Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells

The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Biochemical studies of mammalian oogenesis: Metabolic cooperativity between granulosa cells and growing mouse oocytes

Freeze fracture and lanthanum tracer experiments have shown that gap junctions exist throughout folliculogenesis between granulosa cells and growing mouse oocytes (Anderson and Albertini, J. Cell Biol.71, 680–686, 1976). The following lines of experimentation in the present study suggest that metabolic cooperativity exists between granulosa cells and their enclosed oocytes, i.e., gap junctions are functional, and that in most cases examined, greater than 85% of the metabolites present in follicle-enclosed oocytes were originally taken up by the granulosa cells and transferred to the oocyte via gap junctions: (1) When incubated with various radiolabeled compounds, follicle-enclosed oocytes contained more intracellular radioactivity than did oocytes with no attached granulosa cells (denuded oocytes); (2) for two radiolabeled ribonucleosides examined, the distribution of phosphorylated metabolites in follicle-enclosed oocytes resembled that of granulosa cells and differed significantly from that in denuded oocytes; (3) pulse-chase experiments with radiolabeled ribonucleosides revealed that during the chase period more radioactivity became associated with the follicle-enclosed oocyte; (4) treatments known to disrupt gap junctions in other cell types were effective in reversibly uncoupling metabolic cooperativity between granulosa cells and oocytes; and (5) a series of control experiments using (a) medium conditioned by granulosa cells and (b) cocultures of denuded oocytes and granulosa cells in which physical contact between the two cell types was not permitted demonstrated that contact between follicle cells and oocytes was necessary for observing metabolic cooperativity. Metabolic cooperativity was also found between follicle cells and oocytes in the two culture systems which support growth of mouse oocytes in vitro. The fact that oocytes do not grow well, if at all, in the absence of follicle cells and the large contribution of nutrients apparently furnished to the oocyte by the granulosa cells is consistent with the concept that gap junction mediated metabolic cooperativity between follicle cells and their enclosed oocytes is vital for mammalian oocyte growth.     

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.     

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.     

MAPK和p90rsk在大鼠卵泡发育中的表达与活性

利用免疫组织化学方法研究丝裂原激活蛋白激酶(mitogen-activated protein kinases, MAPK)及其底物之一p90rsk在大鼠卵泡发育过程中的表达与活性.结果表明,非活性形式的MAPK存在于大鼠各生长期卵泡的卵母细胞和颗粒细胞中,但磷酸化活性形式的MAPK只存在于部分具有分裂增殖活性的颗粒细胞中.MAPK的作用底物p90rsk只在各期卵泡的卵母细胞中表达,在颗粒细胞中无着色,说明MAPK信号级联在卵母细胞和颗粒细胞中具有不同的作用方式.另外,胎鼠卵巢的免疫组化染色结果显示,MAPK在卵原细胞增殖过程中具有活性,表明MAPK信号级联在这一过程中起作用.     

Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.     

Stage-dependent effects of oocytes and growth differentiation factor 9 on mouse granulosa cell development: advance programming and subsequent control of the transition from preantral secondary follicles to early antral tertiary follicles

The development of an ovarian follicle requires a complex set of reciprocal interactions between the oocyte and granulosa cells in order for both types of cells to develop properly. These interactions are largely orchestrated by the oocyte via paracrine factors such as growth differentiation factor 9 (GDF9). To examine these interactions further, a study was conducted of the effects of oocytes at different stages of development on proteins synthesized by mouse granulosa cells during the transition of granulosa cells (GCs) from preantral, secondary (2 degrees ) follicles (2 degrees GCs) to mural granulosa cells (3 degrees GCs) of antral tertiary (3 degrees ) follicles. The ability of recombinant GDF9 to mimic the effects of oocytes was also determined. Effects were evaluated by high- resolution, two-dimensional protein gel electrophoresis coupled to computer-assisted, quantitative gel image analysis. Coculture of the 2 degrees GCs with growing oocytes (GOs) from 2 degrees follicles brought about many of the changes in granulosa cell phenotype associated with the 2 degrees to 3 degrees follicle transition. GDF9 likewise brought about many of these changes, but only a subset of GDF9-affected protein spots were also affected by coculture with GOs. Coculture of 2 degrees GCs with the nearly fully grown oocytes (FGOs) from 3 degrees follicles had a reduced effect on 2 degrees GC phenotype, in comparison with coculture with GOs. For some proteins, oocyte coculture or GDF9 treatment appeared to have opposite effects on 2 degrees GCs and 3 degrees GCs. Additional effects of GDF9 and oocytes were seen in cultures of 2 degrees GCs for proteins other than those that differed between untreated control 2 degrees and 3 degrees GCs. These results indicate that GOs and GDF9 can each induce 2 degrees GCs to shift their phenotype toward that of 3 degrees GCs. The ability of the oocyte to produce this effect is diminished with oocyte development. The transition in the GC phenotype promoted by oocytes appears stable because differences in 2 degrees GCs promoted by oocytes and GDF9 were observed in untreated 3 degrees GCs. We conclude that the influence of the oocyte on GCs changes with the progression of their development, and so too does the response of the GCs to the oocyte. Moreover, by acting on the 2 degrees GCs, GOs are able to influence stably the phenotype of 3 degrees GCs. Thus, at or near the 2 degrees to 3 degrees follicle transition, signals from the growing oocyte contribute to the development of the mural GC phenotype.     

Mammalian oocyte growth in vitro is stimulated by soluble factor(s) produced by preantral granulosa cells and by Sertoli cells

We have evaluated the possibility that mouse oocyte growth in vitro could be achieved under the influence of soluble compound(s) released by different somatic cell types. For this purpose, zona-free denuded oocytes from 12-day-old mice were cultured on monolayers of NIH-3T3 fibroblasts, which are able to establish gap junctional communications with them, in the presence or absence of media conditioned by preantral granulosa cells or by Sertoli cells, plated at increasing concentrations from 0.3–1 × 10⁶ ml⁻¹ cells. After 3 days, no increase in vitellus diameter was recorded from fibroblast-coupled oocytes maintained in culture medium or in the presence of media conditioned by 0.3 × 10⁶ ml⁻¹ Sertoli cells. By contrast, increasing proportions of coupled oocytes grew, provided the continuous presence of media conditioned by 0.5 or 1 × 10⁶ ml⁻¹ Sertoli cells, or by 0.3, 0.5, and 1 × 10⁶ ml⁻¹ preantral granulosa cells. Since the ligand of c-kit, the growth factor KL, promotes the growth in vitro of oocytes cultured in follicles from 8-day-old mice, an antibody against mouse KL was used to evaluate whether in our culture conditions KL might also be responsible for the growth of oocytes from 12-day-old mice. No inhibition of growth was evident in oocytes cultured directly on preantral granulosa or Sertoli-cell monolayers. Furthermore, the growth of fibroblast-coupled oocytes cultured in media conditioned by preantral granulosa cells was not significantly affected by the presence of this antibody during culture. By contrast, a high percentage of oocytes cultured on fibroblasts in the presence of media conditioned by Sertoli cells showed a significant inhibition of growth and no metabolic cooperativity. It was concluded that, besides KL, other bioactive factor(s) released by either preantral granulosa or Sertoli cells can induce a significant stimulation of mouse oocyte growth in vitro. © 1996 Wiley-Liss, Inc.     

SPIN,a substrate in the MAP kinase pathway in mouse oocytes

The newly cloned gene Spin encodes a 30-kDa protein, a well-defined abundant molecule found in mouse oocytes and early embryos. This protein SPIN undergoes metaphase-specific phosphorylation and binds to the spindle. To understand the role of SPIN in oocyte meiosis, oocytes were treated with drugs that affect the cell cycle by activating or inactivating specific kinases. The posttranslational modification of SPIN in the treated oocytes was then investigated by one- and two-dimensional gel electrophoresis. Modification of SPIN is inhibited by treatment with 6-dimethylaminopurine (DMAP), suggesting that SPIN is phosphorylated by a serine-threonine kinase. Furthermore, SPIN from cycloheximide-treated oocytes that lack detectable MAP kinase activity is only partially phosphorylated, indicating that SPIN may be phosphorylated by the MOS/MAP kinase pathway. To confirm this observation, SPIN was analyzed in Mos-null mutant mice lacking MAP kinase activity. Normal posttranslational modification of SPIN did not occur in Mos-null mutant oocytes. In addition, there is reduced association of SPIN with the metaphase I spindle in Mos-null mutant oocytes, as determined by immunohistochemical analysis. These findings suggest that SPIN is a substrate in the MOS/MAP kinase pathway and further that this phosphorylation of SPIN may be essential for its interaction with the spindle. Mol. Reprod. Dev. 50:240–249, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: insights into regulation of spermatogenic progress

The development of follicles in the mammalian ovary involves a bidirectional communication system between the follicular cells and oocyte that is now beginning to be characterized. Little is known about the mechanisms underlying the beginning of the oocyte growth and the acquisition of the competence to resume meiosis by the growing oocyte. In the present study, we devised a multistep culture system for mouse oocytes obtained from 15.5- to 16.5-days postcoitum embryos (mean diameter +/- SEM, 9.7 +/- 1.3 microm), allowing three stages of the oocyte growth to be identified: (i) an early stage in which the oocyte growth is induced by direct stimulation of a soluble growth factor, namely stem cell factor (SCF), independent of the formation of gap junctions with granulosa cells; (ii) a second phase in which the oocyte growth depends on the combined action of SCF and contacts with granulosa cells; and (iii) a third phase of granulosa cell-dependent, SCF-independent growth. At each stage, key events of oocyte development and differentiation, such as the c-kit reexpression, the early zona pellucida assembly, and the beginning of follicologenesis, were observed to occur independently by the presence of SCF. At the end of the in vitro growing phases, lasting 18-20 days, oocytes reached a size (50 +/- 2.5 microm) and a chromatin differentiation (stage I-II) equivalent to those of 9- to 10-day-old preantral oocytes and were unable to complete the growth phase. About 50% of the in vitro-grown oocytes were induced to resume meiosis by okadaic acid (OA) treatment. However, a significant fraction of them (48%) showed inability to maintain the chromosome condensation in M-phase. When in vitro-grown oocytes were treated with UO126, a specific MEK inhibitor that prevents activation of mitogen-activated protein kinases (ERK-1 and ERK-2), for 1 h before, during, and following OA treatment, only 22% of oocytes underwent germinal vesicle breakdown after 24 h from the OA treatment. These studies demonstrate that SCF alone can induce the onset of the oocyte growth. This is, however, not sufficient to fully activate the mechanisms governing the acquisition of the meiotic competence previously described as a 15-day oocyte-autonomous clock starting at the onset of growth. The inability of oocytes to progress into the last stages of growth and the lack of synchrony between nuclear and cytoplasm maturation showed by a subset of them resemble the characteristics of oocytes from connexin-37- and -43-deficient mice and indicate the preantral/antral transition point as a critical stage of oocyte development requiring the coordinated differentiation of the oocyte with granulosa cells and the maintenance of adequate communication between these two cell types to assure the correct oocyte meiotic maturation.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.