Abstract
Background and aims: The changes in cytoplasmic inclusions during meiotic maturation have only been examined in porcine oocytes. In the present study, the amount and the number of cytoplasmic inclusions (glycogen granules, lipid droplets and fibrous structures) were examined in mouse oocytes in the process of in vivo and in vitro maturation. For those inclusions that changed in amount during maturation, we also examined their content in oocytes treated with olomoucine, an inhibitor of cyclin‐dependent kinase, in order to clarify the relationship between nuclear maturation and changes in the inclusions.
Methods: Nuclear maturation in the oocytes cultured for various periods and those collected from antral follicles and oviducts was examined after staining with aceto‐orcein. For the demonstration of glycogen granules and lipid droplets, oocytes were stained with periodic acid‐Schiff or Sudan IV. Fibrous structures in the oocytes were observed under an electron microscope.
Results: The amount of glycogen granules, Sudanophilic lipid droplets and fibrous structures did not change in the oocytes matured in vivo and in vitro, whereas the number of the lipid droplets increased during maturation. In the oocytes treated with olomoucine, the resumption of nuclear maturation was inhibited, whereas the increase in the number of Sudanophilic lipid droplets was not inhibited.
Conclusion: Present findings suggest that the increase in the number of Sudanophilic lipid droplets occurs in the cytoplasm of mouse oocytes during maturation, regardless of in vivo or in vitro maturation, and that such the change in the inclusion is not related to nuclear maturation. (Reprod Med Biol 2004; 3: 231–236)
Keywords: cytoplasmic inclusion, maturation, mouse oocyte, olomoucine
INTRODUCTION
THE FOLLICULAR OOCYTES and tubal embryos of mammals contain glycogen granules and/or lipid droplets, which are used as an energy source for the growth of the oocytes or development of the embryos, and the amount of such inclusions in an embryo varies with the stage of development. 1 , 2 The presence of fibrous or lamellar structures, one of the cytoplasmic proteinaceous inclusions, has been electron microscopically reported in the oocytes and the embryos of many species of rodents, 1 , 2 , 3 , 4 and in those of rhesus monkeys 5 and humans. 6 Although the physiological significance of these structures is not clearly known, the structures are considered to be used as an energy source for the oocyte growth and the embryo development as well as glycogen granules and lipid droplets.
Electron microscopic studies have revealed that the lipid droplets contained in porcine oocytes markedly increase in number from 30 to 50 h after human chorionic gonadotropin (hCG) injection, whereas the total amount of lipids in the oocytes remained unchanged from 0 to 50 h after the injection, suggesting that the increase in the number of lipid droplets is related to the resumption of meiotic maturation. 7 The authors have also reported that the amount of proteins, glycogen granules and Sudanophilic lipid droplets does not change in porcine oocytes from immediately after collection until 44 h after culture, whereas the lipid droplets diminish in the size and increase in the number after 22 h of culture, and that the number of Sudanophilic lipid droplets of different sizes is comparable between ovulated oocytes and those cultured for 44 h. 8 However, the changes in cytoplasmic inclusions during maturation have only been examined in porcine oocytes, 7 , 8 and the changes in cytoplasmic inclusions with oocyte maturation in other mammals have not yet been examined.
In the present study, glycogen granules, lipid droplets and fibrous structures were histochemically or electron microscopically demonstrated in mouse oocytes in the process of in vivo and in vitro maturation to investigate the changes in the amount and the number of these inclusions with oocyte maturation. For those inclusions that changed in the amount and the number during maturation, we also examined their content in oocytes treated with olomoucine, 9 an inhibitor of cyclin‐dependent kinase, which is a maturation‐promoting factor, in order to clarify the relationship between nuclear maturation and changes in the inclusions.
MATERIALS AND METHODS
Animals
TWO HUNDRED and eighty‐one female mature mice of ICR strain were used in the present study. They were housed in autoclaved metal cages and were given a standard chow (MF, Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum in an air‐conditioned room (24°C), under controlled‐lighting conditions (the light : dark cycle was 14:10 h). They received humane care as outlined in the Guide for the Care and Use of Laboratory Animals (Niigata University Animal Care Committee). These mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotrophin (PMSG, Serotropin; Teikoku Hormone Manufacturing Co. Ltd, Tokyo, Japan).
Collection and culture of oocytes
In the present investigation, mice were killed under anesthesia by diethyl ether to collect oocytes. In order to observe oocytes in the process of in vitro maturation, immature oocytes covered with cumulus cells (COC) were collected from antral follicles 48 h after the PMSG injection and cultured in TYH medium 10 containing 5% fetal bovine serum (FCS; Gibco BRL, NY, USA) and 10 IU/mL PMSG at 37°C in a CO2 incubator (5% CO2 in air). Conversely, PMSG‐injected females were further intraperitoneally injected with 5 IU of hCG (Gonatropin; Teikoku Hormone Manufacturing Co. Ltd) 48 h after the PMSG injection to obtain oocytes in the process of maturation in vivo. COC were collected from antral follicles 4 and 8 h after the hCG injection, and from oviducts 14 and 20 h after the hCG injection.
In order to observe fertilized oocytes, recently ovulated oocytes with cumulus cells 14 h after the hCG injection and those cultured for 14 h were inseminated in vitro. Sperm suspension was prepared by minutely cutting caudal epididymis of mature males in TYH medium for 1 h at 37°C in a CO2 incubator. A small volume of the sperm suspension was introduced into 100 µL droplets of TYH medium, so that the final concentration of spermatozoa was adjusted to 3–5 × 105/mL. The COC were introduced into the droplets of sperm suspension and cultured for 6 h at 37°C in a CO2 incubator. Conversely, in order to obtain in vivo fertilized oocytes, some of the superovulated females were mated with mature males of the same strain immediately after the hCG injection, and fertilized oocytes at the pronuclear stage were collected from oviducts 20 h after the hCG injection.
Demonstration of lipid droplets and glycogen granules
At 0, 4, 8, 14 and 20 h after the hCG injection, and at 4, 8, 14 and 20 h after maturation culture, COC were immersed in phosphate buffered saline (PBS; pH 7.4) 11 containing 0.1% hyaluronidase (Sigma‐Aldrich, St. Louis, MO, USA) to disperse their cumulus cells. In order to demonstrate Sudanophilic lipids, these denuded oocytes and oocytes fertilized in vivo and in vitro were fixed in PBS containing 10% formalin, and then stained with Sudan IV. 12 The same procedures of staining were repeated three times. For the demonstration of glycogen granules, denuded oocytes and fertilized oocytes were further immersed in PBS containing 0.2% pronase (Sigma‐Aldrich) to dissolve their zonae pellucidae, fixed in Bouin solution 12 and stained with periodic acid‐Schiff. 13 Glycogen granules which appeared in the cytoplasm of the oocytes were confirmed with a salivary test. 12 The same procedures for the demonstration of glycogen granules were applied three times to 30 oocytes from each culture or from each period after the hCG injection.
After staining, oocytes were washed in PBS, and were placed on glass slides to be photographed under a light microscope (Nikon, Tokyo, Japan). Degenerated oocytes were eliminated from the observation.
Demonstration of fibrous structures
The oocytes matured in vivo and in vitro, and those fertilized in vivo and in vitro were fixed in a 0.1 m cacodylate buffer solution (pH 7.4) containing 4.0% glutaraldehye and 2.0% paraformaldehyde for 3 h at 4°C, rinsed three times in a 0.1 m cacodylate buffer solution (pH 7.4) overnight, and were postfixed in a 0.1 m cacodylate buffer solution (pH 7.4) containing 1% osmium tetroxide for 1 h at 4°C. The fixed oocytes were dehydrated through an acetone series, and then embedded in Quetol 812. Some of the embedded samples were stained with toluidine blue (pH 7.0) after being sectioned, and then observed under a light microscope. The other samples were cut with an ultramicrotome, stained with uranium acetate and lead nitrate, and then photographed under a CM‐200 electron microscope (Philips Electron Optics, Eindhoven, Netherlands). The amount of fibrous structures was examined in 10 oocytes from each culture or from each period after the hCG injection. Degenerated oocytes were eliminated from the observation.
Observation of nuclear maturation
In order to investigate nuclei, oocytes cultured for various periods and those collected from antral follicles and oviducts were fixed in 25% (v/v) acetic acid in ethanol for 24 h at room temperature. The fixed oocytes were stained with 1.0% (v/v) aceto‐orcein and examined for evidence of nuclear maturation under a light microscope (Nikon, Tokyo, Japan).
Demonstration of Sudanophilic lipids and observation of nuclear maturation in olomoucine‐treated oocytes
To observe the amount of Sudanophilic lipids in oocytes in which resumption of meiotic division was inhibited, COC collected from antral follicles 48 h after the PMSG injection were cultured for 8 h at 37°C in TYH medium containing 400 µm olomoucine (Sigma‐Aldrich). A certain amount of olomoucine was previously dissolved in dimethyl sulfoxide (DMSO) and then diluted with TYH medium to adjust the final concentration of 400 µm. The concentration of DMSO in the culture medium was adjusted to 0.37% (v/v). Oocytes cultured in the medium containing DMSO at 0.37% for 8 h were used as controls. After culture, demonstration of lipids droplets was performed as mentioned previously, and then the amount of the lipids was determined under a light microscope. To ensure the inhibition of resumption of meiotic division by olomoucine treatment, oocytes cultured for 8 h with olomoucine were fixed in 25% (v/v) acetic acid in ethanol, stained with 1.0% aceto‐orcein and then examined under a light microscope (Nikon, Tokyo, Japan). To determine the viability of the olomoucine‐treated oocytes, progression of nuclear maturation was also observed in those further cultured for 14 h in the medium containing no olomoucine.
Statistical analysis
The rates of nuclear maturation were statistically analyzed by χ2‐test. The number of Sudanophilic lipid droplets was statistically analyzed by anova.
RESULTS
Nuclear maturation
AT 0 h AFTER the hCG injection, nuclei of oocytes were in the germinal vesicle (GV) and diakinesis stages, mostly in the GV stage (83%). The percentage of oocytes at the GV stage decreased over time of maturation in vivo and in vitro and reached 0% after culture for 4 h, or 8 h after the hCG injection, respectively. Nuclei of oocytes cultured for 8 h or 8 h after the hCG injection were almost in the metaphase I (MI) stage, 73 and 76%, respectively. At 14 and 20 h after maturation culture, the percentages of oocytes with the MII stage nuclei were 83 and 86%, respectively. Of oocytes 14 and 20 h after the hCG injection, 76 and 77%, respectively, were in the MII stage.
Changes in the amount of lipids
When the oocytes were stained with Sudan IV, the presence of Sudanophilic lipids was observed as reddish–orange droplets of different sizes in the cytoplasm (Fig. 1a,b). In the present study, these Sudanophilic lipids were classified into three groups using a micrometer under a microscope: small droplets less than 1.0 µm in diameter, medium ones 1.0–2.4 µm and large ones more than 2.5 µm. Since the number of lipid droplets with small size was not able to count owing to their extremely large numbers, the amount of lipid droplets with such size was estimated on a 3‐point scale: large (+ + +), moderate (+ +) and small (+). The number of lipid droplets with medium and large sizes was counted under a microscope.
Figure 1.
Cytoplasmic inclusions in mouse oocytes 0 h after the human chorionic gonadotropin injection (a, c) or cultured for 14 h (b, d). (a) Sudanophilic lipid droplets in the cytoplasm (magnification: ×300). (b) Sudanophilic lipid droplets in the cytoplasm (magnification: ×300). (c) Periodic acid‐Schiff reactive glycogen granules in the cytoplasm (magnification: ×300). (d) Fibrous structures (arrows) in the cytoplasm (magnification: ×10 000).
As shown in Table 1, the oocytes 0 h after the hCG injection (Fig. 1a) contained a small amount of Sudanophilic lipid droplets with small size. Average numbers of Sudanophilic lipid droplets with medium and large sizes were 81.9 and 28.4, respectively.
Table 1.
The amount of Sudanophilic lipid droplets of different sizes in mouse oocytes matured in vivo and in vitro
| Maturation | Hours after hCG injection or culture | Number of oocytes examined | The amount and number of lipid droplets | ||
|---|---|---|---|---|---|
| Small size (<1.0 µm) | Medium size (1.0–2.4 µm) | Large size (≥2.5 µm) | |||
| In vivo | 0 | 33 | +† | 81.9 ± 7.6b ‡ | 28.4 ± 4.7a |
| 4 | 35 | + + | 105.1 ± 7.5a | 4.7 ± 0.9b | |
| 8 | 37 | + + + | 102.6 ± 10.5ab | 3.3 ± 0.9b | |
| 14 | 30 | + + + | 100.8 ± 6.9ab | 1.1 ± 0.2c | |
| 20 | 30 | + + + | 118.7 ± 10.9a | 1.2 ± 0.2c | |
| In vitro | 4 | 35 | + + | 105.4 ± 6.6a | 4.6 ± 1.5b |
| 8 | 37 | + + + | 108.5 ± 6.1a | 2.6 ± 0.9bc | |
| 14 | 30 | + + + | 101.6 ± 5.5ab | 2.4 ± 0.4bc | |
| 20 | 30 | + + + | 106.8 ± 5.5a | 1.2 ± 0.4c | |
hCG, human chorionic gonadotropin. †The symbols +, + + and + + + represent a small, moderate and large amount, respectively. ‡Mean ± S.E. Values with different superscripts in the same column are significantly different (P < 0.01).
In the oocytes matured in vivo and in vitro, although the amount of Sudanophilic lipid droplets with small size increased, the number of those with large size significantly decreased (P < 0.01) (Fig. 1b). The number of lipid droplets with medium size did not change remarkably.
The amount of Sudanophilic lipid droplets in fertilized oocytes did not differ from that in oocytes cultured for 20 h or 20 h after the hCG injection. In fertilized oocytes, there were no differences in the amount of lipid droplets of different sizes among three groups: (i) oocytes matured and fertilized in vivo; (ii) those matured and fertilized in vitro; and (iii) those matured in vivo and fertilized in vitro (Table 2).
Table 2.
The amount of Sudanophilic lipid droplets of different sizes in fertilized mouse oocytes
| Maturation | Fertilization | Number of fertilized oocytes examined | The amount and number of lipid droplets | ||
|---|---|---|---|---|---|
| Small size (<1.0 µm) | Medium size (1.0–2.4 µm) | Large size (≥2.5 µm) | |||
| In vivo | In vivo | 33 | + + + † | 119.9 ± 6.9a ‡ | 0.9 ± 0.3a |
| In vivo | In vitro | 35 | + + + | 119.2 ± 5.4a | 0.9 ± 0.2a |
| In vitro | In vitro | 37 | + + + | 113.0 ± 3.2a | 0.9 ± 0.2a |
The symbol + + + represents a large amount.
Mean ± S.E. Values with different superscripts in the same column are significantly different (P < 0.05).
Changes in the amount of glycogen granules and fibrous structures
When the mouse oocytes were stained with periodic acid‐Schiff, fine red–purple granules appeared in the cytoplasm (Fig. 1c). These granules were confirmed as glycogen, because they disappeared with a salivary test. The amount of glycogen granules was large in the every oocyte collected from antral follicles and oviducts and in those cultured for 4–20 h.
Fibrous structures were abundantly distributed throughout the cytoplasm (Fig. 1d) when the mouse oocytes 0 h after the hCG injection were observed under an electron microscope. The inclusions consisted of groupings of individual fibrous elements with about 150 Å in diameter and 1.0 µm in length. In the oocytes 4–20 h after the hCG injection, although the inclusion slightly shortened in length, the amount of the inclusion did not change. The amount and the figure of fibrous structures in cultured mouse oocytes were similar to those in the oocytes matured in vivo.
The amount of glycogen granules and fibrous structures in fertilized oocytes did not differ from that in oocytes cultured for 20 h or 20 h after the hCG injection. The amount of these inclusions was not different among fertilized oocytes of three different groups.
Amount of Sudanophilic lipids and nuclear maturation in olomoucine‐treated oocytes
As shown in Table 3, the olomoucine‐treated oocytes had a large amount of Sudanophilic lipid droplets with small size and 100.8 and 2.6 of those with medium and large sizes. The number and amount of the droplets of different sizes in the treated oocytes did not differ from those in the control oocytes.
Table 3.
The amount of Sudanophilic lipid droplets of different sizes in mouse oocytes cultured with olomoucine
| Concentrations of olomoucine (µM) | Number of oocytes examined | The amount and number of lipid droplets | ||
|---|---|---|---|---|
| Small size (<1.0 µm) | Medium size (1.0–2.4 µm) | Large size (≥2.5 µm) | ||
| 400 | 34 | + + +† | 100.8 ± 6.2a ‡ | 2.6 ± 0.8a |
| 0 | 32 | + + + | 110.3 ± 6.0a | 1.8 ± 0.5a |
The oocytes were observed after 8 h of culture. †The symbol + + + represents a large amount. ‡Mean ± S.E. Values with different superscripts in the same column are significantly different (P < 0.05).
Nuclei of the olomoucine‐treated oocytes were all in the GV stage, while those of the control oocytes were in the diakinesis to telophase I stages, mostly in the MI stage (83%, 25/30). From the results, it was confirmed that olomoucine significantly inhibits the resumption of nuclear maturation.
When treated oocytes were further cultured in the medium without olomoucine for 14 h, most of their nuclei were in the MII stage (90%, 28/31), suggesting that the ability of the treated oocytes to mature was sustained.
DISCUSSION
AS MENTIONED IN the Introduction, Cran 7 reported that porcine oocytes contain many lipid droplets. Although the amount of the lipids does not alter over time after hCG injection, the number of droplets increases from 30 h after the injection, suggesting that nuclear maturation is related to the increase in lipid droplets because GV breakdown occurred in oocytes with increased lipid droplets. In a previous study 8 we carried out to show Sudanophilic lipids in cultured porcine oocytes, it was revealed that the lipid droplets diminished in size after 22 h of culture, and that the number of Sudanophilic lipid droplets of different sizes was comparable between ovulated oocytes and those cultured for 44 h. Furthermore, we reported 8 that the resumption of nuclear maturation is completely inhibited in the olomoucine‐treated oocytes, and the reduction in the size of the droplets is also inhibited. Therefore, we suggested that nuclear maturation in porcine oocytes is closely associated with the size of the Sudanophilic lipid droplets, and the transformation of the larger ones into smaller droplets could serve as a marker for the resumption of meiotic maturation.
In the present study, the amount of glycogen granules, lipid droplets and fibrous structures did not change with oocyte maturation, whereas the lipid droplets diminished in size after 4 h of culture or 4 h after the hCG injection. The change in the lipid droplets occurred similarly in the oocytes matured in vivo and in vitro. Because nuclear maturation was found to progress in the oocytes observed in the present study, as in previous reports, 14 , 15 nuclear maturation was considered to have progressed normally. From these findings, it is suggested that transformation of large lipid droplets into small ones in the cytoplasm (i.e. the increase in the number of lipid droplets) is related to the resumption of meiotic maturation, regardless of in vivo or in vitro maturation. Since it has been reported that the change in size of Sudanophilic lipid droplets occurs in porcine oocytes matured in vivo and in vitro, 7 , 8 we consider the increase in number of lipid droplets is a common phenomenon in mammalian oocytes during maturation.
In the present study, we also attempted to show Sudanophilic lipid droplets which had diminished in size with maturation, as mentioned above, using olomoucine‐treated oocytes to determine the relationship between nuclear maturation and changes in size of lipid droplets in the cytoplasm. The resumption of nuclear maturation was completely inhibited in the olomoucine‐treated oocytes, while the size and the number of lipid droplets were comparable between the treated oocytes and the control oocytes. Therefore, we consider that nuclear maturation in mouse oocytes is not associated with the change in the size of Sudanophilic lipid droplets in the cytoplasm.
In the present study, it was suggested that lipid droplets accumulate in mouse oocytes by transformation of the large droplets into smaller ones to prepare for the succeeding fertilization and early development following fertilization. However, the amount and the number of Sudanophilic lipid droplets in in vivo and in vitro fertilized oocytes were similar to those in unfertlized oocytes 14 and 20 h after maturation culture or 14 and 20 h after the hCG injection. Therefore, the results of the present study could not provide the information needed to identify the utilization of lipid droplets for fertilization as energy sources. The physiological significance of the change in size of lipid droplets with oocyte maturation should be further studied.
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