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Meiotic arrest in incompetent rat oocytes is not regulated by cAMP
S Goren1, Y Piontkewitz, N Dekel
1Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Fully grown, but not growing, mammalian oocytes spontaneously resume meiosis in vitro. Resumption of meiosis, also known as oocyte maturation, is associated with a drop in intraoocyte concentrations of cAMP followed by activation of the maturation promoting factor (MPF). Microtubule-associated-protein (MAP) kinase has been suggested as a substrate for the active p34cdc2 kinase, the catalytic subunit of MPF. Our study was designed to explore the mechanism of regulation of meiotic arrest in growing rat oocytes. Confirming previous observations we showed that in our rat colony oocytes do not acquire the competence to spontaneously resume meiosis earlier than 22 days postpartum. We further demonstrated that follicle-enclosed oocytes from 20-day-old female rats fail to resume meiosis in response to luteinizing hormone, follicle-stimulating hormone, a gonadotropin-releasing hormone analog, or forskolin, all of which are known to induce maturation in competent oocytes. Immunoblot analysis using highly specific anti p34cdc2 antibodies revealed that incompetent oocytes express the catalytic subunit of MPF at amounts that are not different from that found in competent oocytes. In addition, highly specific anti MAP kinase antibodies detected the presence of similar quantities of two isoforms (42 and 44 kDa) of MAP kinase in competent and incompetent oocytes. Measurements of cAMP revealed that as compared to competent oocytes, incompetent oocytes contain somewhat lower levels of this nucleotide (1.42 +/- 0.3 and 1.17 +/- 0.07 fmole/oocyte, respectively). However, considering the difference in protein content, the calculated concentrations seem to be similar. Furthermore, similar to competent oocytes, intracellular concentrations of cAMP in incompetent oocytes dropped significantly (from 1.17 +/- 0.07 to 0.77 +/- 0.12 fmole/oocyte) 2 hr after isolation from the follicle. We hereby suggest that (a) in mammals, similar to amphibians, the term meiotic incompetence can be extended to include inability to resume meiosis in response to hormonal stimulation; (b) it is not the lack of p34cdc2 or downstream regulatory elements, such as MAP kinase, that prevents growing oocytes from resuming meiosis; and (c) unlike fully grown oocytes, resumption of meiosis in growing oocytes is not subjected to negative regulation by cAMP.
Insights
Growing rat oocytes are meiotically incompetent due to factors other than cAMP levels or the presence of maturation promoting factor (MPF) and MAP kinase. These oocytes do not resume meiosis even with hormonal stimulation, suggesting a unique regulatory mechanism.
Area of Science:
- Reproductive Biology
- Cellular Biology
- Developmental Biology
Background:
- Mammalian oocyte maturation involves resuming meiosis, triggered by a drop in cyclic adenosine monophosphate (cAMP) and activation of maturation promoting factor (MPF).
- Growing oocytes often remain arrested in meiosis I, exhibiting meiotic incompetence until a specific developmental stage.
Purpose of the Study:
- To investigate the regulatory mechanisms underlying meiotic arrest in growing rat oocytes.
- To determine if hormonal stimulation can induce meiosis resumption in immature oocytes.
- To identify key molecular players involved in regulating oocyte meiotic competence.
Main Methods:
- Oocyte isolation and in vitro culture from rats of different postpartum ages.
- Hormonal stimulation assays using luteinizing hormone, follicle-stimulating hormone, GnRH analog, and forskolin.
- Immunoblot analysis for p34cdc2 (MPF catalytic subunit) and MAP kinase.
- Measurement of intracellular cAMP concentrations.
Main Results:
- Rat oocytes gain meiotic competence around 22 days postpartum.
- Immature oocytes (20-day-old) failed to resume meiosis in response to hormonal stimuli.
- Both competent and incompetent oocytes expressed similar levels of p34cdc2 and MAP kinase.
- While cAMP levels were slightly lower in incompetent oocytes, they dropped significantly upon isolation, similar to competent oocytes.
Conclusions:
- Meiotic incompetence in growing oocytes extends to hormonal stimulation, not just spontaneous resumption.
- The absence of p34cdc2 or MAP kinase does not explain the meiotic arrest in growing oocytes.
- Unlike mature oocytes, cAMP does not appear to exert negative regulation on meiosis resumption in growing oocytes.