Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine.
TL;DR: It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.
Abstract: during the period of oocyte growth preceding nuclear maturation. This phase of oocyte development is characterized by 3 To whom correspondence should be addressed the synthesis and storage of RNA and translational products It has been postulated that premature shortening of the (Rossant and Pederson, 1986) which are subsequently used oocyte growth phase due to the recovery of oocytes from for meiotic and early embryonic developmental events (Kastrop small diameter follicles may be responsible for the et al., 1991; Wickramasinghe and Albertini, 1993; Fair developmental anomalies associated with in-vitro matura- et al., 1995). tion. 6-Dimethylaminopurine (DMAP) was used to artifi- In vivo, each human menstrual cycle is characterized by the cially lengthen the pre-maturation period of oocyte growth, recruitment and growth of numerous follicles. One or two in vitro, by inhibiting germinal vesicle breakdown in mouse selected follicles continue growth until the day of ovulation, and human oocytes. DMAP inhibited the meiotic matura- whilst most follicles undergo atresia, terminating their growth. tion of mouse and human oocytes and the inhibition was By the time of ovulation the dominant follicle measures fully reversible. The timing of polar body extrusion was approximately 20 mmol/l in diameter and may have been accelerated in mouse oocytes following the withdrawal of growing for up to 14 days (Calderon and Healy, 1993). DMAP; however, the kinetics of nuclear maturation in Human oocytes obtained for in-vitro maturation are aspirated human oocytes was unaffected by exposure to DMAP. All from 2–12 mmol/l antral follicles early in the follicular phase mouse and human DMAP-treated oocytes that matured of the menstrual cycle and are matured for only 48 h in culture. to metaphase II expressed histone H1 kinase activity. Hence, at the time of aspiration for in-vitro maturation, Fertilization rates in both DMAP-treated and control human oocytes have not fully completed their phase of oocyte mouse and human oocytes were comparable, and human growth and may be retrieved during the process of atresia. embryonic development was similar in control and Therefore, all human oocytes retrieved for in-vitro maturation DMAP-treated oocytes. However, blastocyst development have an artificially truncated growth phase. Consequently, it was significantly reduced in DMAP-treated mouse oocytes is possible that the maturational and developmental anomalies (P < 0.05). It is concluded that lengthening the pre- observed in in-vitro matured oocytes are attributable to their maturation growth phase, by temporarily inhibiting kinase shortened growth phase and thus the inability to complete
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TL;DR: It is demonstrated that supplementing IVM media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development.
Abstract: Oocyte in vitro maturation (IVM) is an important reproductive technology that generates mature oocytes that are capable of supporting preimplantation embryo development and full development to term. There is great clinical and commercial incentive to improve the efficiency of the technology, however, progress has been slow over the past decade. A critical challenge is to understand what constitutes oocyte developmental competence and the mechanisms governing it. We have taken the approach of studying in detail oocyte-somatic cell interactions; including, oocyte-cumulus cell (CC) gap-junctional communication, and bidirectional paracrine signalling between the two cell types. It is becoming clear that, compared to oocytes matured in vivo, IVM oocytes undergo maturation prematurely as they are still in the process of acquiring developmental competence in vivo, and the molecular cascade reinitiating meiosis differs entirely to that in vivo. Attempts to enhance oocyte developmental competence by attenuating the spontaneous meiotic resumption of oocytes in vitro have been met with mixed success. Kinase inhibitors that prevent maturation-promoting factor activity have, in general, been ineffectual on promoting oocyte developmental potential post-IVM. In contrast, agents that modulate oocyte cAMP during IVM show greater potential, possibly as these compounds extend oocyte-CC gap-junctional communication. An important concept that is now emerging is that the oocyte secretes potent growth factors that regulate fundamental aspects of CC function and thereby determine the distinctive phenotype of the cumulus-oocyte complex. The capacity of an oocyte to regulate its own microenvironment by oocyte-secreted factors (OSFs) may constitute an important component of oocyte developmental competence. In support of this notion, we have recently demonstrated that supplementing IVM media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development.
333 citations
Cites background from "Regulation of human and mouse oocyt..."
...6-DMAP Bovine Decreased [21–23] Human Unchanged [24] Murine Decreased [24] Table 3 Effect of phosphodiesterase inhibitors during IVM on embryo production...
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...These studies show that controlling spontaneous meiotic progression by manipulating MPF activity, in general either adversely affects [20–24] or has no positive effect [20,25–28] on subsequent oocyte developmental potential, although there is one report of an actual improvement in oocyte quality [29]....
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TL;DR: Successful introduction of a new approach to IVM will reduce the requirement of fertility hormones and will be less invasive to the patient's daily life by reducing the need for monitoring of serum hormone levels and intravaginal ultrasound.
Abstract: An innovative approach to in vitro maturation (IVM) for application in infertility treatment and fertility preservation is required to bring this patient-friendly treatment into routine practice. Current approaches to IVM never report more than a 10 to 15% implantation rate per embryo transferred, which is two to three times lower and early pregnancy losses are higher than in conventional in vitro fertilization/intracytoplasmic sperm injection. The cornerstone of such an innovative culture technique is the use of pharmacological compounds that allow synchronization of nuclear and cytoplasmic maturation processes within the oocyte. The rationale of a prolonged oocyte maturation period is to promote a longer interaction between the immature oocyte with adequately conditioned cumulus cells. Successful introduction of a new approach to IVM will reduce the requirement of fertility hormones and will be less invasive to the patient's daily life by reducing the need for monitoring of serum hormone levels and intravaginal ultrasound. The new IVM conditions will reduce a whole range of minor and major complications in assisted reproductive technology and finally will also reduce the total cost for treatment. The minimal invasiveness of this procedure will benefit cancer patients who want to store gonadal tissue before undergoing therapy that devastates subsequent germ-cell competence.
142 citations
TL;DR: PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs, and this resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development.
Abstract: Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6–12 mm) were retrieved 34–36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro mat...
117 citations
Cites background or result from "Regulation of human and mouse oocyt..."
..., PMC) might alleviate this asynchrony by allowing time for completion of cytoplasmic maturity [12, 24, 25, 29–34]....
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...Hypothetically, prematuration culture (PMC) of oocytes designed to retard nuclear maturation could allow time for ooplasmic maturation to catch up [12–14] and, therefore, better synchronize the nuclear and cytoplasmic compartments....
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...Inhibition of meiosis by kinase inhibitors has been used as an attempt to improve oocyte developmental competence; however, inconsistent results have been obtained in large mammalian oocytes [29–34] and proved not to be successful in human oocytes [12]....
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TL;DR: The role of cAMP hydrolytic isoenzyme phosphodiesterase type 3 (PDE 3) modulation on human oocyte maturation in vitro is addressed and reversal from pharmacological arrest leads to a progression through meiosis in a normal time frame with formation of a well-aligned metaphase plate.
Abstract: This study addresses the role of cAMP hydrolytic isoenzyme phosphodiesterase type 3 (PDE 3) modulation on human oocyte maturation in vitro. Presence of phosphodiesterase type 3 A (PDE 3A) mRNA was confirmed in human germinal vesicle-stage (GV) oocytes. Making use of a selective PDE 3 inhibitor, Org 9935 (10 μM), oocytes retrieved from immature follicles were arrested in prophase I with a high efficiency for up to 72 h. Cumulus oocyte complexes (COCs) were retrieved in the follicular phase of the cycle before or after exposure to endogenous LH or hCG administration in vivo and randomly distributed into maturation medium with or without the PDE 3 inhibitor. Previous exposure of small follicles to LH activity in vivo had no influence on the arresting capacity of the PDE 3 inhibitor. Reversal from pharmacological arrest leads to a progression through meiosis in a normal time frame with formation of a well-aligned metaphase plate. Ultrastructure analysis of COC derived from follicles between 8 and 12 mm showed that the induced extension of prophase I arrest in vitro resulted in cytoplasm changes but not in apparent nuclear changes during culture.
99 citations
Cites background from "Regulation of human and mouse oocyt..."
...[43] reported that H1 kinase, which is related to chromatin condensation, was activated after gonadotropin stimulation and DMAP did not inactivate this process....
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TL;DR: It is suggested that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.
Abstract: In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 μM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% ± 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.
99 citations
Cites background from "Regulation of human and mouse oocyt..."
...There exists evidence that, once hormonal stimulation has been carried out in vivo, the process of chromatin condensation and transcription repression in fully grown oocytes is activated [8, 12, 13] and inhibitor of protein phosphorylation (such as 6-dimethylaminopurine) does not reverse this process [13]....
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...The inhibitor of protein phosphorylation, 6-dimethylaminopurine, has been shown to alter the kinetics of nuclear maturation of mice and to compromise the subsequent developmental competence [13]....
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...It has been hypothesized that an in vitro prematuration period of oocytes retrieved from small antral follicles might lead to an improved oocyte cytoplasmic maturation [13]....
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References
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TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
232,912 citations
"Regulation of human and mouse oocyt..." refers methods in this paper
...Proteins were separated on a 12% polyacrylamide gel (SDS–PAGE) as described (Laemmli, 1970)....
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...…two pronuclei and the extrusion of the second polar groups of 30 murine oocytes in 10 µl of Laemmli sample bufferbody 8–10 h after insemination. followed by one-dimensional SDS gel electrophoresis of oocyteIn-vitro matured human oocytes were fertilized by intracytoplasmic proteins (Laemmli, 1970)....
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...SDS–PAGE was performed using a 12% (w/v) polyacrylamide gel (Laemmli, 1970)....
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Journal Article•
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
203,017 citations
TL;DR: Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle, and anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.
Abstract: Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle. When a region in the N terminus of cyclin is fused to a foreign protein, it produces a hybrid protein susceptible to proteolysis at mitosis. During the course of degradation, both cyclin and the hybrid form conjugates with ubiquitin. The kinetic properties of the conjugates indicate that cyclin is degraded by ubiquitin-dependent proteolysis. Thus anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.
2,372 citations
"Regulation of human and mouse oocyt..." refers background in this paper
...Consequently, the kinase dependent pathway requiring intact microtubules (Glotzer et al., 1991; Kubiak et al., 1993)....
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TL;DR: Fully grown oocytes of the frog (Rana pipiens) undergo cytoplasmic and nuclear maturation when treated with progesterone after the follicular envelopes have been removed, and the arrest of mitosis and cleavage can be attributed to a specific “cytostatic factor” in the cy toplasm of the secondary oocyte.
Abstract: Fully grown oocytes of the frog (Rana pipiens) undergo cytoplasmic and nuclear maturation when treated with progesterone after the follicular envelopes have been removed. The mechanism of this maturation was investigated by injection of cytoplasm from progesterone-treated oocytes at various stages of maturation into fully grown but immature oocytes. The injected cytoplasm becomes effective in inducing maturation by 12 hours after progesterone administration, reaches a maximum effectiveness around 20 hours, and then declines after the donor oocytes complete maturation. However, even cytoplasm from early embryos retains some capacity to induce oocyte maturation. The frequency with which maturation is induced is proportional to the volume of the injected cytoplasm. Progesterone itself is not directly responsible for the maturation-producing effect of injected cytoplasm since injected progesterone does not promote maturation. However, externally applied progesterone does induce the completion of the first meiotic division, presumably by releasing a cytoplasmic “maturation promoting factor.” The production of this cytoplasmic factor was not affected by removal of the nucleus.
After completion of the first meiotic division, oocytes cease further development at the metaphase of the second meiotic division, where they remain until fertilized or activated to develop. Cytoplasm from such secondary oocytes when injected into one of the blastomeres at the two-cell stage of development suppresses mitosis as well as cleavage. Mitosis is usually arrested at metaphase. No such inhibition was brought about by injection of cytoplasm from cleaving blastomeres. Thus, the arrest of mitosis and cleavage can be attributed to a specific “cytostatic factor” in the cytoplasm of the secondary oocyte. Activation of donor secondary oocytes by insemination or pricking with a glass needle soon destroys the cytostatic factor. Likewise, addition of cortical cytoplasm to endoplasm from the secondary oocyte rapidly destroys the cytostatic capacity. This result implies that cortical material is involved in the process of removing the cytostatic factor at the time of normal activation or fertilization. Enucleation of oocytes demonstrated that production and removal of the cytostatic factor is independent of the nucleus.
1,508 citations
TL;DR: The crystal structure of the human cyclinA-cyclin-dependent kinase2-ATP complex has been determined at 2.3 A resolution and activates the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft.
Abstract: The crystal structure of the human cyclinA-cyclin-dependent kinase2 (CDK2)-ATP complex has been determined at 2.3 A resolution. CyclinA binds to one side of CDK2's catalytic cleft, inducing large conformational changes in its PSTAIRE helix and T-loop. These changes activate the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft.
1,426 citations
"Regulation of human and mouse oocyt..." refers background in this paper
...The maturational changes associated with atresia are believed to be due, in part, to a decline oractivity (Jeffrey et al., 1995)....
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...The maturational changes associated with atresia are believed to be due, in part, to a decline or activity (Jeffrey et al., 1995)....
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