[A new system of classification and symbols for the evaluation of the maturity and nutritional status of newborns using an infant maturity-nutrition index].
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Birth Weight↗
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The histochemistry of the jaw muscles of the mature and maturing rat [proceedings].
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Age Factors↗
The effect of glucose on the progression of the nuclear maturation of pig oocytes.
The progression of the nuclear maturation of oocytes is a useful marker for the estimation of the subsequent developmental competence of oocytes. In this study, we examined the effect of energy substrates in an in vitro maturation medium on the progression of the nuclear maturation of oocytes. In experiment 1, the supplementation of the maturation medium with 0, 5 and 10 mM of glucose lead to increase in the total cell number of the blastocysts. In experiments 2 and 3, the maturation phase was divided into two stages (germinal vesicle (GV) stage: 0-20 h and nuclear maturation stage: 20-44 h), and the effects of glucose or pyruvate added at each stage on the kinetics of nuclear maturation were examined. The addition of glucose at the nuclear maturation stage rather than at the GV stage of maturation effected greater acceleration in the progression of nuclear maturation. However, the addition of pyruvate at both stages had the same effect on the progression of nuclear maturation was the same. In addition, when glucose was added to the medium containing pyruvate, an additive effect on the progression of nuclear maturation was observed (experiment 4). In experiment 5, the inhibitors of glucose-6-phosphate dehydrogenase (G6PD), dehydroepiandrosterone (DHEA) and 6-aminonicotinamide (6-AN) decreased the rate of the final maturation of oocytes and reduced the difference between the rates of the final maturation of oocytes cultured with glucose and those cultured with pyruvate. In the experiment 6, when the activator of G6PD, brilliant cresyle blue (BCB), was added to the maturation medium, the progression of nuclear maturation was significantly accelerated. The results of this study suggested that in addition to the role of an energy substrate, glucose or its metabolites play a role in nuclear maturation. This role was more pronounced at the second stage of maturation (transition from GV breakdown (GVBD) to M2), probably due to the metabolism of glucose via the pentose phosphate pathway (PPP) rather than the glycolysis pathway.
6-Aminonicotinamide↗
The pattern of auditory brainstem response wave V maturation in cochlear-implanted children.
OBJECTIVE: Maturation of acoustically evoked brainstem responses (ABR) in hearing children is not complete at birth but rather continues over the first two years of life. In particular, it has been established that the decrease in ABR wave V latency can be modeled as the sum of two decaying exponential functions with respective time-constants of 4 and 50 weeks [Eggermont, J.J., Salamy, A., 1988a. Maturational time-course for the ABR in preterm and full term infants. Hear Res 33, 35-47; Eggermont, J.J., Salamy, A., 1988b. Development of ABR parameters in a preterm and a term born population. Ear Hear 9, 283-9]. Here, we investigated the maturation of electrically evoked auditory brainstem responses (EABR) in 55 deaf children who recovered hearing after cochlear implantation, and proposed a predictive model of EABR maturation depending on the onset of deafness. The pattern of EABR maturation over the first 2 years of cochlear implant use was compared with the normal pattern of ABR maturation in hearing children. METHODS: Changes in EABR wave V latency over the 2 years following cochlear implant connection were analyzed in two groups of children. The first group (n=41) consisted of children with early-onset of deafness (mostly congenital), and the second (n=14) of children who had become profoundly deaf after 1 year of age. The modeling of changes in EABR wave V latency with time was based on the mean values from each of the two groups, allowing comparison of the rates of EABR maturation between groups. Differences between EABRs elicited at the basal and apical ends of the implant electrode array were also tested. RESULTS: There was no influence of age at implantation on the rate of wave V latency change. The main factor for EABR changes was the time in sound. Indeed, significant maturation was observed over the first 2 years of implant use only in the group with early-onset deafness. In this group maturation of wave V progressed as in the ABR model of [Eggermont, J.J., Salamy, A., 1988a. Maturational time-course for the ABR in preterm and full term infants. Hear Res 33, 35-47; Eggermont, J.J., Salamy, A., 1988b. Development of ABR parameters in a preterm and a term born population. Ear Hear 9, 283-9] of normal hearing children: a sum of two decaying exponential functions, one showing an early rapid decrease in latency and the other a slower decrease. Remarkably, the time-constants fell well within the ranges described by Eggermont and Salamy (i.e., 3.9 and 68 weeks), consistent with the time-course of the neurophysiological mechanisms presumably involved in auditory pathway maturation during the first 2 years of life: i.e., myelination and increased synaptic efficacy. In contrast, relatively little change in wave V was evident in children with late-onset deafness. In agreement with the notion that EABR maturation follows an apex-to-base gradient as described for ABR, we observed that wave V latencies were longer for the basal than the apical end of the implant electrode array and remained so throughout the study period, whatever the time of onset of deafness. CONCLUSIONS: The findings in the early-onset of deafness group support the theory that auditory pathways remain "frozen" during the period of sensory deprivation until cochlear implant rehabilitation restores the normal chronology of maturational processes. In children with late-onset deafness, however, some maturational processes may occur before the onset of deafness, and thus less additional maturation is required during the first two years of implant use resulting in no significant EABR latency changes being observed in this period. The results suggest that the rehabilitation-induced plasticity of the auditory pathways is, in case of late auditory deprivation, unlikely to result in neurophysiological outcomes similar to those observed in children with early auditory deprivation. SIGNIFICANCE: Changes in EABR wave V latency over the first 2 years of cochlear implant use were found to be well fitted by the sum of two decaying exponential functions in children with early-onset deafness. This is in line with the maturation of ABR wave V latency in normal-hearing children over the first two years of life. Further studies are needed to assess whether the differences observed in terms of auditory pathways maturation are associated with consistent differences in terms of language development.
Adolescent↗
Effects of polyunsaturated fatty acids and prostaglandins on oocyte maturation in a marine teleost, the European sea bass (Dicentrarchus labrax).
The effects of the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and prostaglandins (PGs) on oocyte maturation were investigated in a marine teleost, the sea bass (Dicentrarchus labrax). Follicle-enclosed postvitellogenic, preovulatory oocytes were cultured in vitro and maturation was verified by assessing volume increase, lipid droplet coalescence, yolk clarification, and germinal vesicle migration and breakdown. Human chorionic gonadotropin was administered as the maturation-inducing gonadotropin (GTH) and was capable of inducing maturation in a time- and dose-dependent manner. Free AA induced maturation in a dose- and time-dependent manner and enhanced GTH-induced maturation, while EPA, DHA, and oleic acid were ineffective. Maturation induced by GTH was significantly suppressed by a phospholipase A(2) blocker, suggesting that mobilization of AA was involved in GTH-induced maturation. Moreover, EPA and DHA exhibited a significant, dose-dependent attenuation of GTH-induced maturation. Maturation induced by GTH was inhibited in the presence of a cyclooxygenase inhibitor, indomethacin, and this inhibition was reversed by addition of AA, PGE(2), or PGF(2alpha). PGE(2) and PGF(2alpha) alone were both effective stimulators of maturation, while PGE(1) and PGE(3) were ineffective. The effect of PUFAs on oocyte maturation in vitro were corroborated with studies in vivo. Oocytes were obtained from females fed a commercial, PUFA-enriched diet (RD) and maturational behavior was compared with oocytes from females fed a natural diet (ND) with a higher EPA content and n-3:n-6 ratio. Although no significant difference was observed in the rate of spontaneous oocyte maturation, a higher percentage of GTH-induced maturation and lower percentage of atresia were observed in RD oocytes. Moreover, while basal PGE production from oocytes from both groups was the same, RD oocytes produced significantly higher levels of PGE in the presence of hCG. The results from this study provide evidence for the participation of AA metabolism in GTH-induced oocyte maturation, and suggest that other PUFAs and PGs may play important roles in the induction of maturation in a marine teleost.
Animals↗
An analysis of body weights and maturing patterns in western range ewes.
The objectives were to determine the association of maturing patterns with growth rates and body weights and to estimate heritabilities and genetic and phenotypic correlations among these characters of sheep. Records of 1,109 range ewes from the Montana Agricultural Experiment Station Red Bluff Research Ranch at Norris were analyzed. Body weight and degree of maturity of body weight at birth, weaning, 12 mo of age, 18 mo of age and maturity (body weight only), and absolute growth rate (AGR), absolute maturing rate (AMR) and relative growth rate (RGR) over various age intervals were examined. Mature weight, required to calculate degree of maturity and AMR, was estimated by the average of the fall weights taken at 42, 54, 66 and 78 mo of age. Heritability estimates were .53 +/- .12 for mature weight and from .26 to .46 for immature weights. Genetic correlations among body weights at all ages were positive and generally large between immature weights and mature weights. Heritability estimates for degree of maturity ranged from .63 +/- .12 at 12 mo of age to .19 +/- .11 at 30 mo of age, at which time maturity was being approached. Genetic correlations between degree of maturity and body weight at the same age were positive; however, degree of maturity at all ages was negatively correlated with mature weight. Animals more mature at any age or stage during growth tended to be more mature at later stages, to be lighter at maturity, and to grow faster and weigh more up to 12 mo of age. Heritability estimates for AGR, RGR and AMR were moderate to high and were similar for the same age intervals. Selection for any one of the measures of growth rate would tend to expand the shape of the growth curve toward heavier weights and lower degree of maturity for any interval.
Animals↗
Sexual development in early- and late-maturing Bos indicus and Bos indicus x Bos taurus crossbred bulls in Brazil.
Two experiments were conducted to evaluate sexual development in early- and late-maturing Nelore (Bos indicus) and Canchim (3/8 Bos indicus x 5/8 Bos taurus crossbred) bulls and to determine predictors of sexual precocity, and pubertal and maturity status. In Experiment 1, 12 Nelore bulls where examined from 300 to 900 days of age. Puberty was characterized by an ejaculate containing > or =50 million sperm with > or =10% motile sperm, and maturity by an ejaculate containing > or =70% morphologically normal sperm. In Experiment 2, 28 Canchim bulls where examined from 295 to 488 days of age and puberty was characterized by an ejaculate containing > or =30% motile sperm. In both experiments, bulls were classified as early- or late-maturing based on age at puberty. Early-maturing bulls were younger (P < 0.05) than late-maturing bulls at puberty (527 days versus 673 days in Experiment 1 and 360 days versus 461 days in Experiment 2) and at maturity (660 days versus 768 days in Experiment 1). In general, early-maturing bulls were heavier and had greater scrotal circumference (SC), testes, and testicular vascular cone diameter than late-maturing bulls during the experimental period. Scrotal circumference adjusted for 365 days of age was a good predictor of sexual precocity; minimum yearling SC of 19 and 24 cm for Nelore and Canchim bulls, respectively, had the best predictive values. Early-maturing bulls were lighter and had smaller SC at puberty than late-maturing bulls; therefore, sexual precocity was not related to the attainment of a threshold body weight or testicular size earlier, but to lower thresholds in early-maturing bulls. When predictors of pubertal status were evaluated, SC had the best sensitivity/specificity relationship in Nelore bulls, and high sensitivity and specificity in Canchim bulls. When predictors of sexual maturity were evaluated in Nelore bulls, age, weight, and SC had similar sensitivity, specificity, and predictive values. At puberty, approximately 60% of the sperm present in the ejaculate were morphologically defective. Changes in semen quality after puberty in Nelore bulls were characterized by increased motility and proportion of morphologically normal sperm, with a decrease in the proportion of major sperm defects. In conclusion, early-maturing bulls were more developed in the pre-pubertal period and attained puberty at earlier stages of body and testicular development than late-maturing bulls. Yearling SC could be used to select bulls for sexual precocity and SC was the best predictor of pubertal status. Age, weight, and SC were equally good predictors of sexual maturity in B. indicus bulls.
Age Factors↗
Spindle positions and their distributions in in vivo and in vitro matured mouse oocytes.
BACKGROUND: This study was carried out to compare spindle locations and their developmental competencies both in vivo and in vitro in matured mouse oocytes. Spindle locations were identified using a polscope. Since meiotic spindles in living oocytes are highly birefringent, their structures can be viewed non-invasively by using a polscope. METHODS: In vivo matured metaphase II oocytes were collected from the oviducts of mice. Immature oocytes were collected from mouse ovaries, and then cultured in YS medium until the first polar body (PB) extrusion. In vitro and in vivo matured oocytes were classified into four categories according to their spindle positions relative to the first PB (0 degrees , 0-90 degrees , 90-180 degrees and without a spindle image), and rates of fertilization and blastocyst formation were assessed. In vivo matured oocytes with a 0 degrees spindle disposition relative to PB were cultured in vitro for 24 h, and then their spindle positions were re-assessed. RESULTS: Most in vivo matured oocytes (89.1%) had a 0 degrees spindle position. Only 6 and 3% of oocytes had spindle positions of 0-90 degrees and 90-180 degrees , respectively. No spindle image was observed in 2%. However, most in vitro matured oocytes (83.1%) had a 0-90 degrees spindle position and, in contrast, only 6.5% of these oocytes had a 0 degrees spindle position. The rate of fertilization and blastocyst rate were significantly higher for in vivo matured oocytes than in vitro matured oocytes (87.1 versus 64.9% and 76.1% versus 66.0%, respectively, P<0.05 for each). We also observed that 71.7% of the in vivo matured oocytes with the 0 degrees spindle position showed a spindle position change to 0-90 degrees after 24 h of culture. These oocytes had a poor fertilization rate (43%) and a zero blastocyst rate. CONCLUSION: In vitro matured mouse oocytes showed quite different spindle positions compared with in vivo matured oocytes. Moreover, in vivo matured oocytes cultured for 24 h had a spindle position distribution that was similar to that of in vitro matured oocytes. The different spindle positions observed in in vivo and in vitro matured oocytes may reflect differences in their cytoplasmic maturation processes. These findings have implications regarding the lower developmental competency of in vitro matured oocytes.
Animals↗
Body size versus gonad maturation form in under-yearling precocious males of the sea trout (Salmo trutta m. trutta L.).
The study was aimed at analysing body size in relation to form of gonad maturation (amount of mature germ cells) in 329 under-yearling sea trout males. The fish, aged 7 months, were caught in late October-early November in 3 streams located in north-western Poland. Each stream supported fish belonging to a different sib group. Standard histological techniques and a computer image analysis programme were used to detect the class of gonad maturation and percentage of the gonad area occupied by tubules with active spermatogenesis. Gonad maturation forms were distinguished based on the latter criteria. Gonads with developing germ cells occupying less than 90% of gonad area were classified as incomplete forms of gonad maturation, others as complete maturation forms. In each sib-groups analysed, even the smallest individual were already precocious, their gonads being incompletely mature. The smallest maturing male measured 7.1 cm in length. The average size of an incompletely maturing individual was slightly smaller than that of the completely mature one but the difference lacked statistical significance (P > 0.05). The sib-group of smaller fish contained less precocious, and the gonads of the more precocious were incompletely mature, compared to the sib-group of larger fish (P < 0.001). It seems that the incomplete form of gonad maturation (defected maturation) occurs at a smaller critical fish size than the complete gonad maturation form. Incomplete maturation is more frequent smaller individuals and possibly in among slow-growing groups of fish.
Animals↗
Reduced oxygen concentration improves the developmental competence of mouse oocytes following in vitro maturation.
Reduced atmospheric oxygen concentration is beneficial to embryo development; however, optimal oxygen concentration for oocyte maturation remains undetermined. Likewise, there is no consensus of appropriate medium supplementation during maturation. The objective of this study was to determine whether oxygen tension (20% or 5% O2) and epidermal growth factor (EGF) affect oocyte metabolism and subsequent embryo development. Cumulus-oocyte complexes (COCs) were collected from 28-day-old equine chorionic gonadotropin (eCG) primed or unprimed F1 (C57BL/6xCBA) mice. COCs were matured in defined medium in one of four groups: 20% O2, 20% O2 + EGF, 5% O2, 5% O2 + EGF. In vivo matured COCs were also collected for analysis. COCs from unprimed mice, matured in 5% O2 +/- EGF or 20% O2 + EGF had higher metabolic rates than COCs matured in 20% O2 (P < 0.05). COCs from primed mice had higher metabolic rates when matured in the presence of EGF, regardless of oxygen tension (P < 0.01). Oxygen uptake and mitochondrial membrane potential were higher for in vivo matured oocytes and oocytes matured under 5% O2 compared to oocytes matured under 20% O2 (P < 0.05). Blastocyst formation was not different between maturation groups (primed or unprimed); however, embryo cell numbers were 20-45% significantly higher when COCs were matured at 5% O2 (P < 0.05). Results suggest that oocytes matured in physiological concentrations of oxygen have improved development and metabolic activity, more closely resembling in vivo maturation. These findings have implications for oocyte maturation in both clinical and research laboratories.
Animals↗
In vitro maturation of collagen fibrils modulates spreading, DNA synthesis, and collagenolysis of epidermal cells and fibroblasts.
Collagen fibrils were maturated in vitro by incubating them in a serum-containing culture medium at 37 degrees C for varied lengths of time. Epidermal cells and fibroblasts were cultured on these maturated collagen gels to see the effects of maturation on cellular morphology and physiology. The spreading and DNA synthesis of both types of cells on the maturated collagen gels were significantly enhanced compared to those on fresh gels. The maturation did not affect the cellular adhesiveness to the substrate. The secretion of collagenase by epidermal cells was suppressed on the maturated collagen gels, the extent of the suppression being related to the length of maturation of the gels. These maturation-related effects of collagen were also observed when collagen was incubated in the medium without serum, indicating that the effects are not due to deposition of serum proteins to collagen gels during maturation. Physical and chemical characterizations of the maturated collagen were performed: the mechanical strength of collagen gels increased in maturated collagen gels, the amounts of insoluble collagen increased with the maturation. These changes in the chemical and physical nature of the maturated collagen gel strongly suggested that there was an increase in intermolecular crosslinks during the process of maturation. These maturation-induced changes in collagen were marked when collagen gels were incubated in the presence of glucose, indicating that a glucose-protein reaction such as the Maillard reaction is involved in this phenomenon.
Animals↗
Study of evaluation criteria used for in vitro bovine oocyte selection and maturation.
Production of bovine preimplantation embryos in vitro requires beneficial maturation conditions and high quality oocytes at the germinal vesicle stage. The current classification of oocytes is based on character of the cumulus cell investment around the oocyte. We wished to study the nuclear stage of immature oocytes selected for in vitro maturation according to cumulus cell character and, in the other hand, to compare the relationship among 3 parameters utilized to evaluate in vitro maturation of bovine oocytes (degree of cumulus expansion, meiotic maturation rate and in vitro fertilization rate) when fetal calf serum, steer serum and bovine follicular fluid supplementation were used. Ovaries were collected at an abattoir and the oocytes harvested. As regards selection criteria, immature oocytes were classified as Class A, B, C and D according to the character of the cumulus cells. A high percentage of Class A oocytes (87.7%) were in the germinal vesicle stage with respect to the other classes (p < 0.05). Significant differences were found in the meiotic maturation rate in Class A oocytes (76.5%) versus those of the other classes (p < 0.05). The meiotic maturation rate diminished to 47.5% when Class A oocytes were denuded and then matured in vitro (p < 0.05). As regards maturation criteria, there was no cumulus expansion when oocytes were matured in TCM-199 without supplementation, partial expansion with the addition of fetal calf serum and full expansion when supplemented with steer serum or bovine follicular fluid. No significant differences were found in the meiotic maturation rate for the various treatments. In vitro fertilization rate was significantly lower in media without supplementation versus supplemented media (p < 0.05), but no significant differences were found between the supplemented media inter se. There is no direct relationship between the three studied parameters to evaluate in vitro maturation. Class A oocytes are the most likely to mature in vitro as they not only have a close association with their surrounding cumulus cells, but are also very numerous in the germinal vesicle stage. The degree of cumulus expansion and the meiotic maturation rate have a relative importance in evaluating in vitro maturation, as oocyte maturation implies not only nuclear events but also at other cellular levels, as evaluated by in vitro fertilization.
Animals↗
Low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine cumulus-oocyte complexes.
The effects of carbohydrates on meiotic maturation and ATP content of bovine oocytes under low oxygen tension (5%) were investigated. Furthermore, the developmental competence or intracellular H(2)O(2) contents of the oocytes matured under 5% or 20% O(2) was assessed. In vitro maturation of bovine cumulus-oocyte complexes was performed in synthetic oviduct fluid (SOF) containing 20 amino acids and hormones (SOFaa). The proportion of the oocytes that matured to the metaphase II stage in SOFaa containing 1.5 mM glucose, 0.33 mM pyruvate, and 3.3 mM lactate under 5% O(2) was dramatically lower than that of oocytes matured under 20% O(2) (P < 0.01). Similarly, the ATP content of the oocytes that matured under 5% O(2) was much lower than that of oocytes matured under 20% O(2) (P < 0.05). Under 5% O(2) the proportion of metaphase II oocytes increased with increasing glucose concentration (0-20 mM) in SOFaa without pyruvate or lactate. In addition, the ATP content of oocytes cultured in 20 mM glucose was higher (P < 0.05) than that of oocytes cultured in 1. 5 mM glucose. Two glucose metabolites (pyruvate and lactate) and a nonmetabolizable glucose analog (2-deoxy-glucose), however, had no noticeable effects on meiotic maturation under 5% O(2). These results suggest that ATP production under 5% O(2) is not dependent on the TCA cycle. Addition of iodoacetate, a glycolytic inhibitor, to SOFaa containing 20 mM glucose significantly reduced (P < 0.01) the proportion of metaphase II and ATP content. Moreover, the proportion of the development to the blastocyst stage of oocytes matured under 5% O(2) was higher (P < 0.05) than that of oocytes matured under 20% O(2). H(2)O(2) contents of oocytes matured under 5% O(2) was lower (P < 0.05) than that of oocytes matured under 20% O(2). The results of the present study demonstrate that glucose plays important roles in supporting the completion of meiotic maturation in bovine cumulus-oocyte complexes under low oxygen tension and that low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine oocytes.
Adenosine Triphosphate↗
Prediction of nuclear maturity from cumulus-coronal morphology: influence of embryologist experience.
PURPOSE: A majority of in vitro fertilization (IVF) programs continues to evaluate oocyte maturity on the basis of cumulus-coronal morphology (CCM) even though marked asynchrony has been reported between CCM and nuclear maturity. This study was designed to examine changes in embryologists' ability to correctly predict nuclear maturity from CCM as a function of increasing experience. Nuclear maturity was assessed by inverted microscopy with a modified spreading technique at follicular aspiration. A second objective was to determine the percentage of oocytes which displayed asynchrony between CCM and nuclear maturity as assessed by embryologists with extensive experience in oocyte maturity evaluation. RESULTS: The three participating embryologists had directly evaluated 1304, 75, and 0 oocytes for nuclear maturity and CCM at study initiation and correctly predicted nuclear maturity from CCM in 74, 64, and 47% of oocytes, respectively. Embryologist 1 did not significantly change in predictive ability during the 17-month study period. Embryologist 2 significantly improved in predictive ability during the first 9 months of the study (841 oocytes evaluated) and plateaued thereafter, at a similar percentage of correct predictions as embryologist 1. Embryologist 3 continued to improve in predictive ability throughout the study period, reaching 61% correct predictions at the close of the study after evaluating 223 oocytes. Once embryologists had plateaued in their predictive ability, 72% of oocytes evaluated received the correct nuclear maturity classification based on CCM. Significantly fewer oocytes (54%; 375/690) evaluated by embryologists who had not plateaued in their predictive ability received the correct nuclear maturity classification based on CCM. CONCLUSIONS: These results indicate that embryologists' ability to predict oocyte nuclear maturity correctly from CCM continues to change over several months even when pretraining video recordings are used before beginning direct evaluations. After embryologists plateaued in their predictive ability, nuclear maturity still could not be correctly predicted from CCM in 28% of oocytes due to asynchrony between nuclear and CCM maturity. Based upon this, circumstances in which the spreading technique should be used for direct assessment of nuclear maturity as opposed to assessment of CCM only are discussed.
Cell Differentiation↗
Effects of external pH on hormonally regulated ovarian follicle maturation and ovulation in Atlantic croaker.
In vitro studies of ovarian follicle maturation and ovulation in teleost fishes typically are conducted within a narrow range (7.5-7.8) of constant external (medium) pH, although there is evidence that pH can influence ovulation. Therefore, this study with Atlantic croaker investigated the effects of external pH on hormonally regulated in vitro maturation and ovulation as well as changes in the pH of ovarian fluid during in vivo maturation and ovulation. For the in vitro experiments, follicles were first incubated with human chorionic gonadotropin (hCG) to induce maturational and ovulatory competencies, and then with maturation-inducing hormone (MIH) to induce completion of maturation and ovulation. At a constant external pH within the range of 7.0-8.2, the lower pH levels (7.0-7.3) generally inhibited or slowed down hormonally induced maturation and ovulation whereas higher pH (7.6-8.2) facilitated these processes. When ovarian follicles were incubated at a constant pH of 7.6 during the priming incubation with hCG, changing the external pH during the incubation with MIH had relatively little effect on oocyte maturation or ovulation. Thus, the inhibitory effect of constant low levels of external pH (7.0-7.3) on maturation and ovulation may be primarily due to disruptions in the gonadotropin-dependent acquisition of maturational and ovulatory competencies. The pH of ovarian fluid remained constant at 8.5 during in vivo ovarian follicle maturation and ovulation. Subsequent in vitro tests showed that external pH of 8.5 enhances hormonally induced maturation and ovulation relative to pH of 7.6. These observations suggest that attention should be paid to the pH of incubation media used in basic research and in biotechnological applications relying on in vitro maturation and ovulation in teleosts. Further, an understanding of the physiological significance of the enhancing effect of alkaline pH on maturation and ovulation will require determination of the intrafollicular pH around the oocyte during the periovulatory period.
Animals↗
Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws.
Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37 degrees C. Oocytes were loaded into straws in approximately 2 microL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37 degrees C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342. Maturation of oocytes to MII after thawing was similar (P>0.05) among groups within species. All equine treatment groups had lower (P<0.01) maturation rates than bovine groups. Live/dead status did not differ among vitrification treatments within species. The percentage of oocytes that survived and reached MII did not differ (P>0.05) within treatment groups of each species. Rates of mature cortical granule distribution did not differ (P>0.05) within species; however, more bovine oocytes (P<0.05) had mature cortical granule distribution and nuclear maturation than equine oocytes. When concurrent cortical granule distribution and nuclear maturation were examined, there was no difference within species; however, only 30% of equine oocytes had nuclear and cytoplasmic maturation compared with 70% of bovine oocytes (P<0.05). In summary, both immature and mature equine and bovine oocytes survived cryopreservation using vitrification in open-pulled straws. However, survival rates were lower for equine than for bovine oocytes.
Animals↗
Fertilization and development of Caprine oocytes matured over granulosa cell monolayers.
The aim of the present study was to compare the two oocyte maturation systems, i.e. a granulosa cell (GC) monolayer from small (<4mm) or large (>4mm) follicles and a granulosa co-culture for their effects on in vitro maturation (IVM), fertilization and developmental competence of caprine oocytes. A total of 1945 oocytes were used for studies on maturation, fertilization and embryo development. Monolayers were primed with maturation medium, 18-24h before the onset of maturation. Nuclear studies of 263 fertilized oocytes, 18h post-fertilization, revealed that the rate of sperm penetration was not affected by any of the maturation culture systems. Penetration rate was 66.30% versus 69.59% for the control and GC monolayers. On the other hand, progression of fertilization, i.e. sperm head decondensation (32.70% versus 9.78%) and pronucleus formation (8.76% versus 2.17%) were significantly (P<0.05) enhanced in the oocytes matured over GC monolayers, compared to those with GC co-culture respectively. Cleavage rate was significantly (P<0.05) higher in the oocytes matured and cultured over GC monolayers (27.59%) compared to those in oocytes matured and cultured with the GC co-culture (19.28%). Proportionately more embryos derived from oocytes matured and cultured with the GC co-culture blocked (16.53 and 25.92%) at early developmental stages (2-cell and 4-cell, respectively), compared to those derived from oocytes matured and cultured over GC monolayers (7.61% versus 10.56%; 2-cell versus 4-cell). It was concluded that GC monolayers better support cytoplasmic maturation of growing caprine oocytes, which is evident by a better maturation rate, active fertilization, an improved cleavage rate and subsequently a higher rate of morula formation. Granulosa cells from small and large follicles can be used for IVM and IVC with approximately the same efficiency after conditioning them with maturation medium and embryo development medium 18-24h before the onset of culture.
Journal Article↗
Culture environment modulates maturation and metabolism of human oocytes.
BACKGROUND: The clinical use of oocytes matured in vitro for IVF is increasing, but little is known about the effect of culture conditions on oocyte maturation. METHODS: Denuded immature oocytes identified following superovulation prior to ICSI were individually matured in one of two commercial media: tissue culture medium (TCM) 199 or modified Eagle medium with Earle's modified salts (MEME). During maturation, depletion of pyruvate and accumulation of lactate in the culture medium were non-invasively measured. RESULTS: Maturing oocytes took up pyruvate (20-30 pmol/oocyte/h) and produced lactate (2-10 pmol/oocyte/h). Oocytes matured faster in MEME, with significantly more oocytes reaching metaphase II by 24 h after oocyte retrieval compared with TCM 199 (P = 0.03). The oocytes that matured more quickly in MEME had significantly lower lactate production than oocytes that matured more slowly (P = 0.02). In TCM 199, pyruvate uptake by rapidly maturing oocytes was lower than by slowly maturing oocytes (P = 0.05). During the second incubation, from 24 to 48 h post-oocyte retrieval, pyruvate uptake in MEME was 30% lower than in TCM 199 (P = 0.007). Pyruvate uptake and lactate production differed depending on the stage of nuclear maturation: pyruvate uptake and lactate production were greater during germinal vesicle breakdown than during polar body extrusion in MEME (P < 0.05). CONCLUSIONS: We have shown that: (i). pyruvate is a major energy source during oocyte maturation; (ii). the composition of the culture medium can affect the rate of maturation; and (iii). the culture medium and stage of nuclear maturation can affect pyruvate uptake and lactate production.
Cells, Cultured↗