 ± 1.15 versus 122.83 ± 0.74 μm). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [³⁵S]methionine and [³⁵S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence. Mol. Reprod. Dev. 49:168–175, 1998. © 1998 Wiley-Liss, Inc.     

Developmental competence of morphologically poor oocytes in relation to follicular size and oocyte diameter in the pig

The objective of this study was to assess the practical usefulness of morphologically poor oocytes (MPCOCs) in relation to follicular size and oocyte diameter. Oocytes collected from medium (3–8 mm in diameter) and small (<3 mm) follicles were classified into five categories of morphologically good oocytes (MGCOCs) from medium follicles (MA, control), MPCOCs with larger and smaller diameters from medium follicles (ML and MS, respectively), and those from small follicles (SL and SS, respectively). The oocytes were examined for maturation and developmental competence after parthenogenesis and somatic cell nuclear transfer (SCNT). Nuclear maturation of ML oocytes (91%) was similar to that of control oocytes (94%), but higher than MS (80%), SL (79%), and SS (63%) oocytes. This pattern was also observed in the intracellular glutathione level, p34^cdc2 kinase activity, and gene (CDK1, PCNA, and ERK2) expression levels in in vitro‐matured oocytes. ML oocytes showed a similar proportion of blastocyst formation (20%) after SCNT to control oocytes (21%). In addition, the use of ML oocytes resulted in a 50% farrowing rate with 1.8% efficiency of piglet production after SCNT embryo transfer, while control oocytes showed a 60% farrowing rate with 2.4% production efficiency. Our results demonstrate that MPCOCs, if appropriately selected, have a comparable ability to MGCOCs in supporting not only in vitro blastocyst formation, but also development to term in vivo after SCNT. These oocytes can be used as a source for in vitro production of embryos with normal in vivo viability in pigs. Mol. Reprod. Dev. 77: 330–339, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Maintenance of meiotic arrest in bovine oocytes in vitro using butyrolactone I: effects on oocyte ultrastructure and nucleolus function

In this study, we have shown that butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, affects oocyte cytoplasmic morphology and nuclear function in terms of nucleolar ultrastructure and immunocytochemistry. Bovine oocytes were recovered from three classes of follicle size: 1.5-2, 2-3, and 3-6 mm. The oocytes were incubated for 40 hr with BL-I, and subsequently processed for transmission electron microscopy or immunocytochemistry. A control group of oocytes were processed immediately upon recovery (0 hr). In general, incubation with BL-I for 40 hr disrupted contact between cells of the cumulus oophorous and the oocyte, caused degeneration of the cortical granules and the peripheral migration of all cytoplasmic organelles. At the level of the nucleus, it induced convolution of the nuclear membrane and caused acceleration of nucleolar compaction in oocytes from follicles < 3 mm and fragmentation of nucleoli, particularly evidenced by immunocytochemistry, in oocytes from follicles > 3 mm. Furthermore, the effects appear to be more profound in fully-grown oocytes.     

Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats

Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality.     

Effect of follicular size on meiotic and developmental competence of porcine oocytes

In several species, the developmental competence of the oocyte is acquired progressively during late follicular growth, after the acquisition of the competence to resume and complete meiosis. In the pig, full meiotic competence of the oocyte is reached in ovarian follicles with a diameter of 3 mm or more. However, there is no information about developmental competence acquisition. We analyzed the ability of oocytes from three foll icular size classes to resume and complete meiosis, to be fertilized, and to develop in vitro to the blastocyst stage. A total of 941 follicles were dissected from slaughterhouse gilt ovaries and classified as small (<3 mm, n = 330), medium (3-5 mm, n = 373), or large (>5 mm, n = 238). The cumulus-oocyte complexes recovered from these follicles were submitted to in vitro maturation for 44 h in TCM199 supplemented with 10 ng/ml EGF, 400 ng/ml pFSH and 570 microM cysteamine; in vitro fertilized for 18 h in mTBM with 10(5) frozen-thawed percoll-selected sperms/ml; and developed for 7 days in mSOF. Samples of oocytes or presumptive zygotes were fixed and stained at the end of maturation and fertilization. Groups of oocytes were cultured for 3 h in the presence of 35S-methionine before or after maturation for SDS-PAGE analysis of protein neosynthesis. More oocytes originating from medium and large follicles were competent for maturation than oocytes from small follicles (77 and 86% of metaphase II, respectively, versus 44%, P < 0.05). More oocytes from medium and large follicles werepenetratedby spermatozoa during in vitro fertilization, resulting in significantly more oocytes presenting two or more pronuclei at the end of fertilization (73 and 77% for medium and large follicles, respectively, versus 53% for small follicles, P < 0.05). More oocytes from medium and large follicles developed to the blastocyst stage (14 and 23%, respectively) than those from small follicles (3%, P < 0.05), even if the development rates were corrected by the maturation or fertilization rates. It is concluded that a high proportion of oocytes harvested from follicles of less than 3 mm in the pig are not fully competent for meiosis and are cytoplasmically deficient for development.     

Quantitative and qualitative analysis of RNA synthesis in stage 6 and stage 4 oocytes of Xenopus laevis

RNA synthesis has been studied in oocytes taken from Xenopus laevis females which have not recently ovulated. Such females contain a population of large (stage 6) oocytes which exhibit white equatorial bands and which are considered to represent the terminal stage of oocyte development. Rates of RNA synthesis in these “banded” oocytes were measured by analyzing the kinetics of incorporation of ³H-guanosine into acid-precipitable, alkaline-labile material, and changes in precursor pool (GTP) specific activity during incubations. In additional experiments, rates of RNA synthesis were measured after ³H-GTP was injected directly into stage 6 oocytes. For comparison, rates of RNA synthesis were measured in lampbrush chromosome stage oocytes (stage 4; 0.5–0.6 mm diameter). The results show that, under the in vitro conditions employed, stage 6 oocytes are not metabolically dormant, but synthesize total RNA at a rate at least as great as the stage 4 oocytes.Qualitative studies on newly synthesized RNA in the two oocyte classes have been performed using sucrose density gradient centrifugation and acrylamide gel electrophoresis. Both stage 4 and stage 6 oocytes exhibited similar patterns, and the bulk of the RNA synthesized and accumulated during 12-hr pulses appears to be ribosomal. These observations are discussed in terms of existing concepts concerning synthetic activity in stage 6 oocytes.     

Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 +/- 2.1 pmol/oocyte) and medium (13.69 +/- 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 +/- 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 +/- 5.18 nM and 52.25 +/- 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 +/- 68.6 ng/ml) than from medium (40.0 +/- 6.4 ng/ml) and small (41.2 +/- 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 +/- 45.9 ng/ml) and medium (267.5 +/- 38.6 ng/ml) follicles were significantly higher than that (174.7 +/- 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.     

Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes

To investigate the effect of recombinant bovine somatotrophin (rGH) on ovarian folliculogenesis in sheep, 18 mature Scottish Blackface ewes were assigned randomly to two treatment groups. Starting from day 5 of the synchronised oestrous cycle, animals were injected daily with either vehicle (control group) or 12.5 mg rGH (rGH-treated group) for 7 days. Blood samples were collected once daily during the experimental period for the measurement of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, follicle-stimulating hormone (FSH), luteinising hormone (LH) and progesterone. At the end of treatment animals were killed and ovaries collected. All follicles at least 1.0 mm in diameter were dissected out and diameters measured to assess follicular populations for individual animals. Five small follicles (1.0–3.4 mm in diameter) and all the large follicles (at least 3.5 mm) from each animal were incubated in 1 ml of Medium 199 for 1 h. Medium was then changed and incubation continued for a further hour. All medium samples were assayed for IGF-I, oestradiol, testosterone and progesterone.Treatment of ewes with rGH had no effect on the total number of follicles at least 1.0 mm in diameter (control, 34.4 ± 2.6; rGH-treated, 31.3 ± 1.4; P > 0.2). However, when follicles were further classified into different size categories (1.0–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, 5.1–6.0 and over 6.0 mm in diameter), the population of follicles 2.1–3.0 mm in diameter was significantly increased by rGH treatment (control, 9.2 ± 0.7; rGH-treated, 13.8 ± 1.1; P = 0.02). The number of follicles of 3.1–4.0 mm diameter in the rGH-treated group tended to be increased (P = 0.09), whilst the population of follicles 1.0–2.0 mm in diameter was reduced (P = 0.07). Treatment of ewes with rGH significantly increased peripheral concentrations of GH (P < 0.01), IGF-I (P < 0.01), insulin (P < 0.01) and progesterone (P < 0.05). There was no effect of rGH treatment on circulating concentrations of FSH and LH. Both large and small follicles from rGH-treated ewes secreted significantly (P < 0.001) more IGF-I (37.8 ± 2.2 ng ml h⁻¹, n = 50) than follicles from the control group (26.7 ± 1.6 ng ml⁻¹ h⁻¹, n = 73). However, there was no significant effect of rGH treatment on the secretion of oestradiol, testosterone and progesterone by either large or small follicles.It is concluded that treatment of mature ewes with rGH can enhance the development of ovarian follicles to the gonadotrophin-dependent stages. Furthermore, rGH appears to act through increased secretion of ovarian IGF-I, as well as increased peripheral concentrations of IGF-I and insulin.     

Follicular size affects the meiotic competence of in vitro matured prepubertal and adult oocytes in sheep.

Non-atretic follicles dissected from prepubertal and adult ovaries were allocated in three groups: a) < 1 mm; b) 1-2 mm; c) > 2 mm. After 24 h of maturation, a lower percentage of adult oocytes from group a (P < 0.01) reached metaphase II than those from groups b and c (70.4 versus 89.5 and 95.5). Prepubertal oocytes showed similar results (P < 0.01; 27.2 versus 79.8 and 81.8). There was a significant difference (P < 0.01) in meiotic progression between prepubertal and adult oocytes of < 1 mm follicles. The diameter of prepubertal oocytes derived from group a was significantly lower (P < 0.01) compared to groups b and c (138.1 versus 142.1 and 145.6); adult oocytes showed similar results (P < 0.01; 142.2 versus 157.2 and 158.1). Oocytes with the same diameter derived from different follicles of prepubertal and adult ovaries showed similar meiotic progression rates.