DEVELOPMENTALBIOLOGY
Temporal
152,l-&151 (l%2,
Pattern of Synthesis of the Mouse Cortical Granule Protein, ~75, during Oocyte Growth and Maturation
We previously demonstrated that a protein of :W, 75,000 (~75) is localized to cortical granules (CGs) in mouse oocytes and eggs and is released upon activation or fertilization of eggs (K. E. Pierce, M. C. Siehert, G. S. Kopf, R. M. Schultz, and P. G. Calarco, 1990, Ur~cl. Bid. lA1,381L392).To examine the temporal pattern of synthesis of p75 during the early stages of CG formation, growing oocgtcs, which were isolated from juvenile mice, were incubated for 1 hr in medium containing [““S]methioninc, and radiolaheled proteins \vere immunoprecipitated using an antiserum that detects ~‘75. Synthesis of p75 is detected at low levels in the smallest oocytes examined (120 pm). Synthesis of p75 relative to total protein synthesis increases about 12-fold during oocyte growth from the 20-40 urn size and then remains constant throughout thv remaining period of ooqte growth (40-70 pm). In the fully grown, germinal vesicle (Girl-intact oocyte (70-80 pm), immunoprecipitated p75 comprises approximately 1.5% of the total amount of radiolahelrd protein. Three hours after t hc transfer of these oocptes to a medium that supports resumption of meiosis and GV breakdown irr rilro, oocytes level of p75 synthesis. Uy 15 hr of maturation, suljjected to a 1-hr labeling pulse display a 35’) ;) decrease in the relative pi5 synthesis was reduced to 14% of that in the fully grown, GV-intact oocytr and this is similar to the level of 137.3 synthesis in ovulated eggs. The level of p75 synthesis following itc r*ifro translation of total egg RNA is only 3X’/; lower than that obtained from total oocyte RNA. In addition, synthesis of p75 is observed following i,c ctitro translation of oocptc, hut not egg, poly(A)+ RNA. These results arc consistent with ~75 synthesis during oorytc maturation twing under ‘c’ 1992 Aradrmic Press. Inr translational control.
In the mouse, most oocytes are arrested in the diplotene stage of meiotic prophase shortly after birth (Schultz, 1986). Within a few days, a group of oocytes is selected by some unknown mechanism to begin growth. During the period of oocyte growth, which takes about 2 weeks and occurs while the oocytes are arrested in the first meiotic prophase, oocyte diameters increase from an initial size of -12-20 pm to a final size of -80 pm. Toward the end of this growth phase, oocytes are able to respond to hormonal signals ix ,~ic:o, or under proper conditions it! r>itrq to resume meiosis, and arrest at metaphase II following emission of the first polar body. Cortical granules (CGs) are first detected in growing mammalian oocytes in the region of the Golgi apparatus (Adams and Hertig, 1964; Baca and Zamboni, 1967; Szollosi, 1967; Odor and Blandau, 1969). Based on these electron microscopic studies, it was proposed that increasing amounts of materials are packaged into membranebound vesicles that fuse with one another to form granules of a final diameter of 200-600 nm. These granules are then localized to the cortical cytoplasm of oocytes and are found near the egg plasma membrane in ovulated eggs. The contents of CGs are released upon fertilization or egg activation and are believed to be in-
valved in the modification of proteins and/or carbohydrates of the ,YOVU pdlllcidn (ZP) and the egg surface. These changes are thought to constitute the block to polyspermy (reviewed by Wassarman, 1988a). Few dctails are known, however, about the contents of mammalian CG or the mechanisms by which they alter the ZP and the egg plasma membrane. Recently, we identified a protein of M, 75,000 (~75) that is found within CGs and is released from the egg upon fertilization or activation (Pierce ef (II., 1990). This protein represents to our knowledge the first described mammalian cortical granule protein component. Neither the function of ~75 nor its temporal pattern of synthesis during oocyte growth and maturation has been determined. In the present study, we have used metabolic labeling and immunoprecipitation to examine the expression of p75 in growing oocytes isolated from juvenile mice, in fully grown oocytes, maturing oocytes, and ovulated eggs. The level of ~‘75synthesis as a percentage of total protein synthesis shows a 12-fold increase between oocgtcs of diameters ~20 to 40 pm and stays constant for the remainder of the growth phase. Following the onset of oocyte maturation, the level of p75 sgnthesis decreases about lo-fold. 1rr t~ifrw translation expcriments demonstrate that the amount of p75 synthesized from total egg RNA is decreased about 38’% relative to
146
DEVELOPMENTALBIOLOGY
that synthesized from total oocyte RNA. In contrast, synthesis of ~‘75 is observed following 1:rzP&YJ translation of oocyte, but not egg, poly(A)+ RNA. MATERIALS
AND
METHODS
Collection of Oocytes and Eggs Fully grown oocytes and ovulated eggs were obtained from 6- to %week-old female mice (CF-1, Harlan) as previously described (Bornslaeger et al., 1986; Endo et al, 1987). The collection medium was bicarbonate-free minimal essential medium with Earle’s salts containing polyvinylpyrrolidone (MEM/PVP) (Poueymirou and Schultz, 1987). GV-intact oocytes were collected in MEM/PVP containing 0.2 mM 3-isobutyl-l-methyl xanthine (IBMX) to maintain meiotic arrest (Bornslaeger et al., 1986). Growing oocytes were obtained by culturing ovaries from juvenile mice (l-20 days old) in calcium-free Ml6 medium (Whittingham, 1971) containing 1 mg/ml collagenase (Type III, Worthington Biochemical) and 0.2 mg/ml DNase (Sigma) for 30-60 min (Eppig, 1976). Pieces of ovary were pipetted to separate oocytes from granulosa cells and other ovarian tissue. Oocytes were collected, rinsed three times with Ml6 medium, and radiolabeled as described below. In cases where the age of the mice exceeded 12 days, 0.2 mM IBMX was added to all media to maintain meiotic arrest.
Growing oocytes, fully grown oocytes, and ovulated eggs were metabolically radiolabeled by placing the cells in drops of Ml6 medium containing 1 mCi/ml [35S]methionine (>800 Ci/mmole; Amersham) and 0.2 mM IBMX (where needed for maintenance of meiotic arrest). They were then cultured for 4 hr at 3’7°C in a humidified atmosphere of 5% CO, in air. Following this incubation, oocytes and eggs were rinsed in MEM/PVP without radiolabel and were solubilized in a solution containing 50 mMTris-HCl, pH 8.0,150 mMNaC1,5 mM EDTA, 1% Nonidet P-40 (TNEN), and 0.2 TIU aprotinin. The total amount of cpm in radiolabeled protein was determined by precipitating an aliquot from each sample with ice-cold, trichloroacetic acid (TCA) (5% final concentration) and measuring acid-insoluble radioactivity as previously described (Poueymirou and Schultz, 1987). Oocytes to be labeled during in vitro maturation were collected from adult mice as described above and then transferred to MEM/PVP lacking IBMX to allow meiotic maturation. At various intervals (3-15 hr) after this transfer, groups of oocytes that had undergone GV breakdown were rinsed three times with Ml6 medium
VOLUME 152,1992
and metabolically radiolabeled as described above, except that the labeling incubation was only 1 hr.
An IgG fraction was obtained from the serum (ABL2) of a rabbit immunized with whole mouse blastocysts and adsorbed against adult mouse tissues (Johnson and Calarco, 1980). The IgG fraction from normal rabbit serum (NRS) was used as a control, since the preimmune serum was exhausted when these studies were initiated. No radiolabeled proteins were immunoprecipitated with NRS (Pierce et a,L., 1990 and data not shown). I?nmurloprecipitaI.f%o?l
ud
Quanti~cation
ofp75
Proteins radiolabeled with [35S]methionine were immunoprecipitated as previously described (Pierce et al., 1990). For each experiment, immunoprecipitation was performed using equal numbers of cpm of radiolabeled protein. One-dimensional gel electrophoresis in 7.5% polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE) and fluorography were performed as previously described (Pierce et al., 1990). Following SDSPAGE and fluorography, regions containing radiolabeled p75 were cut from the gel, rehydrated, and solubilized in 0.25 ml of 30% hydrogen peroxide at 70°C in tightly closed vials. Six milliliters of EcoScint (National Diagnostics) were added to each vial and radioactivity was measured by liquid scintillation counting. ~75 synthesis is expressed as the total number of immunoprecipitable cpm in ~75 divided by the total number of cpm of acid-insoluble radioactive material that was subjected to immunoprecipitation. RNA Isolatiw
and in Vitro Translation
Total RNA was isolated by solubilizing oocytes and eggs (about 500 of each) in a solution of 50 mMTris, pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.5 mg/ml glycogen, and 0.5 mg/ml proteinase K. The samples were extracted three times with phenohchloroform (l:l, v/v), and nucleic acid was precipitated by the addition of potassium acetate (0.3 Mfinal concentration) and 2.5 vol of 100% ethanol. Following an overnight incubation at -2O”C, nucleic acids were pelleted by centrifugation, rinsed with 807r ethanol, and air dried. Pellets were resuspended in the desired volume of diethyl pyrocarbonate-treated water. Poly(A)’ RNA was isolated using the FastTrack kit (Invitrogen) according to the manufacturer’s instructions. RNA pellets were rinsed in 80% ethanol, air dried, and resuspended as described above. RNA samples were translated it1 &TO in the presence of 2.5 mCi/ml [35S]methionine using a rabbit reticulo-
MI X 1O-3
123456
‘**II
am 69-
FIG. 1. Fluorogram of [“‘Slmethionine-radiolabcled proteins from grou-ing ooeytes. Lanes l-3, total radiolabeled proteins in oocytes with average diameters of 17, 26, and 34 pm, respectively. Each lane contains 10.000 cpm of acid-insoluble radioactive material. Lanes l-6, rZ-HL2 immrlnoyrecipitates of radiolabeled proteins from the respective samples shown in lanes 1X1. Each of the immunoprecipitates was from lOt~,OOO cpm of T(‘A-precipitable material. Exposure times were 2 days for lanes l-3 and 5 days for lanes 4-6.
cyte lysate (Promega) according to the manufacturer’s instructions; the final incubation volume was 20 ~1. The translation was terminated after 2 hr at 30°C by the addition of 2 ~1 of a buffer containing 10 mM Tris, pH 7.5, 50 pug/ml RNase, and 1 mM EDTA. Samples were then incubated for an additional 20 min at 37°C. Twenty-eight microliters of TNEN was added to each sample. The total amount of radioactivity incorporated into protein was measured by TCA precipitation and immunoprecipitation was performed with samples containing equal amounts of TCA-precipitable radioactivity.
made and the amount of radioactivity in immunoprecipitated p75 was measured. Immunoprecipitated ~75 contained approximately the same fraction of the total radiolabeled protein from samples diluted over a range of two orders of magnitude (data not shown). Nonlinear recovery of radiolabeled p75 was found only from the most highly concentrated samples (>lOO fully grown oocytes per 100 ~1 of sample) and such concentrations were avoided in the experiments presented below. To analyze protein synthesis in the growing oocyte, juvenile mice were used to ensure that a large group of oocytes with uniform sizes could be obtained and to avoid possible hormonal variations that might be present with cycling adults. Ovaries from 2-day-old mice contained a small number of oocytes with diameters of 20-25 pm, indicating that oocyte growth from an initial size of 12-20 pm (Schultz, 1986) had likely already begun. The largest oocytes obtained from 5-day-old mice measured 45 pm in diameter, although the growing oocytes averaged approximately 38 pm. To determine if ~75 was synthesized at these stages, oocytes were metabolically radiolabeled with [““SJmethionine and radiolabeled proteins were then immunoprecipitated and analyzed by SDS-PAGE and fluorography. Oocytes were grouped according to size so that the range of diameters within a group usually was less than 5 pm from the mean. The synthesis of ~75 was detected in the smallest size of oocytes used (~20 pm) and its synthesis increased about 12-fold during oocyte growth (Fig. 1, compare lanes 4-6; Fig. 2). It could not be determined if the synthesis of ~75 in the smallest oocytes represented a basal level in the nongrowing oocyte
2 a
2.0
(18)
(17)
(14) T
(11)
(4)
30-39
40-49
50-59
60-69
z-70
I
RESIJLTS
~7.5 is localized to mouse CGs and is synthesized in the fully grown, GV-intact oocyte (Pierce et al., 1990). The current study was undertaken to examine the expression of this protein during other stages of oogenesis. Since CGs can be first detected during early stages of oocyte growth (Gulyas, 1980), ~75 synthesis was examined in growing oocytes. Preliminary experiments were done to determine if immunoprecipitation reflected an accurate quantification of the amount of radiolabeled p75 in samples from metabolically radiolabeled oocytes. Dilutions of metabolically radiolabeled, fully grown oocgte samples were
Temporal
152,l-&151 (l%2,
Pattern of Synthesis of the Mouse Cortical Granule Protein, ~75, during Oocyte Growth and Maturation
We previously demonstrated that a protein of :W, 75,000 (~75) is localized to cortical granules (CGs) in mouse oocytes and eggs and is released upon activation or fertilization of eggs (K. E. Pierce, M. C. Siehert, G. S. Kopf, R. M. Schultz, and P. G. Calarco, 1990, Ur~cl. Bid. lA1,381L392).To examine the temporal pattern of synthesis of p75 during the early stages of CG formation, growing oocgtcs, which were isolated from juvenile mice, were incubated for 1 hr in medium containing [““S]methioninc, and radiolaheled proteins \vere immunoprecipitated using an antiserum that detects ~‘75. Synthesis of p75 is detected at low levels in the smallest oocytes examined (120 pm). Synthesis of p75 relative to total protein synthesis increases about 12-fold during oocyte growth from the 20-40 urn size and then remains constant throughout thv remaining period of ooqte growth (40-70 pm). In the fully grown, germinal vesicle (Girl-intact oocyte (70-80 pm), immunoprecipitated p75 comprises approximately 1.5% of the total amount of radiolahelrd protein. Three hours after t hc transfer of these oocptes to a medium that supports resumption of meiosis and GV breakdown irr rilro, oocytes level of p75 synthesis. Uy 15 hr of maturation, suljjected to a 1-hr labeling pulse display a 35’) ;) decrease in the relative pi5 synthesis was reduced to 14% of that in the fully grown, GV-intact oocytr and this is similar to the level of 137.3 synthesis in ovulated eggs. The level of p75 synthesis following itc r*ifro translation of total egg RNA is only 3X’/; lower than that obtained from total oocyte RNA. In addition, synthesis of p75 is observed following i,c ctitro translation of oocptc, hut not egg, poly(A)+ RNA. These results arc consistent with ~75 synthesis during oorytc maturation twing under ‘c’ 1992 Aradrmic Press. Inr translational control.
In the mouse, most oocytes are arrested in the diplotene stage of meiotic prophase shortly after birth (Schultz, 1986). Within a few days, a group of oocytes is selected by some unknown mechanism to begin growth. During the period of oocyte growth, which takes about 2 weeks and occurs while the oocytes are arrested in the first meiotic prophase, oocyte diameters increase from an initial size of -12-20 pm to a final size of -80 pm. Toward the end of this growth phase, oocytes are able to respond to hormonal signals ix ,~ic:o, or under proper conditions it! r>itrq to resume meiosis, and arrest at metaphase II following emission of the first polar body. Cortical granules (CGs) are first detected in growing mammalian oocytes in the region of the Golgi apparatus (Adams and Hertig, 1964; Baca and Zamboni, 1967; Szollosi, 1967; Odor and Blandau, 1969). Based on these electron microscopic studies, it was proposed that increasing amounts of materials are packaged into membranebound vesicles that fuse with one another to form granules of a final diameter of 200-600 nm. These granules are then localized to the cortical cytoplasm of oocytes and are found near the egg plasma membrane in ovulated eggs. The contents of CGs are released upon fertilization or egg activation and are believed to be in-
valved in the modification of proteins and/or carbohydrates of the ,YOVU pdlllcidn (ZP) and the egg surface. These changes are thought to constitute the block to polyspermy (reviewed by Wassarman, 1988a). Few dctails are known, however, about the contents of mammalian CG or the mechanisms by which they alter the ZP and the egg plasma membrane. Recently, we identified a protein of M, 75,000 (~75) that is found within CGs and is released from the egg upon fertilization or activation (Pierce ef (II., 1990). This protein represents to our knowledge the first described mammalian cortical granule protein component. Neither the function of ~75 nor its temporal pattern of synthesis during oocyte growth and maturation has been determined. In the present study, we have used metabolic labeling and immunoprecipitation to examine the expression of p75 in growing oocytes isolated from juvenile mice, in fully grown oocytes, maturing oocytes, and ovulated eggs. The level of ~‘75synthesis as a percentage of total protein synthesis shows a 12-fold increase between oocgtcs of diameters ~20 to 40 pm and stays constant for the remainder of the growth phase. Following the onset of oocyte maturation, the level of p75 sgnthesis decreases about lo-fold. 1rr t~ifrw translation expcriments demonstrate that the amount of p75 synthesized from total egg RNA is decreased about 38’% relative to
146
DEVELOPMENTALBIOLOGY
that synthesized from total oocyte RNA. In contrast, synthesis of ~‘75 is observed following 1:rzP&YJ translation of oocyte, but not egg, poly(A)+ RNA. MATERIALS
AND
METHODS
Collection of Oocytes and Eggs Fully grown oocytes and ovulated eggs were obtained from 6- to %week-old female mice (CF-1, Harlan) as previously described (Bornslaeger et al., 1986; Endo et al, 1987). The collection medium was bicarbonate-free minimal essential medium with Earle’s salts containing polyvinylpyrrolidone (MEM/PVP) (Poueymirou and Schultz, 1987). GV-intact oocytes were collected in MEM/PVP containing 0.2 mM 3-isobutyl-l-methyl xanthine (IBMX) to maintain meiotic arrest (Bornslaeger et al., 1986). Growing oocytes were obtained by culturing ovaries from juvenile mice (l-20 days old) in calcium-free Ml6 medium (Whittingham, 1971) containing 1 mg/ml collagenase (Type III, Worthington Biochemical) and 0.2 mg/ml DNase (Sigma) for 30-60 min (Eppig, 1976). Pieces of ovary were pipetted to separate oocytes from granulosa cells and other ovarian tissue. Oocytes were collected, rinsed three times with Ml6 medium, and radiolabeled as described below. In cases where the age of the mice exceeded 12 days, 0.2 mM IBMX was added to all media to maintain meiotic arrest.
Growing oocytes, fully grown oocytes, and ovulated eggs were metabolically radiolabeled by placing the cells in drops of Ml6 medium containing 1 mCi/ml [35S]methionine (>800 Ci/mmole; Amersham) and 0.2 mM IBMX (where needed for maintenance of meiotic arrest). They were then cultured for 4 hr at 3’7°C in a humidified atmosphere of 5% CO, in air. Following this incubation, oocytes and eggs were rinsed in MEM/PVP without radiolabel and were solubilized in a solution containing 50 mMTris-HCl, pH 8.0,150 mMNaC1,5 mM EDTA, 1% Nonidet P-40 (TNEN), and 0.2 TIU aprotinin. The total amount of cpm in radiolabeled protein was determined by precipitating an aliquot from each sample with ice-cold, trichloroacetic acid (TCA) (5% final concentration) and measuring acid-insoluble radioactivity as previously described (Poueymirou and Schultz, 1987). Oocytes to be labeled during in vitro maturation were collected from adult mice as described above and then transferred to MEM/PVP lacking IBMX to allow meiotic maturation. At various intervals (3-15 hr) after this transfer, groups of oocytes that had undergone GV breakdown were rinsed three times with Ml6 medium
VOLUME 152,1992
and metabolically radiolabeled as described above, except that the labeling incubation was only 1 hr.
An IgG fraction was obtained from the serum (ABL2) of a rabbit immunized with whole mouse blastocysts and adsorbed against adult mouse tissues (Johnson and Calarco, 1980). The IgG fraction from normal rabbit serum (NRS) was used as a control, since the preimmune serum was exhausted when these studies were initiated. No radiolabeled proteins were immunoprecipitated with NRS (Pierce et a,L., 1990 and data not shown). I?nmurloprecipitaI.f%o?l
ud
Quanti~cation
ofp75
Proteins radiolabeled with [35S]methionine were immunoprecipitated as previously described (Pierce et al., 1990). For each experiment, immunoprecipitation was performed using equal numbers of cpm of radiolabeled protein. One-dimensional gel electrophoresis in 7.5% polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE) and fluorography were performed as previously described (Pierce et al., 1990). Following SDSPAGE and fluorography, regions containing radiolabeled p75 were cut from the gel, rehydrated, and solubilized in 0.25 ml of 30% hydrogen peroxide at 70°C in tightly closed vials. Six milliliters of EcoScint (National Diagnostics) were added to each vial and radioactivity was measured by liquid scintillation counting. ~75 synthesis is expressed as the total number of immunoprecipitable cpm in ~75 divided by the total number of cpm of acid-insoluble radioactive material that was subjected to immunoprecipitation. RNA Isolatiw
and in Vitro Translation
Total RNA was isolated by solubilizing oocytes and eggs (about 500 of each) in a solution of 50 mMTris, pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.5 mg/ml glycogen, and 0.5 mg/ml proteinase K. The samples were extracted three times with phenohchloroform (l:l, v/v), and nucleic acid was precipitated by the addition of potassium acetate (0.3 Mfinal concentration) and 2.5 vol of 100% ethanol. Following an overnight incubation at -2O”C, nucleic acids were pelleted by centrifugation, rinsed with 807r ethanol, and air dried. Pellets were resuspended in the desired volume of diethyl pyrocarbonate-treated water. Poly(A)’ RNA was isolated using the FastTrack kit (Invitrogen) according to the manufacturer’s instructions. RNA pellets were rinsed in 80% ethanol, air dried, and resuspended as described above. RNA samples were translated it1 &TO in the presence of 2.5 mCi/ml [35S]methionine using a rabbit reticulo-
MI X 1O-3
123456
‘**II
am 69-
FIG. 1. Fluorogram of [“‘Slmethionine-radiolabcled proteins from grou-ing ooeytes. Lanes l-3, total radiolabeled proteins in oocytes with average diameters of 17, 26, and 34 pm, respectively. Each lane contains 10.000 cpm of acid-insoluble radioactive material. Lanes l-6, rZ-HL2 immrlnoyrecipitates of radiolabeled proteins from the respective samples shown in lanes 1X1. Each of the immunoprecipitates was from lOt~,OOO cpm of T(‘A-precipitable material. Exposure times were 2 days for lanes l-3 and 5 days for lanes 4-6.
cyte lysate (Promega) according to the manufacturer’s instructions; the final incubation volume was 20 ~1. The translation was terminated after 2 hr at 30°C by the addition of 2 ~1 of a buffer containing 10 mM Tris, pH 7.5, 50 pug/ml RNase, and 1 mM EDTA. Samples were then incubated for an additional 20 min at 37°C. Twenty-eight microliters of TNEN was added to each sample. The total amount of radioactivity incorporated into protein was measured by TCA precipitation and immunoprecipitation was performed with samples containing equal amounts of TCA-precipitable radioactivity.
made and the amount of radioactivity in immunoprecipitated p75 was measured. Immunoprecipitated ~75 contained approximately the same fraction of the total radiolabeled protein from samples diluted over a range of two orders of magnitude (data not shown). Nonlinear recovery of radiolabeled p75 was found only from the most highly concentrated samples (>lOO fully grown oocytes per 100 ~1 of sample) and such concentrations were avoided in the experiments presented below. To analyze protein synthesis in the growing oocyte, juvenile mice were used to ensure that a large group of oocytes with uniform sizes could be obtained and to avoid possible hormonal variations that might be present with cycling adults. Ovaries from 2-day-old mice contained a small number of oocytes with diameters of 20-25 pm, indicating that oocyte growth from an initial size of 12-20 pm (Schultz, 1986) had likely already begun. The largest oocytes obtained from 5-day-old mice measured 45 pm in diameter, although the growing oocytes averaged approximately 38 pm. To determine if ~75 was synthesized at these stages, oocytes were metabolically radiolabeled with [““SJmethionine and radiolabeled proteins were then immunoprecipitated and analyzed by SDS-PAGE and fluorography. Oocytes were grouped according to size so that the range of diameters within a group usually was less than 5 pm from the mean. The synthesis of ~75 was detected in the smallest size of oocytes used (~20 pm) and its synthesis increased about 12-fold during oocyte growth (Fig. 1, compare lanes 4-6; Fig. 2). It could not be determined if the synthesis of ~75 in the smallest oocytes represented a basal level in the nongrowing oocyte
2 a
2.0
(18)
(17)
(14) T
(11)
(4)
30-39
40-49
50-59
60-69
z-70
I
RESIJLTS
~7.5 is localized to mouse CGs and is synthesized in the fully grown, GV-intact oocyte (Pierce et al., 1990). The current study was undertaken to examine the expression of this protein during other stages of oogenesis. Since CGs can be first detected during early stages of oocyte growth (Gulyas, 1980), ~75 synthesis was examined in growing oocytes. Preliminary experiments were done to determine if immunoprecipitation reflected an accurate quantification of the amount of radiolabeled p75 in samples from metabolically radiolabeled oocytes. Dilutions of metabolically radiolabeled, fully grown oocgte samples were
