Influence of culture medium osmolality on maturation and ovulation of common frog oocytes stimulated in vitro by pituitary extract or progesterone

The influence of diluted Ringer solution on ovulation and maturation of common frog oocytes stimulated in vitro by homologous pituitary extract (0.005 pit/ml) or progesterone (1 μg/ml) was studied. During hibernation, the dilution of Ringer solution led to a decreased percentage of oocytes ovulated and matured under the influence of both inducers. As the season of reproduction approached, the dependence of oocyte maturation and ovulation on the Ringer solution dilution was reduced. Possible causes of different dependence of the ovulation of amphibian and sturgeon oocytes stimulated by gonadotropic hormones or progesterone on the culture medium osmolality is discussed.     

Relations Between Hibernation and Ovarian Functions in a Temperate Zone Frog,Rana temporaria

Adult female frogs were exposed to artificial hibernation in a refrigerator, at about 1–3°C, 1–2 months earlier than in nature, isolated or together with males. Ovarian functional state at the beginning of hibernation was assessed from ovarian biopsies taken in mid-September. All frogs ovulated during hibernation which was interrupted in mid-May, but in seven out of 19 frogs ovulation was incomplete. The number of non-ovulated oocytes varied from a minor fraction to about two thirds of the complement of vitellogenic oocytes, indicating that the follicle size reached at the time of induced hibernation was at the critical level for successful maturation. This critical size was about half the normal oocyte volume at the onset of natural hibernation. There was no vitellogenic growth during hibernation, which presumably constitutes the period of follicle maturation. In five of the frogs, oogenesis sensu stricto had occurred during the period of hibernation, some oogenic events being completely finished at autopsy in mid-May and others still progressing.     

Follicle cell regulation of protein synthesis and developmental competence in sheep oocytes

The developmental capacity of sheep oocytes cultured outside the follicle was greatly increased by the presence of high concentrations of gonadotrophins (10 micrograms/ml) in the medium. However, even under these conditions, the developmental capacity of the oocytes was only half that of oocytes cultured within the intact follicle. The presence of the cumulus was essential for development; nearly all denuded oocytes failed to undergo cleavage. Maturational changes in the oocyte involving increased amino acid uptake increased incorporation and specific changes in protein synthesis were inhibited by the follicle cells; this suppression was alleviated by gonadotrophic hormones. The cumulus cells suppressed amino acid incorporation and, to some extent, the changes in protein synthesis. However, the suppression of amino acid uptake required the presence of the whole follicle. Patterns of protein synthesis by oocytes cultured outside the follicle differed from those in oocytes cultured within the follicle, irrespective of the presence of the cumulus or gonadotrophins. Analysis of single oocytes cultured outside the follicle showed that the protein profiles varied markedly even under identical culture conditions.     

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34^cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13^suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34^cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34^cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.     

AMINO ACID TRANSPORT IN PERIPHERAL NERVE: ENERGY AND SODIUM DEPENDENCE

Abstract— The energy and sodium dependence of the several carrier-mediated mechanisms for amino acid uptake have been studied in frog sciatic nerve. The different transport mechanisms are found to be variable in their dependence on sodium and metabolic energy. Saturable uptakes of lysine, phenylalanine and valine are relatively independent of the presence or absence of sodium in the incubation medium, indicating that uptakes by those mechanisms subserving basic, large neutral amino acids, and those amino acids containing aromatic or heterocyclic ring structures are largely sodium independent. Saturable uptakes of glutamic acid, proline, glycine and β-alanine are considerably reduced in the absence of sodium; thus carrier mechanisms for uptake of acidic, small neutral amino acids, β-alanine and proline are highly sodium dependent. The efficacies of several cations in substituting for sodium is variable; greatest inhibitions are found when potassium is used to replace sodium.
With the exception of proline, those mechanisms found to be sodium dependent are also found to be energy dependent, since they are inhibited by both DNP and lowered temperature. Although proline uptake is sodium dependent, proline uptake is stimulated by DNP and relatively insensitive to lowered temperature.     

In vitro inhibition of oocyte and follicular maturation and spawning in starfish (Asterias forbesi) by 2-4-dinitrophenol

The in vitro effects of 2-4-dinitrophenol (DNP) on spawning and follicular and oocyte maturation in starfish ovaries and its various cellular components were investigated. Spawning and oocyte and follicular maturation induced by starfish gonadotropin radial nerve factor (RNF) in isolated ovarian fragments were all inhibited by appropriate doses of DNP. DNP inhibits processes which occur shortly after addition of the gonadotropin; in ovarian fragments insensitivity to DNP inhibition occurred shortly after addition of RNF but prior to initiation of spawning. Spontaneous follicular and oocyte maturation which occurred following release of ovarian follicles into sea water was prevented by DNP. In non-spontaneously maturing follicles released from the ovary, DNP inhibited both follicle and oocyte maturation induced by the secondary stimulator of spawning and maturation, 1-methyladenine (1-MA). DNP also inhibited 1-MA induced meiotic maturation in isolated immature oocytes incubated in the absence of follicle cells. Inhibition of oocyte maturation was not associated with inhibition of 3H-1-MA incorporation by isolated oocytes. Immature oocytes incubated in the presence of DNP underwent maturation following washing and subsequent exposure to 1-MA. Immature oocytes initially exposed to both 1-MA and DNP, however, showed decreased maturation responsiveness following washing and re-exposure to 1-MA. The results suggest that the inhibitory effects of DNP on spawning and oocyte maturation are the result of direct effects on the oocytes and possibly other cells and tissues within the ovary.     

In vitro Inhibition of Oocyte and Follicular Maturation and Spawning in Starfish (Asterias forbesi) by 2-4-Dinitrophenol

The in vitro effects of 2-4-dinitrophenol (DNP) on spawning and follicular and oocyte maturation in starfish ovaries and its various cellular components were investigated. Spawning and oocyte and follicular maturation induced by starfish gonadotropin radial nerve factor (RNF) in isolated ovarian fragments were all inhibited by appropriate doses of DNP. DNP inhibits processes which occur shortly after addition of the gonadotropin; in ovarian fragments insensitivity to DNP inhibition occurred shortly after addition of RNF but prior to initiation of spawning. Spontaneous follicular and oocyte maturation which occurred following release of ovarian follicles into sea water was prevented by DNP. In non-spontaneously maturing follicles released from the ovary, DNP inhibited both follicle and oocyte maturation induced by the secondary stimulator of spawning and maturation, 1-methyladenine (1-MA). DNP also inhibited 1-MA induced meiotic maturation in isolated immature oocytes incubated in the absence of follicle cells. Inhibition of oocyte maturation was not associated with inhibition of ³H-1-MA incorporation by isolated oocytes. Immature oocytes incubated in the presence of DNP underwent maturation following washing and subsequent exposure to 1-MA. Immature oocytes initially exposed to both 1-MA and DNP, however, showed decreased maturation responsiveness following washing and re-exposure to 1-MA. The results suggest that the inhibitory effects of DNP on spawning and oocyte maturation are the result of direct effects on the oocytes and possibly other cells and tissues within the ovary.     

Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro

Fully grown germinal vesicle-stage oocytes are induced to resume meiosis and acquire the capacity to undergo fertilization in response to a surge of gonadotropins. The present study examined possible direct and indirect roles of gonadotropins in the maturation and fertilization of rat oocytes by determining 1) the effect of exogenous administration of gonadotropins (priming) to immature rats prior to oocyte collection on the capacity of oocytes to undergo maturation and fertilization in vitro, 2) the effect of follicle-stimulating hormone (FSH) in the maturation media on the resumption of meiosis and subsequent capacity of oocytes to undergo fertilization, and 3) the capacity of oocytes to undergo maturation and fertilization following culture in preovulatory follicular fluid or in conditioned media obtained from gonadotropin-stimulated granulosa cell (GC) cultures. In the first experiment, oocytes from unprimed rats underwent spontaneous meiotic maturation in vitro and 17% underwent subsequent fertilization. Priming increased the proportion of oocytes undergoing fertilization. Maturation of oocytes in media supplemented with various concentrations of FSH or for various lengths of time (6-16 h) in medium with 500 ng FSH/ml indicated that FSH slowed the rate of meiotic maturation, but had no effect on the capacity of the oocytes to be fertilized. Oocytes obtained from primed animals and cultured in the presence of preovulatory follicular fluid were fertilized in proportions similar to those cultured in serum-containing medium. In the third experiment, medium conditioned by FSH-stimulated GC for 40 h slowed the rate of meiotic maturation; the addition of luteinizing hormone (LH) to the FSH-stimulated cells produced a medium in which the rate of oocyte maturation was not different from that of control oocytes (in medium from unstimulated cells). Medium conditioned by FSH- or LH-stimulated GC, but not fibroblasts, increased the proportions of oocytes undergoing fertilization following maturation in those media. FSH + LH stimulation of GC increased the fertilization of oocytes to proportions significantly higher than with either gonadotropin alone. These data suggest that GC respond to gonadotropin stimulation by providing a factor(s) that regulates the rate of oocyte maturation and promotes the capacity of oocytes to undergo fertilization.     

Pitfalls in using sodium hypochlorite as a seed disinfectant in C incorporation studies

Seeds sterilized with sodium hypochlorite (NaOCl) retained sufficient amounts to interfere with studies of amino acid metabolism of the sterilized seeds during germination. Repeated washing in water did not remove NaOCl completely. However, soaking the seeds for 10 min in 0.01 n HCl removed NaOCl completely, without reducing germinability.Residual NaOCl reacted with the amino acids and reduced their concentrations in the incubation media. This reaction resulted in high production of CO(2) and low uptake of amino acids by the seeds. Decarboxylation of the amino acids occurred in the incubation medium outside the seed, was independent of the presence of seeds in the reaction, and therefore was not related to amino acid metabolism by the seeds. Effects of NaOCl on uptake, incorporation, and CO(2) production from indoleacetic acid were similar to those of the amino acids studied.     

Translational capacity of sheep oocytes microinjected with messenger RNA

Sheep oocytes were microinjected with tobacco mosaic virus RNA (TMV-RNA) and isotopically labelled with L-[35S]methionine. Total incorporation of labelled methionine was similar in TMV-RNA-injected and in carrier-injected control oocytes, whether injections were performed during the period of high protein synthesis at maturation or during the period of reduced synthesis at a time equivalent to the mid-cleavage transition (48 h after germinal vesicle breakdown). Varying the amount of TMV-RNA injected from 2.5 to 10 pg had little effect on the overall level of amino acid incorporation. Furthermore TMV-RNA appeared to be very stable in oocytes and eggs; the proportion of total polypeptide synthesis directed by TMV-RNA did not diminish during the first 48 h after injection. Synthesis of most endogenous proteins was uniformly reduced to compensate for the synthesis of TMV-polypeptides. Our results suggest, therefore, that the translational capacity of sheep oocytes is fully saturated during maturation.     

Metabolic requirements associated with GSH synthesis during in vitro maturation of cattle oocytes

Glutathione (GSH) concentration increases in bovine oocytes during in vitro maturation (IVM). The constitutive amino acids involved in GSH synthesis are glycine (Gly), glutamate (Glu) and cysteine (Cys). The present study was conducted to investigate the effect of the availability of glucose, Cys, Gly and Glu on GSH synthesis during IVM. The effect of the amino acid serine (Ser) on intracellular reduced/oxidized glutathione (GSH/GSSG) content in both oocytes and cumulus cells was also studied. Cumulus-oocyte complexes (COC) of cattle obtained from ovaries collected from an abattoir were matured in synthetic oviduct fluid (SOF) medium containing 8 mg/ml bovine serum albumin-fatty acid-free (BSA-FAF), 10 microg/ml LH, 1 microg/ml porcine FSH (pFSH) and 1 microg/ml 17 beta-estradiol (17beta-E2). GSH/GSSG content was measured using a double-beam spectrophotometer. The COC were cultured in SOF supplemented with 1.5mM or 5.6mM glucose (Exp. 1); with or without Cys+Glu+Gly (Exp. 2); with the omission of one constitutive GSH amino acid (Exp. 3); with 0.6mM Cys or Cys+Ser (Exp. 4). The developmental capacity of oocytes matured in IVM medium supplemented with Cys and the cell number per blastocyst were determined (Exp. 5). The results reported here indicate (1) no differences in the intracellular GSH/GSSG content at any glucose concentrations. Also, cumulus cell number per COC did not differ either before or after IVM (Exp. 1). (2) Glutathione content in oocytes matured in SOF alone were significantly different from oocytes incubated with SOF supplemented with Cys+Glu+Gly (Exp. 2). (3) Addition of Cys to maturation medium, either with or without Gly and Glu supplementation resulted in an increase of GSH/GSSG content. However, when Cys was omitted from the IVM medium intracellular GSH in oocytes or cumulus cells was less but not significantly altered compared to SOF alone (Exp. 3). (4) Glutathione content in both oocytes and cumulus cells was significantly reduced by incubation with 5mM Ser (Exp.4). (5) There was a significant increase in cleavage and blastocyst rates when Cys was added to maturation medium. In contrast, the cleavage, morula and blastocyst rates were significantly different when 5mM Ser was added to maturation media. There was also a significant difference in mean cell number per blastocyst, obtained from oocytes matured with 5mM Ser (Exp. 5). This study provides evidence that optimal embryo development in vitro is partially dependent on the presence of precursor amino acids for intracellular GSH production. Moreover, the availability of Cys might be a critical factor for GSH synthesis during IVM in cattle oocytes. Greater Ser concentration in IVM medium altered "normal" intracellular GSH in both oocytes and cumulus cells with negative consequences for subsequent developmental capacity.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

Involvement of cAMP-Dependent protein kinase and intracellular free calcium ions in regulation of ovulation of the follicle-enclosed oocytes ofRana temporaria by gonadotropic hormones

The inhibitor of cAMP-dependent protein kinase N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (H-8) (12.5–50 μM) decreased the rate of ovulation of the follicle-enclosedRana temporaria oocytes induced by the homologous pituitary extract in amphibian Ringer solution and in a chloride-free medium. The inhibitor of voltage-dependent calcium channels diltiazem (10 and 100 μM) decreased the rate of ovulation in Ringer solution but did not affect it in a chloride-free medium or decreased the ovulation inhibitory effect of this medium. It was concluded that cAMP-dependent protein kinase and intracellular free calcium ions were involved as second messengers in the gonadotropin regulation not only in maturation of amphibian oocytes but also in ovulation.     

Role of protein kinase A and calcium ions in regulating ovulation of oocytes by gonadotropins in the grass frog (Rana temporaria) in vitro

The inhibitor of cAMP-dependent protein kinase N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) (12.5-50 microM) decreased the rate of ovulation of the follicle-enclosed Rana temporaria oocytes induced by the homologous pituitary extract in amphibian Ringer solution and in a chloride-free medium. The inhibitor of voltage-dependent calcium channels diltiazem (10 and 100 microM) decreased the rate of ovulation in Ringer solution but did not affect it in a chloride-free medium or decreased the ovulation inhibitory effect of this medium. It was concluded that cAMP-dependent protein kinase and intracellular free calcium ions were involved as second messengers in the gonadotropin regulation not only in maturation of amphibian oocytes but also in ovulation.     

The effects of bioregulators upon amino acid transport and protein synthesis in isolated rat hepatocytes

Isolated rat hepatocytes prepared by an enzyme perfusion technique possess a functional amino acid transport system and retain the capacity to synthesize protein. Amino acid transport was studied using the non-metabolizable amino acid analog alpha-aminoisobutyric acid. The transport process was time, temperature and concentration dependent. Similarly, leucine incorporation into protein was time and temperature dependent being optimal at 3m degrees C. Amino acid, fetal calf serum, growth hormone and glucose all produced small, reproducible increases in protein synthesis rates. Bovine serum albumin diminished the uptake of alpha-aminoisobutyric acid and leucine incorporation into protein. The amino acid content on either side of the cell membrane was found to affect transport into or out of the cellular compartment (transconcentration effects). High cell concentrations decreased transport and protein synthesis as a result of isotopic dilution of labelled amino acids with those released by the hepatocytes. This was consistent with the capacity of naturally occurring amino aicds to compete with alpha-aminoisobutyric acid for uptake into the hepatocyte. In order to define more precisely the effects of bioregulators on transport and protein synthesis it will be necessary to define and subfractionate cellular compartments and proteins which are the specific targets of cellular regulation.     

Biochemical research on oogenesis: protein synthesis in whole cells and in cell-free extracts of Xenopus laevis immature ovaries

Nearly all tRNA molecules in previtellogenic oocytes of Xenopus laevis are included in nucleoprotein particles sedimenting at 42S. The tRNA-binding sites of these particles have several properties in common with those of the ribosomes. This suggests that the 42S particles might behave like unprogrammed ribosomes and be the site of a template-independent polymerization of amino acids. We expected this reaction to be insensitive to protein synthesis inhibitors, such as cycloheximide and puromycin. We found that these antibiotics almost completely inhibit the incorporation of labeled amino acids into protein, when added to the incubation medium of whole ovaries or free oocytes. In cell-free extracts of ovaries, the incorporation of amino acids is partially insensitive to cycloheximide and puromycin. When such extracts are fractionated by sucrose density centrifugation and incubated with ATP, a major peak of amino acid incorporation can be detected, which nearly coincides with the 42S particle peak.     

Amino acid uptake and protein synthesis in rat testes: stimulation by dissociable factors

Low concentration (25 mM) of sodium in the incubation medium produced a decrease in the amino acid uptake by the testis tissue as well as a reduction in the response to FSH. In this experimental condition, the basal protein synthesis and the stimulatory effect of FSH was not modified. The subcutaneous administration of testosterone to 15 day old rats increased the protein synthesis in the testis without any modification in the amino acid uptake. The addition of DBcAMP (1 mM) or glucose (14 mM) to the incubation medium increased the protein synthesis in the testes of immature (12 day-old) or prepubertal (32 day-old) rats respectively. The amino acid uptake was not modified. In immature rat testes, with protein synthesis completely inhibited by cycloheximide, the restoration of the sodium concentration in the incubation medium to normal levels produced an increase in amino acid uptake. The results above seem to indicate that protein synthesis and amino acid uptake in rat testes tissue can be regulated, at least partially, by different factors.     

Influence of culture medium osmolality on maturation and ovulation of Rana temporaria oocytes stimulated in vitro by the pituitary gland extract or progesterone

The influence of diluted Ringer solution on ovulation and maturation of common frog oocytes stimulated in vitro by homologous pituitary extract (0.005 pit/ml) or progesterone (1 pg/ml) was studied. During wintering, the dilution of Ringer solution led to a decreased percentage of oocytes ovulated and matured under the influence of both inducers. As the season of reproduction approached, the dependence of oocyte maturation and ovulation on the Ringer solution dilution weakened. Possible causes of different dependence of the ovulation of amphibian and sturgeon oocytes stimulated by gonadotropic hormones or progesterone on the culture medium osmolality is discussed.     

In-vivo and in-vitro maturation rate of oocytes from two strains of mice

Female mice of the KE and CBA strains were used to examine the rate of oocyte maturation in vivo and in vitro. In CBA females killed just before ovulation most preovulatory oocytes were already in the metaphase II stage, while the oocytes of KE mice were arrested at metaphase I until the time of ovulation, and further stages of maturation occurred in the oviduct, reaching the metaphase II stage 3-5 h later. A similar strain difference in oocyte maturation rate was observed from in-vitro culture of cumulus-free oocytes, isolated from the ovaries of PMSG-primed females and intact females killed at the metoestrous phase of the cycle. This indicates that the strain-specific course of maturation is determined in the oocyte by a few days before ovulation. Therefore, if the rate of oocyte maturation is influenced by somatic components of the follicle, this must occur at some earlier stages of follicle development.