Hours Of Maturation Research Articles

281 MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE CUMULUS CELLS

Mitogen-activated protein kinase (MAPK) is involved in many signal processes within eukaryotic organisms. Its active form is phosphorylated. For meiotic resumption in oocytes the MAPK cascade plays a central role, because it participates in the transfer of the extracellular gonadotropin signal into the nucleus. In pigs it could be shown that for a gonadotropin-induced germinal vesicle breakdown (GVBD), an activation of MAPK in oocytes is not essential, but in the surrounding cumulus cells the MAPK has to be phosphorylated (Ohashi et al. 2003 Biol. Reprod. 68, 604–609). Because cumulus cells are very important for signal transfer, the present investigation dealt with the relevance of porcine cumulus cells and the phosphorylation of MAPK for the resumption of meiosis. Oocytes of slaughtered pigs were collected and cultured (medium: TCM 199, insulin, l-glutamine, gentamycin, 20% (v/v) FCS, and with or without 2.5 μg/mL FSH and 5.0 μg/mL LH). The proteins of isolated cumulus cells and oocytes were separated by gel electrophoresis (cumulus cells of 10 cumulus-oocyte complexes and 40 oocytes per lane, respectively) followed by an immunoblot with antibodies against MAPK and p90rsk (ERK 1 (sc-94) and Rsk-1 (sc-231), respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Alternatively the nuclear maturation was determined by orceine staining. The following results were achieved: The phosphorylation of MAPK in cumulus cells began very early during the in vitro maturation period. This was demonstrated already after 0.5 h unlike in oocytes where phosphorylation of MAPK does not occur until 18 h. The phosphorylation in cumulus cells occurs both in the presence and in the absence of FSH/LH, but without FSH/LH almost no GVBD occurs (after 26 h IVM: 86.9% GV oocytes, n = 59). The phosphorylation in the absence of gonadotropins could be caused by components of FCS, but with an exchange against polyvinylpyrrolidone (0.3%), the phosphorylation without FSH/LH still existed. The specificity was examined with the MAPK kinase inhibitor U0126. A concentration of 10 μM U0126 prevented GVBD and phosphorylation of MAPK in oocytes. However, in cumulus cells the phosphorylation of MAPK was reduced only minimally. In the presence of 50 μM U0126, a distinct decrease was observed during the first hours of maturation. But after 26 h phosphorylated MAPK appeared in cumulus cells despite the high concentration of U0126. The p90rsk is an important substrate of MAPK, which is phosphorylated by activated MAPK in oocytes. In our investigations we could detect only unphosphorylated forms of p90rsk in the cumulus cells. It seems that there are different ways for phosphorylation of MAPK to occur in cumulus cells, but they do not have the same consequences. The phosphorylation of MAPK in cumulus cells is necessary for a gonadotropin induced meiotic resumption, but phosphorylation does not always lead to GVBD. Furthermore, the p90rsk appears not to have the same importance as a substrate of MAPK in cumulus cells as in oocytes.

Successful healthy birth in vitro maturation of human immature oocytes · Attempt of the unstimulated menstrual cycles and Frozen-Thawed embryo cycles

Objective: The IVM of human oocytes retrieved during unstimulated cycles is a reproductive technology of increasing interest. This study was examined about IVM in the unstimulated menstrual cycles (Fresh cycle) and frozen-thawed embryo transfer in hormone replacement cycles (Frozen cycle).Design: A retorospective randomized studyMaterials/Methods: Immature oocytes were retrieved transvaginally between days 8–23 of unstimulated menstrual cycles from women. Oocytes were cultured in TCM199 medium containing 20% heat-inactivated Follicular fluid, 10IU/ml FSH, 20IU/ml hCG, 10ng/ml Estradiol for 24 hours at 37°C and under 5% CO2, 5% O2, 90% N2 humidified atmosphere. All oocytes matured in metaphase2 were injected with husband sperm, and then cultured in fertilization medium for the first 16–18 hours. Fertilized embryos were changed medium to cleavage medium for 3 days until embryo transfer in Fresh cycle. In Frozen cycle, Zygotes on day 1 after ICSI were frozen, and then the thawed embryos were transferred at 2–8 cells stage with hormone replacement.Results: 19 cycles in Fresh cycle and 9 cycles in Frozen cycle from our program were treatment this study. The mean of oocytes aspirated per patients was 8.9 ± 5.9. After 24 hours of maturation 51.8% (88/170) of oocytes had a visible first polar body. After insemination, 78.4% (69/88) of the oocytes were fertilized and cleavage rate were 97.4% (37/38). The embryos were cultured for 3 days after insemination. Frozen-thawed embryos, 78.6% (22/28) were recovered and cleavage rate were 100% (22/22). The average of embryo transfer was 2.8 ± 1.5 in Fresh cycle, 2.4 ± 1.0 in Frozen cycle. The clinical pregnancy rate of Fresh cycle and Frozen cycle was 23.1% (3/13) and 22.2% (2/9). The implantation rate was 8.1% (3/37) and 13.6% (3/22). One patient delivered a healthy singleton in Fresh cycle and healthy twins in Frozen cycle.Conclusions: This study demonstrates that human immature oocytes retrieved from women with unstimulated menstrual cycles can undergo maturation, fertilization, pregnancy, and that the resulting embryo can establish healthy birth. The hormone replacement cycle is a good method. This method can solve synchronization to embryo and endometrium. Pregnancy rate is never high, but it is big progress for the PCO patients. We confirm that further studies are good oocyte retrieval timing, IVM medium of high maturation rate and synchronization method.Supported by: None. Objective: The IVM of human oocytes retrieved during unstimulated cycles is a reproductive technology of increasing interest. This study was examined about IVM in the unstimulated menstrual cycles (Fresh cycle) and frozen-thawed embryo transfer in hormone replacement cycles (Frozen cycle). Design: A retorospective randomized study Materials/Methods: Immature oocytes were retrieved transvaginally between days 8–23 of unstimulated menstrual cycles from women. Oocytes were cultured in TCM199 medium containing 20% heat-inactivated Follicular fluid, 10IU/ml FSH, 20IU/ml hCG, 10ng/ml Estradiol for 24 hours at 37°C and under 5% CO2, 5% O2, 90% N2 humidified atmosphere. All oocytes matured in metaphase2 were injected with husband sperm, and then cultured in fertilization medium for the first 16–18 hours. Fertilized embryos were changed medium to cleavage medium for 3 days until embryo transfer in Fresh cycle. In Frozen cycle, Zygotes on day 1 after ICSI were frozen, and then the thawed embryos were transferred at 2–8 cells stage with hormone replacement. Results: 19 cycles in Fresh cycle and 9 cycles in Frozen cycle from our program were treatment this study. The mean of oocytes aspirated per patients was 8.9 ± 5.9. After 24 hours of maturation 51.8% (88/170) of oocytes had a visible first polar body. After insemination, 78.4% (69/88) of the oocytes were fertilized and cleavage rate were 97.4% (37/38). The embryos were cultured for 3 days after insemination. Frozen-thawed embryos, 78.6% (22/28) were recovered and cleavage rate were 100% (22/22). The average of embryo transfer was 2.8 ± 1.5 in Fresh cycle, 2.4 ± 1.0 in Frozen cycle. The clinical pregnancy rate of Fresh cycle and Frozen cycle was 23.1% (3/13) and 22.2% (2/9). The implantation rate was 8.1% (3/37) and 13.6% (3/22). One patient delivered a healthy singleton in Fresh cycle and healthy twins in Frozen cycle. Conclusions: This study demonstrates that human immature oocytes retrieved from women with unstimulated menstrual cycles can undergo maturation, fertilization, pregnancy, and that the resulting embryo can establish healthy birth. The hormone replacement cycle is a good method. This method can solve synchronization to embryo and endometrium. Pregnancy rate is never high, but it is big progress for the PCO patients. We confirm that further studies are good oocyte retrieval timing, IVM medium of high maturation rate and synchronization method. Supported by: None.

Fator de crescimento derivado das plaquetas, retinol e insulina na regulação da maturação nuclear de oócitos bovinos e suas conseqüências no desenvolvimento embrionário

O objetivo deste trabalho foi verificar as ações do fator de crescimento derivado das plaquetas (PDGF; P), da insulina (I), do retinol (R) e de suas associações (PI, PIR, IR e PR) na maturação nuclear (MN) de oócitos bovinos e suas conseqüências no desenvolvimento embrionário (DE). O meio básico para maturação dos oócitos nos diferentes tratamentos foi o TCM-199 modificado acrescido de PVA (controle). No DE, foram utilizados os grupos R, PIR, IR, um controle negativo (PVA) e um controle positivo, contendo soro fetal bovino e gonadotrofinas (SFBHOR). Os fatores P, I, R e suas associações não aceleraram a MN em 7h mas sim após 18h (P<0,001), com exceção dos tratamentos R e PR, nos quais as percentagens de metáfase II foram, respectivamente, de 4,7% e 8,3%, similares à obtida no grupo-controle (0,0%). Considerando um nível de significância de P<0,0001 em comparação ao grupo-controle, os maiores índices de metáfase II foram obtidos na presença das associações IR (19,0%) e PIR (21,3%). No DE, R (18,3%), PIR (13,9%) e IR (10,6%) incrementaram os índices de clivagem, comparados ao PVA (0,0%; P<0,001), porém não atingiram os índices do grupo SFBHOR (53,8%; P<0,001). Conclui-se que insulina e PDGF aceleram a MN e suas ações são potencializadas pelo retinol. Os índices de clivagem de oócitos maturados na presença de R, IR e PIR são superiores aos do PVA, mas significativamente inferiores aos maturados em SFBHOR.

Establishment of Pregnancy after the Transfer of Nuclear Transfer Embryos Produced from the Fusion of Argali (Ovis ammon) Nuclei into Domestic Sheep (Ovis aries) Enucleated Oocytes

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation by exposure to ionomycin (5 microM, 4 minutes) followed by incubation in 6-dimethylaminopurine (0.2 mM, 4 hours) and cultured in microdrops of CR1aa medium. From a total of 166 constructed nuclear donor cell-bovine cytoplasm NT couples, 128 (77%) successfully fused, 100 (78%) developed to 8-16 cell stage, and 2 (1.56%) developed to the blastocyst stage. The presence of argali nuclei in 8-16 cell stage embryo clones was confirmed after observation of Hoechst 33342 stained embryos under UV light and chromosome analysis of metaphase spreads from blastomeres. A total of 127 constructed nuclear donor cell-ovine cytoplasm NT couples were produced, 101 (80%) successfully fused, 81 (80% of fused) developed to the 16- to 32-cell stage. A total of 28 hybrid (argali-sheep) and 21 sheep-sheep NT embryos were transferred into 6 recipients and 4 recipients, respectively. Two of these recipients, 1 carrying argali-sheep and 1 sheep-sheep, were confirmed pregnant at 49 days by ultrasound, but both pregnancies terminated by 59 days. The results of this study demonstrate the possibility of using xenogenic oocytes to produce early-stage embryos and pregnancies from an established fibroblast cell line of an endangered species.

In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients

To determine the maturational and developmental competence of immature oocytes recovered in situ from anovulatory and ovulatory patients with polycystic ovaries (PCO). A newly designed method for recovery of immature oocytes from 2 to 10 mm follicles by transvaginal ultrasound or laparoscopy was used to compare the recovery and maturation of oocytes from 9 anovulatory polycystic ovarian syndrome (PCOS) patients and 10 ovulatory patients without polycystic ovaries (PCO) (experiment 1). In a second study (experiment 2), we compared the maturation, fertilization, and development of oocytes recovered from another 10 anovulatory PCOS and 13 ovulatory PCO patients. Two types of culture methods and time intervals for maturation were also examined. In experiment 1, a significantly higher number of immature oocytes were recovered from PCOS patients (15.3) compared with non-PCO patients (2.8). Sixty-five percent of oocytes cultured in medium with gonadotropins, estrogen, and fetal calf serum matured to metaphase II by 43 to 47 hours, and 81% were mature at 48 to 54 hours of culture. Thirty-four percent of the inseminated oocytes fertilized and 56% of the cultured pronuclear oocytes cleaved to eight cells or more. In experiment 2, there was no significant difference between anovulatory PCOS and ovulatory PCO patients in the number of oocytes recovered or their maturation, fertilization, and development. There was no difference between oocytes matured in medium or in coculture with mature granulosa cells, with or without added hCG. However, significantly fewer oocytes were immature and more fertilized when oocytes were inseminated after 34.5 to 35.5 hours of maturation than 29.5 to 32.5 hours of maturation. A pregnancy and the birth of a normal baby occurred in one of the anovulatory PCOS patient receiving an abbreviated steroid replacement protocol after ET. Immature oocyte recovery could be developed as a new method for the treatment of women with infertility due to PCO because the oocytes of these patients retain their maturational and developmental competence.

Parthenogenetic development of activated in vitro matured bovine oocytes

Bovine oocytes matured in vitro for 26 hours were electrically stimulated 1) by a single pulse (Treatment A); 2) by 3 pulses 30 minutes apart (Treatment B); 3) by a single pulse followed by 5 minutes of incubation in the stimulation medium (Treatment C); or 4) by a single pulse at 27 hours of maturation (Treatment D). The oocytes were then cultured for up to 8 days to assess parthenogenetic activation and development. Each electrical stimulation consisted of a 60-μs square wave pulse of 2.5 or 3.6 kV/cm. Treatment A was less effective than the other treatments (P<0.05), activating 47 or 59% of oocytes at 2.5 or 3.6 kV/cm, respectively. However, there were no differences due to voltage nor among the other treatments, which activated 64 to 78% of the oocytes. The cleavage rate, 28 to 38%, was not affected by the activation treatment, but development to the 8-cell stage or beyond was greater after activation with the higher voltage. While the numbers of morulae or blastocysts resulting from any given treatment were too small to support meaningful statistical comparison, the results indicate that bovine parthenogenotes produced in vitro are capable of development to the blastocyst stage.

Signal Transduction and the Regulation of Actin Conformation During Myeloid Maturation: Studies in HL60 Cells

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F-actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F-actin at 48 to 72 hours of maturation. The PKC inhibitor 1-5-isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F-actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F-actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a "mature" response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.

Signal transduction and the regulation of actin conformation during myeloid maturation: studies in HL60 cells

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl- L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F- actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F- actin at 48 to 72 hours of maturation. The PKC inhibitor 1–5- isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F- actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F- actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a “mature” response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.

Age dependence of bovine oocyte activation

The ability of bovine oocytes to undergo parthenogenetic activation using either a Ca++-Mg++-H+ ionophore (A23187) or electric shock was investigated, as a prelude to understanding activation potential following nuclear transfer into ooplasm. Oocytes were collected from slaughterhouse ovaries by aspiration of 1-5-mm follicles. The time of placement into maturation medium was noted, and maturational age (time in culture) measured from that point. After exposure to activating conditions eggs were cultured for a further 12-16 hours, fixed, and stained with aceto-orcein. Oocytes that progressed to telophase or pronuclear formation were considered activated. Concentrations of A23187 ranging from 100 pM to 100 microM showed that 1-100 microM levels resulted in 94-100% activation at 30 hours maturation. Frequency of activation differed from controls (no ionophore) at 100 nM (49%; P less than 0.05). With A23187 maximum response occurred between 26 and 30 hours of maturation (77% and 92%, respectively). A short pulse electric shock, capable of causing oocyte membrane fusion, gave similar results relative to maturational age (82% and 90% activation for 26 and 30 hours, respectively). Therefore, maximum response to the two activating stimuli occurred in oocytes at similar maturational ages. Exposure to activating conditions prior to onset of activating ability (18 hours) followed by another exposure at 26 hours showed that the oocytes were still fully able to activate upon reaching maturational activation competence. Because cytochalasin B is present in the medium used for nuclear transfer, oocytes were incubated with cytochalasin B prior to exposure to an activating stimulus. Frequency of activation was similar to the control treatment (61% and 73%). The effect of mechanical stress of cytoplasm removal and replacement by electrofusion on activation was also not significant. Overall, maturational age of the oocyte was the main determinant of activation ability.

Protein requirements for germinal vesicle breakdown in ovine oocytes

The regulation of the cell cycle during the transition from prophase to metaphase I was studied by analysing protein changes and introducing protein blocks during the transition phase. The results show that the progression to metaphase in ovine oocytes is totally dependent on new protein synthesis. By delaying the addition of the inhibitor, cycloheximide, for progressively longer periods after the resumption of meiosis it was established that the required synthesis occurs in the 1-2 h immediately preceding germinal vesicle breakdown (GVBD). The action of cycloheximide was fully reversible: removal of the drug resulted in GVBD between 3 and 4 h later. The synthesis and modification of proteins during these first few hours of maturation were studied by short-term radiolabelling of oocytes with [35S]methionine and [32P]phosphate followed by rapid assessment of their precise nuclear configuration. Changes in phosphorylation of two polypeptides were detected 4-5 h after the beginning of culture, but these changes were not dependent upon protein synthesis. The earliest change in synthesis was the appearance of a new polypeptide 6-8 h after explantation, immediately before GVBD. This polypeptide (Mr 47 X 10(3), pI 5.8) was not significantly phosphorylated and was relatively stable. Oocytes released from cycloheximide treatment began to synthesize this molecule 3-4 h later, again coinciding with GVBD. Synthesis of the polypeptide was suppressed by inhibition of transcription with alpha-amanitin.

Hormone-induced loss of surface membrane during maturation of starfish oocytes: Differential effects on potassium and calcium channels

Prior to fertilization, starfish oocytes undergo meiotic maturation, triggered by the hormone 1-methyladenine (1-MA). Maturation involves a variety of complex biochemical, morphological, and electrical changes, many of which are similar to those caused by progesterone in vertebrates. Using voltage-clamp and ultrastructural techniques to study maturation in starfish, we have discovered a novel process by which 1-MA alters the electrical properties of the oocyte. The surface area of the oocyte decreases by more than 50% during the first hour of maturation, due to the elimination of microvilli, but the calcium and potassium currents present are affected differently by the loss of membrane. The amplitudes of both the transient K current (“A-current”) and the inwardly rectifying K current decrease, following the time course of the decrease in surface area, while the Ca current amplitude remains virtually unaffected, and may even increase in some oocytes. The kinetics of the currents do not change. This selective removal of K channels results in a larger and more rapidly rising action potential in the mature egg, which may aid in the fast block to polyspermy. The differential accessibility of various ion channels to mechanisms of membrane removal and insertion may play an important role in the development of excitable cells.

Influence of exogenous ions on the events of maturation in Rana pipiens oocytes.

AbstractFull‐grown ovarian oocytes removed from non‐hormone‐treated Rana pipiens females exhibit a low level of protein synthesis, the rate of which is dependent upon the ionic environment. The highest rates of protein synthesis in these oocytes are obtained in media containing either a divalent cation (Ca++ or Mg++) or high levels of K+. The dependence of protein synthesis on ionic environment persists through about the first 18‐24 hours of maturation (at 18°C). Normal maturation of oocytes in vitro also has specific ionic requirements for the first 24 hours. In this case, the process requires high ionic strength (T/2 = 1.0‐1.2) and divalent cations. The kinetics of K+ exchange suggest that K+ exists in the ovarian oocyte in two compartments; one in equilibrium with the exogenous medium and freely exchangeable; the other in equilibrium with the exogenous medium and freely exchangeable; the other in equilibrium with the first internal compartment and only very slowly exchangeable. The slowly exchangeable (bound) compartment contains about 95% of all endogenous K+. In hormone stimulated oocytes, the kinetics of K+ exchange are essentially the same. Oocyte adaptation to ionic environment is discussed as a possible regulatory mechanism during maturation.

Studies on cestode metabolism. II. The utilization of glycogen by Schistocephalus solidus in vitro

1. 1. The amount of endogenous glycogen used by Schistocephalus plerocercoids, when cultured to maturity, has been measured in a range of media and physical conditions. 2. 2. It was found that nutrient was absorbed only when glucose was added to the medium. Starvation occurred in horse serum, salines, and peptone broth. 3. 3. Above a threshold value, below which no absorption occurred, the rate of entry of glucose was independent of the external concentration. 4. 4. Schistocephalus was found to exhibit all those characters usually associated with anaerobic respiration. 5. 5. During the 48 hours of maturation the amount of endogenous glycogen consumed, when worms were cultured in non-nutrient media, was statistically greater under anaerobic than under aerobic conditions. After this time, and also when worms were cultured in glucose media, the presence or absence of oxygen had no demonstrable effect. 6. 6. In all media a decrease in the glycogen reserve occurred and a correlation was observed between longevity and the exhaustion of reserves. The rate of depletion in glucose media however was found to be similar to that measured in vivo.