Effect of follicle-stimulating hormone on cyclic adenosine monophosphate level and on meiotic maturation in mouse cumulus cell-enclosed oocytes cultured in vitro

We have reported that in vitro treatment with follicle-stimulating hormone (FSH) delays by about 3 h spontaneous meiotic resumption in cumulus cell-enclosed mouse oocytes. In the present paper we show that the temporary meiotic block is accompanied by a transient increase of cAMP concentration in the oocyte. In cumulus cell-oocyte complexes stimulated with 1 microgram/ml FSH, cAMP significantly increases within 1 h both in the whole complex (from a basal value of 1.9 +/- 0.2 to 169 +/- 13 fmol) and in the enclosed oocyte (from 0.9 +/- 0.2 to 2.4 +/- 0.2 fmol), then progressively decreases to basal values. Stimulation by FSH does not cause any cAMP increase in denuded oocytes. As the concentration of cAMP in the cells decreases, the percentage of oocytes escaping the meiotic block imposed by FSH increases. If the complexes are cultured in the presence of 1 microgram/ml FSH plus 1 mM isobutyl-1-methylxanthine (1BMX), cAMP concentration increases approximately 250-fold in the complex, and 10-fold in the enclosed oocyte; the level of cAMP in the oocyte drops very rapidly (50% degradation in less than 2 min) if the oocyte is then transferred to IBMX-free medium. The data are discussed in terms of the possible role of cAMP transfer from cumulus cells to the oocyte in the regulation of meiotic progression in mouse oocytes.     

Early preantral mouse follicle in vitro maturation: oocyte growth, meiotic maturation and granulosa-cell proliferation

Mechanically isolated early preantral mouse follicles were cultured singly for 16 d and fully grown oocytes were obtained from these follicles. We then compared in vitro and in vivo follicle growth by trypsinising the follicles and counting their cell numbers in a Neubauer-counting chamber and recording the diameter and meiotic status of oocytes under an inverted microscope. As long as the granulosa cells were within the basal membrane, proliferation was slow. From Day 6, when granulosa cells had broken through the basal membrane, the proliferation rate progressed up to Day 10 and decreased thereafter to approximately 12,000 cells per culture droplet. Incorporation of BrdU revealed that proliferating cells were evenly distributed throughout the follicle until antrum formation. As granulosa cell differentiation progressed, proliferation of mural-granulosa cells ceased, while cells around the oocytes continued dividing. Oocyte diameter increased discontinuously in relation to follicle remodelling. During the first growth phase, diameters increased from 56.5 (+/- 4.4 microns) to 67 (+/- 4.1 microns) until the onset of antral-like cavity formation. The last growth phase started after Day 10, and by Day 14 oocyte diameters were not significantly different from those of 26-d-old in vivo control oocytes. The potential to resume meiosis after mechanical removal of granulosa cells was first reached on Day 8; thereafter, removal of the corona showed that all oocytes cultured with FSH remained arrested at the GV stage up to Day 16. After Day 8, approximately 70% of all oocytes underwent GVBD as a result of granulosa-cell removal, but only 23% of these reached MII after 24 h. The in vivo controls reached a comparable GVBD rate (66%) when the granulosa was removed, but most of the oocytes (82%) underwent first polar body extrusion 24 h later. These results suggest that although oocyte diameters after IVM are not different from those of the controls, culture conditions are not yet adequate to support complete meiotic maturation.     

Effect of gonadotrophin environment on growth and development of isolated mouse primary ovarian follicles

Collagen gels containing isolated primary follicles devoid of other ovarian tissue were transferred beneath the kidney capsule of 3 types of female recipients: cycling, ovariectomized and hypogonadal, known to have different circulating concentrations of gonadotrophins. After 10 days the gels were recovered and processed for histology or the oocytes were recovered and their diameters measured and their ability to resume meiosis was determined. The growth of isolated primary follicles was positively correlated with the concentrations of circulating gonadotrophins in the recipient mice, but the numbers of oocytes recovered, the rate of oocyte growth and resumption of meiosis did not differ in the 3 types of recipient studied. This indicates that, in the conditions provided, oocyte growth was not related to the extent of follicular development.     

Role of cyclic adenosine monophosphate (cAMP) in vitro on bovine oocyte maturation

This experiment attempted to determine the effect of cAMP on maturation of bovine oocytes in chemically-defined, serum-free medium. Cumulus-oocyte complexes were incubated in modified DME/Ham F-12 medium containing dbcAMP at 0 (control), 10(-6), 10(-4) and 10(-2) M. After 18 and 24 hours of culture, the percentage of oocyte maturation between 0 (control) and 10(-2) M dbcAMP-treated groups were significant. Some oocytes were cultured with dbcAMP (10(-2) M) for 6, 12 and 24 hours followed by incubation in control medium to test the reversibility of inhibition or of any harmful effect of dbcAMP. The inhibitory effect of 10(-2) M dbcAMP on bovine oocyte maturation was reversed by transferring cumulus-oocyte complexes to the control medium. In addition, forskolin (0.12 and 0.24 mM) was effective (P < 0.01) in preventing the resumption of meiosis. The cAMP content of oocytes cultured with forskolin was not increased, although cumulus cells responded to forskolin with an increase in cAMP content. These results indicate that elevated levels of cAMP in the culture medium are important in regulating resumption of meiosis of bovine oocytes in vitro.     

Estrogen synthetase in choriocarcinoma cell culture. stimulation by dibutyryl cyclic adenosine monophosphate and theophylline

The stimulation of estrogen biosynthesis by N⁶, O² -dibutyryl adenosine 3':5'-cyclic monophosphate and theophylline (dbT) in cultures of the JAr line of choriocarcinoma cells was investigated by measuring the specific activity and kinetic constants of estrogen synthetase (aromatase) in the various subcellular fractions after differential centrifugation of homogenized cells in isotonic sucrose. The low speed (900x$\text{g}$) pellet,from cells grown with or without dbT and homogenized in isotonic sucrose,contains the majority of the aromatase activity and the highest aromatase specific activity. The aromatase specific activity in the homogenate of cells grown with dbT and in the various subcellular fractions is 4- to 10-fold higher than in cells grown without dbT. The Vmax of androstenedione (4-androstene-3,17-dione) aromatization in homogenates from dbT-stimulated cells (6.9 pmol estrogen/min per mg protein) is significantly increased over that measured in the absence of dbT (1.5 pmol estrogen/min per mg protein); the Km values, however, are not significantly different (average of 43.8nM in dbT-stimulated fractions; 53.2nM in control fractions). These results suggest that the increased aromatase specific activity in dbT-stimulated cells results from an increase in amount of active enzyme, rather than from an increase in affinity of the enzyme for its substrate.     

Vasoactive intestinal peptide stimulates oocyte maturation, steroidogenesis, and cyclic adenosine 3',5'-monophosphate production in isolated preovulatory rat follicles

Vasoactive intestinal peptide (VIP) is present in the rat ovary and has been shown to stimulate cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone production in cultured rat granulosa cells. In the present study, VIP-stimulated cAMP production has been studied in relation to steroid accumulation and oocyte maturation in isolated preovulatory rat follicles. VIP stimulated resumption of meiosis (oocyte maturation) in up to 60% of the follicle-enclosed oocytes after 6 h at 1 microM (control, 1.8%; luteinizing hormone 99%). The effect was time- and dose-dependent up to 6 h and was seen with both natural and synthetic VIP. VIP also stimulated the accumulation of steroids (estrogen, 2.3-fold; testosterone, 2.0-fold; and progesterone, 1.6-fold increase after 6 h of incubation) and lactate (2.6-fold) by the follicles. VIP-increased tissue levels of cAMP in the follicle were dose- and time-dependent. This effect was potentiated by a phosphodiesterase inhibitor. When isolated oocyte-cumulus complexes were studied, VIP caused a transient inhibition of spontaneous oocyte maturation, and demonstrated no effect on denuded oocytes. These results extend earlier preliminary observations on the ability of VIP to induce meiotic maturation of follicle-enclosed oocytes. Our results also show that VIP can stimulate steroid and lactate accumulation in the isolated follicles. The pattern of steroids produced suggests an effect both on the theca- and granulosa cells. We also show that VIP can delay spontaneous oocyte maturation. These effects appeared, at least partially, to be mediated by cAMP.     

Effect of dibutyryl cyclic adenosine monophosphate on active amino acid transport in Streptomyces hydrogenans

The active uptake of 2-aminoisobutyric acid (AIB) and several other amino acids in resting cells of Streptomyces hydrogenans was found to be stimulated by exogenously added adenosine cyclic monophosphate (cAMP). The uptake of glycerol, sorbose, and pyrimidine nucleosides remained unaffected. Among the various cAMP derivatives tested, the dibutyryl derivative was found to be most effective, followed by monobutyryl cAMP, and cAMP. Dibutyryl cGMP was also found to stimulate AIB transport, and its effectivity was as good as that of dibutyryl cAMP. The effect of dibutyryl cAMP is time dependent and attains its maximum after 40–60 min of incubation at 30°C in K-Na-phosphate buffer. Dibutyryl cAMP-dependent transport stimulation has a high temperature coefficient and is prevented by rifamycin SV or chloramphenicol. The rate of leucine incorporation into protein was rapidly increased upon addition of dibutyryl cAMP. Kinetic studies reveal that the stimulation of AIB transport is characterized by an increase in maximum uptake rate and an unaltered apparent Michaelis constant. Analysis of the unidirectional fluxes show that both influx and efflux are enhanced by dibutyryl cAMP. It is concluded that exogenous dibutyryl cAMP stimulates de novo synthesis of certain protein including the transport catalysts for various amino acids.     

Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro

Described in the present paper is a culture system that preserves oocyte and granulosa cell morphology in bovine preantral follicles during 5 d in vitro. The effects of additional hypoxanthine and energy substrata (i.e., pyruvate and glutamine) on the morphology of cultured preantral follicles were investigated. It was shown that addition of a mixture of pyruvate, glutamine and hypoxantine to the culture medium increased the percentage of follicles with an intact oocyte from 29.4 to 78.6%. Morphological criteria are described to discriminate between normal and degenerated preantral follicles during culture by inverted microscopy. In addition, the importance of histological evaluation to judge the quality of oocyte and granulosa cells is demonstrated.     

Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.     

Differential effects of testosterone and dibutyryl cyclic AMP on the meiotic maturation of mouse oocytes in vitro

Fully grown, meiotically immature mouse oocytes were isolated and cultured under varying conditions with the aim of determining a) whether the inhibitory effects of testosterone on oocyte meiotic maturation require the synthesis of new oocyte proteins and b) if the meiosis-inhibiting effects of testosterone and dibutyryl cyclic AMP (dbcAMP) are distinct and can be differentiated. We found that the inclusion of puromycin in culture medium containing testosterone has no effect on the meiosis-inhibiting potency of testosterone or upon the reversibility of testosterone effects. We conclude that testosterone inhibits oocyte meiosis by a mechanism that is independent of protein synthesis. We also found that oocytes exposed to testosterone recover more rapidly, as evidenced by the timing of germinal vesicle breakdown (GVBD) following placement in a control medium, than do oocytes exposed to dbcAMP. Through further investigation of this phenomenon we have determined the sequence of testosterone and dbcAMP effects relative to the time course of GVBD. A testosterone-sensitive event occurs 20 min prior to GVBD, while the dbcAMP-sensitive event precedes GVBD by 41 min. The nature of this difference may involve the differential interaction of testosterone and dbcAMP with a set of puromycin-sensitive proteins that are required for GVBD. When oocytes were initially cultured in medium containing both puromycin and either testosterone or dbcAMP and then moved to medium containing puromycin alone the incidence of GVBD was reduced relative to oocytes never exposed to puromycin. This observation suggests that mouse oocytes contain proteins that are required for GVBD and that experience a high turnover rate. The degree of reduction in GVBD was a function of the length of puromycin exposure and was significantly greater in dbcAMP- than in testosterone-exposed oocytes. If oocytes were initially cultured in medium containing puromycin and dbcAMP, the rate of GVBD upon removal of dbcAMP was initially slow but increased with time. This observation is consistent with the hypothesis that dbcAMP inhibits oocytes at a point prior to the functioning of the puromycin-sensitive proteins. However, if oocytes were cultured in medium containing puromycin and testosterone the rate of GVBD following testosterone removal was not significantly reduced relative to oocytes that were not exposed to puromycin. This observation suggests that testosterone acts to inhibit meiosis at a site beyond the function of the puromycin-sensitive proteins or that testosterone causes a reduction in the turnover rate of these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral follicles.

During the development of oocyte-granulosa cell complexes from preantral follicles in vitro, oocytes grow and acquire competence to undergo germinal vesicle breakdown (GVB). In the culture system used here, GVB-competent oocytes were maintained in meiotic arrest solely by endogenous physiological mechanisms of the granulosa cells without supplementation with meiosis-arresting substances. Addition of mycophenolic acid, an inhibitor of inosine monophosphate (IMP) dehydrogenase, induced GVB in about 70% of the GVB-competent oocytes grown in vitro. The mechanism for meiotic arrest in this system is, therefore, similar to that for arrest in vivo insofar as it requires the participation of the IMP dehydrogenase pathway. Rp-cyclic adenosine monophosphothioate, a membrane-permeable antagonist to cAMP, induced GVB by about 30% of the competent oocytes. Cyclic AMP-dependent pathways, therefore, participate in the physiological mechanism by which mouse granulosa cells maintain meiotic arrest. Complexes were grown for 10 days in medium containing 0, 1, 5, or 10 ng/ml FSH, were stimulated with either 1 microgram/ml FSH or LH, and were assessed for GVB and cumulus expansion. GVB was stimulated by FSH whether or not the complexes were grown in medium containing FSH, but LH or hCG induced GVB only when the complexes were grown in medium containing FSH. Cumulus expansion occurred in response to either FSH or LH only when complexes were grown in medium containing FSH. FSH, therefore, promotes the differentiation of granulosa cells from preantral follicles in vitro so that LH can stimulate GVB and cumulus expansion.     

Effect of osmotic pressure, ionic strength and dibutyryl cyclic adenosine monophosphate on the adhesion of hen erythrocytes.

In the presence of lysolecithin at physiological pH it was found that the increase of ionic strength facilitates the adhesion of hen erythrocytes. In this medium, dibutyryl cyclic adenosine monophosphate (DBcAMP) increases the adhesion index of the cells. If the osmotic pressure is elevated without a proper increase of ionic strength, the lysolecithin induced hemolysis and adhesion are found to be lacking.     

Effect of adiponectin on bovine granulosa cell steroidogenesis,oocyte maturation and embryo development

Background  

Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor) in bovine ovary and its role on ovarian cells and embryo, remain however to be determined.     

雷公藤多甙对小鼠卵母细胞成熟和体外受精的影响

采用超排卵技术研究雷公藤多甙（GTW）对小鼠卵母细胞的成熟和体外受精以及脏器等的影响,GTW对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。GTW可以破坏卵母细胞成熟,降低卵母细胞的体外受精能力,影响小鼠的正常生殖功能。     

Effects of dibutyryl cyclic AMP on oocyte maturation and ovulation in the perfused rabbit ovary

The involvement of cyclic AMP (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and in-vitro perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without dibutyryl cyclic AMP [Bu)2cAMP) at 10(-3), 10(-4) or 10(-5) M for 4-12 h. At 4 h spontaneous meiotic maturation was significantly inhibited by (Bu)2cAMP (P less than 0.025). With prolonged incubation, spontaneous maturation progressed despite exposure to (Bu)2cAMP. When ovaries were continuously perfused in vitro for 12 h with (Bu)2cAMP (10(-3) M) or medium alone, (Bu)2cAMP stimulated ovarian progesterone production, but did not affect ovulation or maturation of follicular oocytes. When ovaries were perfused in vitro with or without (Bu)2cAMP (10(-3), 10(-4) or 10(-5) M) for the first 2 h and then transferred to medium without (Bu)2cAMP for an additional 10 h, ovulation did not occur, but transient exposure to (Bu)2cAMP stimulated a dose-related increase in maturation of follicular oocytes. Degeneration of follicle-enclosed oocytes and cumulus-oocyte complexes was not affected by exposure to (Bu)2cAMP. These results suggest that transient, but not continuous, elevation of cAMP after the gonadotrophin surge may be required for the initiation of oocyte maturation.     

Induction of alkaline phosphatase activity by synergistic action of dibutyryl cyclic adenosine monophosphate, prednisolone, butyrate and sodium chloride in cultured cells

Human urinary bladder carcinoma cells (JTC-32) retain a low alkaline phosphatase activity. Prednisolone or a hypertonic concentration of NaCl caused a moderate increase in the activity (10- to 15-fold of control), but dibutyryl cAMP or butyrate did not. Examination of the combined effect of these four agents revealed that they acted synergistically in any combination. When the cells were incubated with the four agents together, the enzyme activity increased 60- to 250-fold. Serum also contributed to this synergistic increase. These agents slightly inhibited cell growth and protein synthesis. The enzyme induction was completely inhibited by cycloheximide or actinomycin D. The synergistic effect of the four agents on the enzyme activity was also observed in other strains of carcinoma cells, human urinary bladder carcinoma cells (JTC-30) and monkey hepatocarcinoma cells (NCLP-6E). Thus, it is concluded that the coexistence of the four agents provides general and superior conditions for the induction of alkaline phosphatase in cultured carcinoma cells.     

Calcium effects on progesterone accumulation and oocyte maturation in cultured follicles of Rana pipiens

Pituitary homogenates (FPH) provoke a cascade of responses in the amphibian ovarian follicle, culminating in progesterone biosynthesis and oocyte maturation (GVBD). Calcium may play an important role as an intracellular second messenger in regulating these physiological responses. Experiments were carried out on cultured, isolated follicles of Rana pipiens to assess the effects of varying extracellular calcium on follicular progesterone accumulation and oocyte maturation. In hormonally unstimulated follicles, an increase in extracellular Ca2+ alone produced a significant increase in progesterone in methanol extracts of follicles after 4 hours of culture, and in some cases also provoked oocyte maturation assessed after 24 hours of culture. In no case did elevated Ca2+ alone stimulate maximal progesterone accumulation as compared with FPH-stimulated follicles, although the time-course of accumulation was similar. The calcium ionophore, A-23187, similarly increased progesterone accumulation in a dose-dependent manner when introduced in amphibian Ringer's (1.35 mM Ca2+), but inhibited progesterone elevation caused by increasing calcium concentrations in the culture media and FPH stimulation. Depleting free calcium from the culture medium with graded doses of the chelator EGTA decreased FPH-induced progesterone accumulation and inhibited FPH- and progesterone-induced GVBD. The calcium channel blocker, verapamil, also inhibited FPH-induced progesterone accumulation and GVDB in a dose-dependent manner, while having no effect on progesterone-induced meiotic resumption. These data strongly implicate intracellular calcium levels regulating progesterone production by ovarian follicle cells and subsequent oocyte maturation.     

Effect of hypophysectomy on mouse oocyte maturation in vitro.

There was no difference in frequency of maturation of oocytes obtained from mice hypophysectomized for 2 weeks compared to those from sham-operated or untreated (control) animals of the same age. By 7 weeks, and also at 12 and 17 weeks, the incidence of polar body formation in vitro was significantly reduced. The number of oocytes which remained meiotically inactive in culture was increased at 7, 12 and 17 weeks after hypophysectomy. This decrease in spontaneous oocyte maturation in vitro could be partly overcome by administering exogenous PMSG, oestradiol-17beta or PMSG + oestradiol-17beta, but not progesteron or hCG, to hypophysectomized mice.