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PubMed2003

Causes of developmental failure of in-vitro matured rhesus monkey oocytes: impairments in embryonic genome activation.

BACKGROUND: Understanding the causes of developmental failure of in-vitro matured primate oocytes may lead to viable strategies for improving their developmental competence. The aims of this study were to determine whether the timely onset of embryonic genome activation among individual blastomeres of preimplantation macaque embryos is impaired by in-vitro maturation (IVM) of oocytes and whether these impairments are associated with developmental failure during the embryonically controlled period of preimplantation development. METHODS: Genome activation among individual blastomeres was assessed using expression of fibrillarin as a marker of nucleolar transcription. Immature oocytes were obtained from rhesus monkeys following treatment with recombinant human FSH and matured in-vitro in one of two IVM media (CMRLa or CMRLb). In-vivo matured oocytes were obtained from FSH treated monkeys following administration of hCG. Oocytes were fertilized in vitro and either cultured for developmental studies or processed at the 8-12-cell stage for expression of fibrillarin. RESULTS: Developmental competence of embryos derived from in-vitro matured CMRLa oocytes was markedly (P < 0.05) impaired compared with those derived from in-vivo matured or in-vitro matured CMRLb oocytes. Developmental profiles were similar among the groups prior to the 8-cell stage. However, in embryos derived from in-vitro matured CMRLa oocytes, developmental failure increased significantly (P < 0.05) after the time of genome activation compared with those derived from in-vivo matured or in-vitro matured CMRLb oocytes. The mean percentages of non-activated blastomeres per embryo, as well as the proportions of embryos with at least one non-activated blastomere, or with no activated blastomeres, were all significantly (P < 0.05) greater in embryos derived from in-vitro matured CMRLa oocytes than in those derived from in-vivo matured or CMRLb oocytes. CONCLUSIONS: The relatively poor developmental competence of in-vitro matured primate oocytes is likely caused in part by failure in the timely onset of embryonic genome activation resulting from incomplete cytoplasmic maturation.

Animals↗
PubMed1996

Effects of follicular fluid during in vitro maturation of bovine oocytes on in vitro fertilization and early embryonic development.

In vivo, oocytes mature while bathed in preovulatory follicular fluid, so we studied maturation of oocytes in vitro in media supplemented with such fluid. Bovine oocytes were obtained from 2-to 6-mm follicles from slaughterhouse ovaries and were matured for 24 h in tissue culture medium 199 + 10% estrous cow serum supplemented with bovine follicular fluid (BFF) obtained from a preovulatory follicle either before (0 h) or 8 or 20 h after a GnRH-induced LH surge. After in vitro fertilization, oocytes matured in 10% or 40% 20-h BFF had higher cleavage rates (p < 0.05) than those matured in 10% or 40% 0-h BFF (80% and 86% vs. 46% and 51%, respectively); oocytes matured in 8-h BFF were intermediate. A greater percentage of oocytes showed cumulus expansion with 20-h BFF than with 0-h BFF (p < 0.05). In experiment 2, more (p < 0.05) embryos from oocytes matured in 20% or 40% 20-h BFF developed to blastocysts after culture of embryos compared to those matured in 20% or 40% 0-h BFF (29% and 29% vs. 7% and 14%, respectively) or those matured in no BFF (12%). Embryos from oocytes matured in a mixture of 0-h and 20-h BFF developed to the blastocyst stage similarly (27%) to those matured in 20-h BFF, suggesting that positive factors in 20-h BFF overcame any inhibitory effect of 0-h BFF. To summarize, bovine oocytes matured with BFF collected 20 h after LH surge result in higher cleavage rates after in vitro fertilization and more blastocysts than those matured in 0-h BFF. When the factors responsible are identified, they might be used to enhance maturation of oocytes in vitro.

Animals↗
PubMed2006

Glycosylation of recombinant proforms of major house dust mite allergens Der p 1 and Der f 1 decelerates the speed of maturation.

BACKGROUND: The efficient manufacture of recombinant Der p 1 and Der f 1 has been an important bottleneck in the study of house dust mite allergies and the development of applications for allergen engineering. While Der f 1 has only one N-glycosylation motif in the mature sequence, Der p 1 has two motifs, one in the prosequence and the other in the mature sequence. To test whether inefficient maturation of a recombinant Pro-Der p 1 versus Pro-Der f 1 is due to N-glycosylation, the maturation speed of N-glycosylation motif mutants was compared. METHODS: Expression vectors for the mutants, in which the motif in the Der p 1 prodomain was disrupted or a motif was created within the Der f 1 prodomain, were constructed by site-directed mutagenesis of preproforms with or without the motif within the mature portion. Culture supernatants of yeast Pichia pastoris transfectant cells containing proforms were buffer exchanged by gel filtration and incubated for maturation. Samples from the reactions were collected every 20 min and subjected to electrophoresis. The maturation speed was compared based on the band densities of the pro- and mature forms. RESULTS: Disruption of the motif in the mature portion decreased the productivity and accelerated the maturation. Maturation was also accelerated by disruption of the other motif in the Der p 1 prodomain and slowed down by introduction of the motif into the Der f 1 prodomain. CONCLUSIONS: Maturation systems using Pro-Der p 1 without the prodomain glycosylation are useful for the efficient preparation of a recombinant mature allergen. In addition, these results demonstrated that the maturation of cysteine protease could be controlled through glycosylation of the prodomain.

Amino Acid Sequence↗
PubMed1984

Tissue factor activity. A marker of alveolar macrophage maturation in rabbits. Effects of granulomatous pneumonitis.

Experiments were carried out to examine relationships between alveolar macrophage maturity and amounts of tissue factor (Clotting Factor III) in these cells under physiologic conditions and during immunologically induced pneumonitis. Using discontinuous density gradient centrifugation, alveolar macrophages from healthy rabbits were rapidly isolated into five subpopulations at different stages of maturation, as demonstrated by morphologic and morphometric evaluation. Very large amounts of tissue factor activity were found in fully mature cells that were purified in the lowest density subpopulation and assayed without preliminary in vitro stimulation or culture. In the remaining four subpopulations of increasing density, amounts of tissue factor were found to progressively diminish in direct correlation with declines of cell maturity. These differences at mean levels were as great as 35-fold. In addition, blood monocytes had less than 1/219 and less than 1/6 of the activity of the fully mature and the least mature subpopulations, respectively. After 16 h culture of the five isolated subpopulations in the absence of lymphokines or of significant numbers of lymphocytes, tissue factor activity increased in inverse correlation with the preincubation stage of cell maturity (2,387 and 109% in the least mature and most mature subpopulations, respectively). These increases required protein synthesis and were accompanied by morphologic and morphometric changes which indicated cellular maturation during the period of tissue factor activity generation in vitro, thus further demonstrating relationships between macrophage maturity and tissue factor content. In additional experiments, direct correlations between cell maturity and tissue factor activity content were also found in activated alveolar macrophage populations from rabbits with Bacillus Calmette Guering (BCG)-induced granulomatous pneumonitis. However, as compared with controls, the BCG populations had increased total amounts of tissue factor activity due to the presence of large numbers of mature alveolar macrophage forms that had high levels of the procoagulant. Thus, tissue factor activity in alveolar macrophages is a marker of cellular maturation in vivo and in vitro. Increased amounts of this initiator of the extrinsic clotting pathway, as found in alveolar macrophage populations from animals with granulomatous pneumonitis induced by BCG hypersensitivity, suggest that alveolar macrophage tissue factor may contribute to the pathology of immune lung diseases.

Animals↗
PubMed1994

Differences in pronucleus formation and first cleavage following in vitro fertilization between pig oocytes matured in vivo and in vitro.

To elucidate the developmental differences occurring after in vitro fertilization (IVF) of pig oocytes matured either in vitro (n = 1934) or in vivo (n = 1128), the present experiment investigated the morphological changes from penetration to the two-cell stage. Oocytes were examined every 2-4 h from 2 to 32 h after in vitro insemination to study sperm penetration, male and female pronucleus formation, synkaryosis and first cleavage. The penetration rate was significantly higher (P < 0.05) for in vivo matured oocytes (69.8%) than for in vitro matured oocytes (35.0%). Penetration of spermatozoa into the ooplasm was first recorded 6 h (in vitro matured oocytes) and 4 h (in vivo matured oocytes) after addition of the spermatozoa to the oocytes. For both in vivo and in vitro matured oocytes, 2 h were required for sperm head decondensation. However, maximum sperm head decondensation occurred 2 h later in in vitro matured oocytes. Within 6 h, 41.7 +/- 5.6% of the in vivo matured oocytes had completed second meiotic division, whereas only 20.8 +/- 6.5% of the in vitro matured oocytes reached this developmental stage (P < 0.01). For in vitro matured oocytes, male pronucleus formation was retarded 2-4 h after onset of insemination and development of the female pronucleus was enhanced compared with in vivo matured oocytes. Synchronized opposing pronuclei were observed 14 h after insemination in in vitro matured oocytes and after 8 h in in vivo matured oocytes. Synkaryosis was first observed at 16 and 18 h in in vivo and in vitro matured oocytes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Maturation of human promyelocytic leukemia cells induced by nicotinamide: evidence of a regulatory role for ADP-ribosylation of chromosomal proteins.

We have studied the role of ADP-ribosylation of chromosomal proteins in the regulation of myeloid cell maturation using the HL-60 cell line as a model. Nuclei isolated from this human promyelocytic leukemia cell line contained (ADP-ribose)n synthetase activity, whereas little or no enzymatic activity was detectable in normal human blood neutrophils. Furthermore, the activity of (ADP-ribose)n synthetase was decreased in HL-60 cells when they were induced to mature with retinoic acid (RA). To determine whether reduced (ADP-ribose)n synthetase activity is simply a result of induced maturation or whether it is a necessary precedent event for the maturation process, we evaluated the effects of nicotinamide (NAm) and its methyl derivative, N'-methylnicotinamide (N'-Met-NAm), agents which decrease ADP-ribosylation. Treatment of HL-60 cells with these drugs caused the cells to undergo maturation and to acquire certain of the morphologic, functional, and biochemical characteristics of normal neutrophils. N'-Met-NAm was more potent than NAm in inducing maturation; at a concentration of 0.8 mM, it caused greater than 80% of the cells to mature, whereas a tenfold greater concentration of NAm was required to induce a similar degree of maturation. NAm and N'-Met-NAm also potentiated the maturation of HL-60 cells induced by RA. Exposure of cells to noninducing concentrations of these compounds caused a leftward shift in the dose-response curve for RA; maturation was observed at 10(-11) M RA in the presence of either 2 mM NAm or 0.2 mM N'-Met-NAm while 10(-9) M RA was required to induce maturation in their absence. A leftward shift in the dose response curve for maturation in the presence of low doses of NAm or N'-Met-NAm did not occur with another inducer, dimethyl formamide (DMF). Two enzymes, NAD glycohydrolase and tissue transglutaminase, that are abundant in macrophages, were induced by RA but not by NAm. N'-Met-NAm decreased by about 75% the amount of endogenous (ADP-ribose)n in a selected fraction of chromosomal proteins which included histone H1 and the nonhistone high mobility group proteins. The results of this study support the concept that ADP-ribosylation of chromosomal proteins influences the regulation of human myeloid cell maturation.

Acyltransferases↗
PubMed1993

Differential surface expression of cell adhesion molecules during granulocyte maturation.

Individual steps of granulocyte maturation, such as lineage commitment, proliferation, maturation, and migration from the marrow to the peripheral blood, may be influenced by distinct interactions with the bone marrow stroma. To identify candidates of membrane components involved in maturational stage-specific interactions, we studied changes in the expression of cell adhesion molecules along the granulocyte maturational pathway. Three-color flow cytometric measurements were used to measure levels of cell adhesion molecules along this pathway. The alpha chains of VLA-4 (CD49d) and VLA-5, the integrin beta 1 chain (CD29), and CD31 (PECAM-1) were expressed in high density on all early myeloid cells but down-modulated during postproliferative maturation. CD44 and L-selectin were expressed on CD34+ myeloid progenitor cells and mature granulocytes but down-modulated during the intermediate stages of maturation. The granulocyte receptor for endothelial selectins, sLex, was specifically expressed by myeloid progenitor cells. sLex was down-modulated during the intermediate stages of granulocyte maturation but up-regulated again during terminal maturation. In contrast, CD67, a putative granulocyte adhesion molecule, was negative on progenitors, transiently up-regulated during the intermediate stages of maturation, and almost absent from the surface of mature granulocytes. These results show that each stage of granulocyte maturation is associated with the expression of a unique combination of cell adhesion molecules. L-selectin, CD44, and beta 1 integrins were regulated as previously described for immature lymphopoietic cells and may therefore play general roles in the compartmentalization and development of leukocytes. In contrast, sLex and CD67 were specifically expressed by myeloid cells and could be specifically important for compartmentalization of distinct phases of granulocyte maturation.

Antigens, CD↗
PubMed1986

Postnatal effects on reproduction and maternal care in early- and late-maturing gerbils.

In previous papers, we have identified a number of differences in both the reproductive profiles and patterns of maternal care shown by female gerbils exhibiting vaginal opening either before or after weaning. Early-maturing females delivered their first litters when younger, produced a greater percentage of female offspring/litter, and a greater percentage of early-maturing daughters than late-maturing females. Further, early-maturing females exhibited reduced levels of maternal care, as assessed by time spent nursing, time pups were gathered in the nest, and probability of retrieval of displaced young. In the present experiments, we found that equating age at first parity of early- and late-maturing females (Experiment 1) eliminated differences between them in pup gathering, but not in pup retrieval or nursing. Further, equating age at first parity affected neither sex-ratios of litters at birth nor rate of maturation of daughters. Equating litter size at birth, sex-ratio of litters at birth, and maternal age at first parturition (Experiment 2) eliminated differences in the nursing behavior of early- and late-maturing females and reduced differences in retrieval behavior, but left rate of maturation of daughters unaffected. Last (Experiment 3), both daughters born to early-maturing females, reared by late-maturing females, and daughters born to late-maturing females, reared by early-maturing females, exhibited rates of maturation typical of daughters reared by their natural mothers.

Age Factors↗
PubMed2005

Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum.

Recently, piglets have been obtained from in vitro-produced blastocysts by using in vitro maturation systems in which oocytes have been matured in North Carolina State University (NCSU) solution supplemented with porcine follicular fluid (PFF). However, PFF is not available commercially. To prepare PFF from the ovaries required time and effort and there is substantial variation in quality among batches. Furthermore, PFF is considered a potential source of infectious agents. We evaluated another commercially available potential protein source, fetal bovine serum (FBS), for in vitro maturation, to produce embryos and piglets. Cumulus-oocyte complexes were matured in NCSU-37 with PFF or with one of four batches of FBS. The proportions of oocytes with expanded cumulus cells were lower in all FBS batch groups (P < 0.05, 15-41%) than that in the PFF group (74%). The proportions of oocytes that matured were also lower in all FBS batch groups (P < 0.05, 26-41%) than in the PFF group (73%), irrespective of cumulus expansion. However, the proportions of oocytes that underwent germinal vesicle breakdown were almost the same in all groups (76-96%). After in vitro fertilization, the rate of sperm penetration into matured oocytes was higher in the PFF group (P < 0.05, 63%) than in one batch of FBS (22%) and removal of the compacted cumulus cells after maturation did not affect fertilization status (21%). Subsequent in vitro embryo culture of the PFF and FBS groups for 6 day resulted in similar rates of blastocyst formation (17 and 19%, respectively) and similar numbers of cells per blastocyst (43 and 46 cells, respectively). When blastocysts obtained from oocytes matured with FBS were transferred into two recipients, one became pregnant and farrowed seven piglets. Transfer of blastocysts obtained from oocytes matured with PFF into two other recipients resulted in one pregnancy and production of four piglets. These data suggested that porcine in vitro maturation in NCSU-37 supplemented with FBS reduced the maturational ability of oocytes, but once oocytes have matured, they have the same ability to develop to term after in vitro fertilization and embryo transfer as those matured with PFF.

Animal Husbandry↗
PubMed2002

Effect of 17beta-estradiol on the in vitro maturation of bovine oocytes.

Although 1 microg/ml of 17beta-estradiol (E2) is often used in routine in vitro maturation (IVM) and in vitro fertilization (IVF), its effect remains controversial. The objective of our study was to investigate the effects of E2 on bovine oocyte IVM and subsequent embryo development, using a defined medium. Bovine cumulus oocyte complexes (COCs), aspirated from 2 to 8 mm follicles of slaughterhouse ovaries, were matured in TCM199 in the presence of 1 microg/ml E2 with or without 0.05 IU/ml recombinant hFSH. Cultures without E2, FSH or both served as controls. COCs were matured for 22 h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. To investigate the effect of E2 with and without FSH on nuclear maturation, COCs were fixed after maturation and the nuclear stage was assessed following DAPI staining. Similarly, denuded oocytes (DO) were matured in the presence of E2 and the nuclear stage assessed after 22 h. To investigate the effect of E2 with and without FSH during IVM on subsequent embryo development, in vitro matured COCs were fertilized in vitro and after removal of the cumulus cells, the presumed zygotes were cocultured on BRL monolayer for 11 days. At Day 4, the number of cleaved embryos, and at Days 9 and 11, the number of blastocysts, were assessed. Addition of 1 microg/ml E2 to TCM199 significantly decreased the percentage of Metaphase II (MII) compared to control (56.3 and 74.0%, respectively), and increased the percentage of nuclear aberrations compared to control (13.3 and 2.1%, respectively). The negative effect of E2 on nuclear maturation was stronger when DO were matured; 25.1 and 60.0% of the oocytes reached MII stage for the E2 and control groups, respectively. When COCs were matured in TCM199 supplemented with FSH, the addition of 1 microg/ml E2 did not influence the proportion of MII oocytes, although a higher percentage of nuclear aberrations as compared to control was observed. Presence of E2 during IVM also decreased the blastocyst rate (14.4 and 10.0% for control and E2 groups, respectively). However, when FSH was present, the addition of E2 had no effect on the cleavage rate and blastocyst formation (20.3 and 21.7% for control and E2 groups, respectively). In conclusion, supplementation of 1 microg/ml E2 to a serum free maturation medium negatively affects bovine oocyte nuclear maturation and subsequent embryo development. Although these effects are attenuated in the presence of FSH, we strongly suggest omission of E2 in routine maturation protocols of bovine oocytes.

Animals↗
PubMed1996

Maturation of hamster oocytes under chemically defined conditions and sperm penetration through the zona pellucida.

This study aimed to achieve high frequencies of nuclear maturation and penetrability through the zona pellucida of hamster oocytes cultured under protein-free conditions. Completion of nuclear maturation by cumulus-intact, immature oocytes (79% metaphase II stage) was depressed (37%; p < 0.05) by adding four amino acids (glutamine, isoleucine, methionine and phenylalanine) reported necessary for nuclear maturation of cumulus-free oocytes. Following in vitro maturation, cumulus cells were removed and oocytes were inseminated with capacitated sperm, but after 6 h sperm:egg co-incubation, only 24% of in vitro matured oocytes were penetrated compared with 60% of in vivo matured oocytes (p < 0.05). Time required for zona lysis by alpha-chymotrypsin was not significantly different among in vitro and in vivo matured oocytes and 1-cell embryos. Addition to the maturation medium of soybean trypsin inhibitor or fetuin, both known to inhibit the zona reaction in vitro, did not improve penetrability of in vitro matured oocytes, implying that in hamsters, unlike other rodent species, a premature zona reaction is unlikely to be responsible for inhibiting sperm penetration. When oocytes were incubated with 20% periovulatory oviductal fluid (OF) for another 3 h after maturation, penetration was significantly improved (60% vs 37% with and without OF, respectively; p < 0.05), but was not equivalent to penetration of in vivo matured follicular oocytes similarly treated with OF (84%, p < 0.05). However, zona penetration was further improved by increasing sperm concentration from 1.0 x 10(4) (66%) to 5.0 or 10.0 x 10(4) sperm/ml (89%, p < 0.05). This study shows that nuclear maturation of hamster oocytes can occur in chemically defined medium, and indicates that a deficiency in the zona of in vitro matured oocytes can be overcome by preincubation with OF and insemination at high sperm concentration.

Amino Acids↗
PubMed2000

Births of kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro.

In Experiment 1, cleavage frequency and in vitro development of domestic cat embryos produced after in vitro maturation of oocytes obtained from ovaries after ovariohysterectomy (in vitro) with that of oocytes retrieved from follicle-stimulating hormone-treated donors at 24 h after administration of luteinizing hormone (in vivo) and fertilization by intracytoplasmic sperm injection (ICSI) or IVF were compared. In each group presumptive zygotes were assessed for cleavage on IVC Days 1 and 4 and for development to blastocysts on IVC Day 7. In vitro matured oocytes had lower frequencies of meiotic maturation (59.2% v. 66.5%), cleavage at Day 1 (41.4% v. 64.9%) and development to the morula stage at Day 4 (65.8% v. 87.9%) than did in vivo matured oocytes, after ICSI and IVF. Development to the blastocyst stage was lower in in vitro matured oocytes (19.0%) than in vivo matured oocytes (29.5%) after ICSI. In Experiment 2, we evaluated the capacity of sperm injected oocytes without a visible polar body to undergo cleavage and in vitro development. More in vivo matured than in vitro matured oocytes underwent cleavage at Day 1 (46.6% v. 12.6%) and developed to the morula stage by Day 4 (66.7% v. 46.1%), but no blastocysts were obtained at Day 7 in either group. In Experiment 3, we evaluated the in vivo viability of domestic cat embryos derived from ICSI of in vitro matured oocytes. Morula stage embryos were transferred to 18 domestic cat recipients either on Day 4 or 5 after oocyte recovery. A total of 3 domestic cat recipients were pregnant after transfer to recipients on Day 5. Two pregnant cats delivered two normal and healthy live male kittens on Day 68 of gestation and the remaining cat delivered a male kitten on Day 62 that died during the last two days of gestation. These results demonstrate that: (1) inadequate cytoplasmic maturation of in vitro matured domestic cat oocytes is the main cause of deficient oocyte activation; (2) the injection of oocytes without a visible polar body is a useful technique to evaluate oocyte cytoplasmic maturation; and (3) blastocysts obtained after ICSI of in vitro matured oocytes are viable and not a result of parthenogenesis.

Animals↗
PubMedDate not supplied

RUS skeletal maturity of children in Beijing.

BACKGROUND: RUS (radius-ulna-short bones) maturity of children has been estimated in two provinces in China, but not in the capital. In order to obtain a clearer picture of skeletal maturity in China, it is indispensable to include children from Beijing in the study. AIM: The aims of this study were three-fold: (1) to establish RUS skeletal maturity of children in Beijing, (2) to compare RUS skeletal maturity in Beijing with the Tanner-Whitehouse 3 (TW3) standard and the Tokyo standard, and (3) to examine regional variation in skeletal maturity in China. METHODS: Hand and wrist radiographs of 631 boys and 642 girls, aged 6-18, were obtained in Beijing in 1997, and their RUS skeletal maturity was estimated using the TW3 method. RESULTS: RUS maturity of the Beijing children was very similar to the TW3 standard until the age of 10 years in the girls, and the age of 12 years in the boys. The girls attained full maturity at 15 years of age, matching the TW3 and Tokyo standards, while the boys reached full maturity at the age of 16 years, as in Tokyo boys, but 1 year earlier than the TW3 standards. Beijing children showed progressively more rapid bone maturation than the Harbin and Changsha children, and attained full maturation 1 year earlier, in both girls and boys. CONCLUSION: The RUS maturity of the Beijing children was comparable with that of Tokyo children, except for the boys between 11 and 13 years. Variability of the RUS maturation among the Chinese children was greater than that found among the TW3, Tokyo and Beijing children.

Adolescent↗
PubMed1998

Differential effects of cryopreservation on nuclear or cytoplasmic maturation in vitro in immature mouse oocytes from stimulated ovaries.

The aim of this study was to develop a maturation protocol for immature oocytes and assess the protocol with cryopreserved oocytes. Nuclear maturation (mature spindle and aligned chromosomes) occurred irrespective of the treatment regime: 71-89% of oocytes matured in vitro had a normal spindle and chromosomes compared with 87% matured in vivo. Fertilization rates were not significantly different from those of in-vivo matured oocytes. Of the maturation treatment regimes investigated, the initial treatment producing best development to blastocyst (cytoplasmic maturation) involved a 2 h incubation in standard maturation medium (SMM) containing 7.5 IU follicle stimulating hormone (FSH) followed by 14 h in SMM plus 7.5 IU FSH:luteinizing hormone with follicular cells [62% (range 49-69)]. The addition of 1 ng/ml epidermal growth factor (EGF) in this protocol resulted in development [75% (range 71-81)] that was not significantly different from in-vivo matured oocytes [82% (range 73-90)]. Exposure of the oocytes to 1.5 M dimethylsulphoxide (DMSO) did not affect fertilization or development rates. Following a slow-cool/thaw freezing regime, 81% (range 74-89) of the oocytes were morphologically normal, i.e. had a spherical shape with an intact zona and oolemma; they had, however, lost their previously attached cumulus and corona cells. Maturation of frozen-thawed oocytes in the presence of EGF gave good fertilization rates but poor development rates [80% (range 77-86) and 37% (range 33-40) respectively]. In conclusion, the best maturation, both nuclear and cytoplasmic, occurred in the presence of a combination of gonadotrophins, EGF and follicular cells. Oocytes cryopreserved using a slow-cool/thaw regime can be matured to produce blastocysts after in-vitro fertilization.

Animals↗
PubMed2002

A cytogenetic study of in-vitro matured murine oocytes after ICSI by human sperm.

BACKGROUND: The purpose of this study was to investigate the chromosomal complement and developmental potential of in-vitro matured murine oocytes following ICSI by human sperm. METHODS: Heterologous ICSI fertilization between mouse oocytes and human sperm was employed in order to overcome the reduced fertilization rates observed after conventional IVF due to zona hardening during in-vitro maturation, and to assess separately maternal and paternal chromosome complements. Cytogenetic analyses were performed in four types of oocytes: (i) in-vitro matured metaphase II (MII) oocytes; (ii) in-vivo matured MII oocytes; (iii) in-vitro matured oocytes after ICSI; (iv) in-vivo matured oocytes after ICSI. RESULTS: Activation rates after ICSI of in-vitro matured oocytes was lower than that of in-vivo matured oocytes (69.9 versus 97.2%, P < 0.01), and premature chromosomal condensation was only observed in in-vitro matured oocytes. However, there were no significant differences in developmental rates after successful activation between in-vivo and in-vitro matured ICSI oocytes (69.7 versus 76.6%). The incidences of aneuploidy and structural aberrations were similar between the ICSI embryos and non-ICSI (MII) oocytes. Furthermore, the frequency of chromosomal aberrations was not associated with in-vitro or in-vivo maturation. Similar analyses of paternal chromosomes indicated that there were no significant differences in the incidence of chromosomal aberrations between the embryos derived from in-vitro and in-vivo matured oocytes. CONCLUSIONS: These results suggest that in-vitro matured oocytes following ICSI do not lead to an increase in the frequency of aneuploidy and structural aberrations when human sperm are injected into mouse oocytes.

Aneuploidy↗
PubMed2002

Gonadotropin and activin enhance maturational competence of oocytes in the zebrafish (Danio rerio).

In most teleosts, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) serves as the most potent maturation-inducing steroid (MIS) to initiate final oocyte maturation. The maturational competence or the responsiveness of oocytes to DHP increases when the ovarian follicles approach the final stage of growth. In the zebrafish, we demonstrated in the present study that full-grown oocytes (approximately 0.7 mm) exhibited the highest maturational competence, which diminished progressively with decreasing size of the follicles. Using midvitellogenic follicles (0.49-0.56 mm), which had little response to DHP, as the material, the present study aimed at investigating the endocrine and paracrine mechanisms that regulate maturational competence of the oocytes. In agreement with the results of studies in other teleost fish, pretreatment of follicles with gonadotropin (hCG) significantly enhanced the responsiveness of midvitellogenic oocytes to DHP in a clear time- and dose-dependent manner. Interestingly, activin, an ovarian growth factor, also had a potent stimulatory effect on the acquisition of oocyte maturational competence. Pretreatment with either recombinant human activin A or goldfish activin B significantly increased the rate of DHP-induced oocyte maturation from approximately 3% to approximately 70%, also in a clear dose-dependent manner. Similar to the results with hCG, pretreatment with activin alone had no effect in inducing maturation of midvitellogenic oocytes without subsequent DHP treatment, although both exhibited a strong effect in promoting maturation of full-grown oocytes. The effect of activin on maturational competence of oocytes could be reduced by cotreatment with follistatin, a potent activin-binding protein. Interestingly, follistatin treatment also significantly reduced the effect of hCG on maturational competence of oocytes, suggesting a mediating role for endogenous activin or activin-related molecules in the action of gonadotropin. The effects of hCG and activin on maturational competence of oocytes could be significantly inhibited by actinomycin D (1 microg/ml) and completely blocked by cycloheximide (1 microg/ml), suggesting that the hCG and activin-induced acquisition of oocyte maturational competence involves de novo protein synthesis at both the transcriptional and translational levels.

Activins↗
PubMed1988

Protein Synthesis during Natural and Precocious Soybean Seed (Glycine max [L.] Merr.) Maturation.

Protein synthesis was studied during precocious and natural soybean seed (Glycine max [L.] Merr.) maturation. Developing seeds harvested 35 days after flowering were precociously matured through controlled dehydration. Total soluble proteins and proteins labeled with [(35)S]methionine were extracted from control, developing seeds and from precociously and naturally matured seeds and were analyzed by one-dimensional PAGE and fluorography. The results demonstrated that several polypeptides which were designated "mature polypeptides," were synthesized de novo during precocious and natural seed maturation. Two of these polypeptides, 31 and 128 kilodalton in mass, also stained intensely with Coomassie blue, suggesting their abundant accumulation during seed maturation. Results from in vitro translation experiments showed that the mRNAs corresponding to these "maturation polypeptides" accumulated during precocious maturation and in naturally matured seeds, but not in seeds freshly harvested 35 days after flowering (control). The role of the "maturation polypeptides" is currently unknown; however, their presence and that of their corresponding mRNAs was coincident with the ability of matured seeds to establish seedling growth. This study has demonstrated that precocious seed maturation treatments may be extremely useful for investigations of metabolic events and molecular control mechanisms affecting soybean seed maturation.

Journal Article↗
PubMed1997

Transfer of immature oocytes to a preovulatory follicle: an alternative to in vitro maturation in the mare?

In the mare, success rates for the in vitro maturation of oocytes are low. Accordingly, we attempted to determine if immature oocytes could be matured in vivo by injecting them into a preovulatory follicle. Groups of 3-9 oocytes collected from donor mares were transferred under ultrasound control into the preovulatory follicle of a recipient mare that was treated with crude equine pituitary gonadotrophin (CEG) to induce ovulation. Just before ovulation (34 h post treatment) the preovulatory follicle of the recipient mare was punctured to collect both the transferred and the indigenous oocytes to analyse the stages of nuclear maturation. The transfer technique did not impair significantly the final maturation of the recipient preovulatory follicle. The indigenous oocytes within the recipient follicles were recognisable by their larger expanded cumulus of yellow colouration due to high hyaluronic acid content; 7/12 of these oocytes were mature (metaphase II). Around half (42/86; 49%) of the oocytes transferred to preovulatory follicles were recovered subsequently. Most of them showed cumulus expansion (41/42, 6 of which were rich in hyaluronic acid), 13 (32%) were mature, 15 (36%) were immature and 13 (32%) were degenerate. When the indigenous oocyte of the recipient mare was mature, 38% of the transferred oocytes were mature, this rate being no different from the in vitro maturation rate of 46%. This study showed that in vivo maturation of immature oocytes by transfer into a preovulatory follicle in a recipient mare is possible. The maturation rate is not different from the in vitro maturation rate. The technique allows the generation of mature oocytes that have an expanded cumulus rich in hyaluronic acid, similar to the situation in preovulatory oocytes. This result has not been obtained in vitro previously.

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