CUMULUS-OOCYTE COMPLEXES FROM FOLLICLES OF DIFFERENT SIZES: THE EFFECT OF CO-MATURATION WITH GRANULOSA CELLS FROM DIFFERENT SOURCES

H. A. Amer

Department of Theriogenology, Faculty of Veterinary Medicine, Zagazig University, Egypt

ABSTRACT

The functional and morphological characteristics that associated with developing follicles and the oocytes they contain were defined in this work. Cows cumulus-oocyte complexes (COCs) were recovered from follicles with different sizes and then classified to 4 categories (cat.I to IV). The COCs with cat.IV were co-matured in vitro with granulosa cells (GCs) from own and >10 mm follicles to investigate their influence on the nuclear maturation. The rate of cat.I COCs was significantly higher (P<0.05) when recovered from 8 – 10 mm and >10 mm follicular groups (42.2 and 50.9%) than 2 – 3 mm groups (12.5%). This observation was converted with cat.IV COCs, it was significantly lower with 8 – 10 and >10 mm follicular groups (24.0 and 19.0%) than with 2 – 3 mm group (50.0%). The number and compactness of cumulus layers enclosing the cat.I COCs in 8 – 10 mm and >10 mm groups showed a higher rate (41.5 and 43.6%) than 2 – 3 mm and 4 – 8 mm groups (0.0 and 26.9%), respectively. A significant (P<0.05) decrease in the number of 3 to 4 cumulus layers was observed with 8 – 10 and >10 mm groups (7.7 and 7.3%) than with 2 – 3 mm and 4 – 8 mm groups (40.0 and 17.1%). The total number of cat.IV COCs with homogeneous ooplasm increased tendentiously compared to those with ooplasmic defects (56.1 and 43.9%). In the group of cat.IV COCs with ooplasmic defects, a significance (P<0.05) was observed in the rate of cumulus denudation in 2 – 3 mm group (25.0%), and in the rate of expanded cumulus with 8 – 10 mm and >10 mm groups (21.6 and 19.0%) as well as with homogeneous ooplasm (5.4 and 9.5%), respectively. After 24 hrs of in-vitro maturation (IVM), the number of COCs with expanded and metaphase II (M-II) stage increased significantly (P<0.05) with increasing the follicular size reaching the highest with cat.I COCs recovered from >10 mm follicles (98.2 and 94.4%). Cat.IV COCs recovered from 2 – 3 mm follicles showed a higher rate of M-II stage (20.0%) only after co-maturation with GCs from >10 mm follicles than own GCs (10.0%), moreover; no significant differences (P>0.05) were observed with 4 – 8 mm and 8 – 10 mm groups. In conclusion, the quality and M-II stage of COCs increased with increasing the follicular size, and GCs from >10 mm follicles could enhance the maturation of cat.IV COCs increasing the possibility to be used for in-vitro culture program.

Keywords:  cows, oocytes, quality, granulosa cells, maturation

INTRODUCTION

Folliculogenesis in mammalian species is a highly selective process. Only a very small proportion of the follicles survive atresia and give rise to dominant follicle, which needs to have the ability to mature before fertilization to occur. During nuclear maturation, meiosis resumes and the oocyte, when ovulated, has reached the M-II stage. During cytoplasmic maturation, complex and not fully understood changes occur in the oocyte. Changes in cortical granules distribution, alterations in the repartition of cytoplasmic organelles, accumulation or destruction of specific mRNA´s or proteins are noted (Fair and Hyttel, 1999). All these changes are of key importance to generate a good quality oocyte. In all mammalian species, there are strong links between follicular growth and oocyte maturation. For example, in cattle, only 1.4% of the oocytes originating from follicles <1 mm in diameter have the ability to reach M-II (Füher et al., 1989). This proportion is at least 10 times lower than the level reached when oocytes are obtained from follicles 1 - 3 mm in diameter. These data were extended by Fair et al. (1999), who showed that 2.5 mm follicles constituted a threshold at which the oocyte became able to complete meiosis to M-II.

The completion of bovine oocytes maturation in vitro could be enhanced with addition of somatic cells to the maturation medium by synthesis of some proteins that are neccessary for the germinal vesicle breakdown (Götze et al., 1990; Hinrichs, 1996), or by secreting soluble factors in the maturation medium that consequently enhance the in-vitro development of produced embryos (Hashimoto et al., 1998). It is known that the GCs from dominant follicles at least from adult animals in comparison to all other follicle sizes exhibited the highest border with gonadotropin receptors (Khatir et al., 1997). On this principle, the co-maturation of COCs in vitro with LH-dependent GCs is probably accompanied by important molecular changes in the oocytes leading to acquisition of  the full capacity to undergo normal maturation (Pavlok et al., 1992; Telfer, 1998). Most of oocytes that co-matured with own GCs degenerated and did not sustain further development during in-vitro culture (Thonon et al., 1993; Telfer, 1998). To understand the dialogue between the follicle and its oocyte will certainly generate informations useful to improve the present IVM technology for mammalian species. A preliminary report, on the work being carried out in our laboratory including the morphological qualification of oocytes recovered from different sized follicles and co-maturation some of these oocytes with GCs from different sources, is presented here.

MATERIAL AND METHODS

Collection of the ovaries:

 For collection of the ovaries, only adult cows were used. The age and breed, the course for selection, number of parturitions or keeping and feeding conditions of donor cows have not been recorded before slaugher. Only these females, which were obviously mature and by good health were choosen. The ovaries were collected in pairs within 30 min after slaughtering and placed in D-Phosphate Buffered Saline (D-PBS) at 30 - 35°C. The collected material were transported to IVF-laboratory within 2 hrs.

Recovery of the COCs:

All the ovarian follicles were qualified according to the parameters described by Pavlok et al. (1992); including vascularization and colour of the wall, transparency of whole follicle and their size. All the visible follicles that found on the ovarian surface were classified according to their size to 2 - 3 mm, 4 - 8 mm, 8 - 10 mm and >10 mm groups. Only healthy appearing follicles were punctured using 20-G needle connected to 5 ml syrings. The aspirates from follicles >10 mm were separately transfered to 35 mm petri dishes. The aspirates from 2 - 3 mm and 4 - 8 mm and 8 - 10 mm follicles were pooled according to size and transfered separately to 60 mm petri dishes. All the aspirates were examined under steriolope for qualification of the COCs (cat.I to IV) as described by De Loos et al. (1989, 1992).

In-vitro maturation:

The selected oocytes were washed twice in warm in TCM-199 with Earl´s salts (Sigma M-7528), 10% steer serum, 100 ug/ml L-glutamine and 50 ug/ml Gentamycin sulfate were added. All the COCs groups were placed for 24 hs in the maturation medium (TCM-199 with Earl´s salts, 20% steer serum, 100 ug/ml L-glutamine and 50 ug/ml Gentamycin sulfate) at 38.5°C, 5% CO2 and 95% Relative Humidity (Gordon, 1995). GCs were added to the COCs in the maturation medium to investigate their influence on the nuclear maturation.

Collection of granulosa cells:

 The collection of GCs was carried out directly after harvesting of COCs from follicular aspirates. The cells were pooled  according  to the follicular diameter into GCs from follicles with 2 - 3 mm, 4 - 8 mm, 8 - 10 mm and  >10 mm. The GCs were washed twice with prewarmed culture medium by centrifugation at 2000 r for 7 - 10 min. The resulting pellet was re-suspended in 400 µl TCM-199 with 20% bull serum and passed several times through a 18-gauge needle to re-desperse the GCs (Gordon, 1995). The cellular concentration was adjusted to 3 - 5 x 106 cells/ml before use.

Experimental design:

 To investigate the physiological effect of GCs on the nuclear maturation, cows COCs different follicles and with different categories were co-matured with GCs originated from either own follicles or >10 mm follicles.

Table (1): In-vitro experimental design:  Cumulus-oocyte complexes from cows recovered post mortem and the respective co-matured granulosa cells

(++ indicate co-maturation with respective granulosa cells)

After 24 hrs,  all the COCs of different groups were examined under steriolope to calculate the rate of maturation depending on the expansion of cumulus complex.

Staining of oocytes:

Using suitable pipettes and/or agitation in 2.9% sodium citrate, all expanded COCs were cleaned in D-PBS from remained cumulus cells. All the oocytes were fixed on slides in washing-bowl with acetic acid (1 part) and ethyle alcohol (3 part). After 24 hrs of fixation, all the oocytes were stained with 2% aceto-orcein that passed in the washing-bowl for about 1 min followed by 30% acetic acid solution to remove the supernatent stain. Under light microscope, oocytes were examined to determine the stage of nuclear maturation (M-I and –II).

Statistical analysis:

The rates of qualification, cumulus compactness and IVM of the recovered COCs were compared using Fisher´s protected least significant difference (PLSD) test following an analysis of variance (ANOVA). P value less than 0.05 was considered to be significant. 

RESULTS

As the oocyte grows within the follicle, a number of factors influence its quality and maturational characteristics. From these factors are the follicle size and level of atresia. For that reasons, these experiments will interpritate the relation between the size of bovine follicle and quality of COCs recovered from it (table 2). A significance (P<0.05) resulted in the recovery rate of COCs from       8-10 mm and >10 mm follicles (79.0 and 84.4%) compared to 2-3 mm and 4-8 mm follicles (46.5 and 43.8%). When quality of recovered COCs were related to the size of follicle, a significant (P<0.05) increase in the number of cat.I COCs was observed with increasing the size of follicle reaching the highest with >10 mm follicles (50.9%) and the lowest with 2 - 3 mm follicles (12.5%). With the four follicular groups, no significant (P>0.05) differences were yielded taking cat.II and III in consideration. Only lower numbers of cat.III oocytes were observed with >10 mm (7.4%) group compared to all other follicular groups (17.5, 15.3 and 13.6%), respectively. However, 2 - 3 mm group showed again higher numbers of cat.IV COCs (50.0%) than 4 - 8 mm, 8 - 10 mm and >10 mm groups (32.4, 24.0 and 19.4%). Within each group of follicular size, the quality and rate of recovered COCs were calculated. Whereas a significant (P<0.05) lower numbers of cat.I COCs than cat.IV (12.5 and 50.0%) were found in 2 - 3 mm group, the groups 8 - 10 mm and >10 mm groups showed higher number of cat.I COCs (42.2 and 50.9%) than cat.IV (24.0 and 19.4%), respectively.

Table (2): Quality of the cumulus-oocyte complexes recovered from adult cows in relation to the follicular size 

a,b: different superscripts in the same column denote significant differences (P<0.05)

The cumulus compactness of the cat.I COCs was compared between all the follicular groups (table 3). An increase in the number and compactness of cumulus layers accompanied by increasing in the size of original follicle. A significant (P<0.05) higher rate of compact cumulus was produced when the cat.I COCs were recovered from >10 mm follicles (43.6%) than from 2 - 3 mm and 4 - 8 mm groups (0.0 and 26.9%). Within each follicular group, differences in the number of cumulus layers were observed,  i.e. 2 - 3 mm group yielded a significant higher rate of 3 to 8 layers than >8 layers (40.0 and 0.0%), while 8 - 10 mm and >10 mm groups produced higher number of compact cumulus (41.5 and 43.6%) than 3 to 4 layers (7.7 and 7.3%) and 5 to 6 layers (9.2 and 16.4%).

However, a notable increase in the number of COCs with cat.I was observed during the successive stages of folliculogenesis and with increasing the size of original follicle. Additionally, the number of cumulus layers forming the cumulus-complex increased with the size of follicle. Statistically, significant correlations were observed between all groups of cat.I COCs with different cumulus layers reaching the highest with 8 - 10 mm and >10 mm follicular groups.

Table (3):  Morphology of the cumulus investment of cat.I COCs recovered from adult cows in relation to the follicular size

a,b,c: different superscripts in the same column denote significant differences (P<0.05)

The morphology of ooplasm and cumulus of cat.IV COCs were related to the size of follicle from which they are originated (table 4). Under this hypophysis, the number of COCs with homogeneous ooplasm and cumulus defects was tendentiously higher (56.1%) than those with ooplasmic and cumulus defects (43.9%).  Regarding the last type of COCs, those originated from 4-8 mm and 8-10 mm follicles produced a number of COCs with compact and expanded cumulus (22.2,16.2 and 16.7, 21.6%) that not highly differ. On the other side, cat.IV COCs with homogeneous ooplasm characterized by a significant (P<0.05) higher rate of incomplete than denuded and expanded cumulus in the most follicular groups.

Table (4): Morphology of cat.IV cumulus-oocyte complexes recovered from adult cows in relation to the follicular size.

a,b,c: different superscripts in the same column denote significant differences (P<0.05)

The cumulus expansion and nuclear matuation of the COCs with different categories and recovered from follicles with different sizes was examined after co-maturation in vitro with GCs from >10 mm follicles (table 5).  In all groups of follicular sizes  2-3 mm,  4-8 mm, 8-10 mm and >10 mm follicles, cat.I COCs showed a significant higher number of expanded cumulus after IVM (80.0, 82.9, 92.3 and 98.2%;P<0.05) than the other oocyte categories.

Reversible results were produced with cat.IV COCs (25.0, 25.0, 40.5 and 47.6%), respectively. In addition, an increase in the rate of M-II was observed with cat.I COCs with increasing of the follicular size, reaching the highest with >10 mm group (94.4%) and the lowest with 2-3 mm group (60.0%).

Table (5): In-vitro maturational characteristics in relation to cumulus expansion and stage of nuclear maturation.

a,b: different superscripts in the same column in each group denote significant differences (P<0.05)

Only cat.IV COCs from >10 mm follicles showed a higher number of M-II (38.0%) than the other sizes (20.0, 27.8 and 27.0%), respectively. Within each group, there was a significant (P<0.05) increase in the rate of M-II with decreasing the quality of in-vitro matured COCs. Moreover, the rate of M-I decresaed with increasing the follicular size. Cat.II and III COCs yielded a variable results of M-I and M-II stages.

Collectively, the COCs recovered from large follicles showed higher rate of cumulus expansion and M-II of nuclear maturation after 24 hs of IVM compared to the small follicles.

Regarding both types of COCs, the total number of recovered oocytes with homogeneous ooplasm increased, and the number of the oocytes with ooplasmic defects decreased with increasing the size of follicle from which they originated. All these observations yielded a significant correlations between the oocyte quality and follicular size.

The influence of co-maturation of cat.IV COCs from different sized follicles with GCs from own or >10 mm follicles was investigated in these experiments. Only a significant (P<0.05) difference was obtained with 2 - 3 mm group of follicular size. The rate of cat.IV COCs with M-II was higher after co-maturation with GCs from >10 mm follicles (20.0%) than with own GCs (10.0%), as shown in table 6. The other groups of follicular sizes did not show any significant differences with two types of GCs. However, a tendential increase in the number of M-II was obtained with oocytes recovered from 8 - 10 mm follicles when co-matured with GCs from >10 mm follicles than with own GCs (27.0 and 24.3%). Thus, the co-maturation with GCs from large preovulatory follicles enhanced the rate of M-II of cattle COCs recovered from small follicles, but no remarkable effect was observed with the COCs recovered from large follicles.

Table (6): Influence of co-maturation with GCs from different sized follicles on nuclear maturation of cat.IV COCs

a,b: different superscripts in the same column and row denote significant differences (P<0.05)

DISCUSSION

Since the follicular diameter is an important parameter for the quality of the COCs, an insight into the relation between follicular diameter and the quality of enclosed COCs is of vital importance to make a selection of the original follicle. The goal of this study was to see whether follicles of different sizes was a meaningful tool to discriminate between different categories of adult cows COCs that also might have different maturational characteristics. A significant (P<0.05) increase in the number of cat.I COCs was observed with increasing the size of follicle reaching the highest with >10 mm follicles (50.9%) and the lowest with 2 - 3 mm follicles (12.5%). Lonergan et al. (1993) classified the follicles with to 2 - 6 mm and >6 mm in diameter. Oocytes were collected and morphologically determined based on the number of layers of surrounding cumulus cells. A significant high number of oocytes with many layers of cumulus cells were obtained from follicles >6 mm than 2 - 6 mm follicles (70.2 and 46.8%). The cat.I COCs recovered from follicles >8 mm follicles characterized by visible lipid droplets which distributed regularly within the ooplasm (Pieterse et al., 1991; Dooney et al., 1997). These lipid droplets indicate the competence of oocytes for maturation and progressive development (Hyttel et al., 1997; 1999). Moreover,  higher rate of COCs with expanded cumulus obtained from the follicles at 5 to 6 day of estrous (³ 6 mm in diameter), whereas a higher rate of denuded oocytes was recovered at 0 to 1 day of estrous (£ 2 mm in diameter) as described by Salamone and Mapletoft (1997). This hypophesis demonstrates that COC morphology changes during the growing, static and regression phases. Additionally, there is a high correlation between follicle morphology and COC quality, Grade I are mainly but not exclusively derive from non-atretic follicles, but grade II and III derive from all classes of atretic follicles and highly atretic follicles (De Wit et al., 2000). This hypophesis was mainly explained by Lonergan et al. (1993). While most of small-or medium-sized follicles showed some atresia (88.0 and 67.0%), fewer of the large follicles were atretic (30.0%). These results of this work come in agreement with the findings of the last authors indicating the fact that large sized follicles produce good quality oocytes that produce high rates of development during in-vitro culture programmes. To answer these questions, most of the retrieved COCs with both cumulus and cytoplasmatic defects in own experiments suggests, that most of all COCs with cat.IV are rather degenerated or pseudomatured prior to aspiration than damaged due to aspiration pressure or other methodical insufficiences.

The experiments be under discussion revealed a significant rate of COCs with compact cumuli increased with increasing the size of follicle. This observation is confirmed by Lonergan et al. (1991). Oocytes recovered from 2 - 6 mm follicles showed a higher number of denuded, expanded, as well as 2 to 3 and 3 to 4 layers of cumulus cells than >6 mm follicles (3.8, 14.5, 12.9, 22.0 and 1.9, 11.6, 6.7, 9.6%), respectively. A significant higher rate of expanded COCs were more frequent during the regressing phase (53.4%) than the growing and static phases (14.4 and 17.8%), while the number of denuded oocytes were higher (P<0.05) in the growing and static phases (Mlodawska and Okolski, 1997; Salamone et al., 1999). These results support the hypophasis that COC morphology change during the growing, static and regression phases of the folliculogenesis.

The data show that there is a great variation in the appearence of immature oocytes, but this does not always indicate a difference in capability to mature in vitro. It is remarkable that a lot of morphological characteristics of cat.III and IV oocytes are also recognized in maturing oocytes. The expeiments yielded a significant results (P<0.05) with cat.I COCs that increased with increasing of the follicular size reaching the highest with 8 - 10 mm and >10 mm groups (89.2 and 94.4%). Under this hypophasis, the nuclear maturation (M-II) of COCs are increased in order grade1>grade2>grade3 (P<0.05) after IVM (Chauhan et al., 1998). De Loos et al. (1989) observed that only cat.IV oocytes have a significantly diminshed potency to mature in vitro (34.0%) up to  M-II than cat.I, II and III (66.0, 78.0 and 76.0%, respect.), and a substantial number of cat.IV oocytes degenerated under  in- vitro conditions. Although there is a great variation in morphology between cat.I, II and III, this is not reflected in different capability to mature in vitro. The same findings were found by Rick et al. (1996), after 24 hs of IVM, cat.IV oocytes yielded a low rate of maturation than cat.I, II and III (16.7, 96.8, 88.1 and 84.4%, respect.). These observations were interprited by Kastrop et al. (1990), who demonstrated that only cat.IV differs obviously from cat.  I-III in protein synthesis patterns of the oocytes. Although a few immature oocytes in cat.IV have protein synthesis patterns resembling those of immature oocytes in cat.I-III, the applied classification system distinguishes cat.IV oocytes as heterogeneous and degenerating oocytes.

In this work, low differences were obseved in the rate of M-II with reference to the increasing in the follicular diameter, but According to the follicular diameter; high levels of M-II (90.6, 86.9 and 94.4%) were resulted among 1 - 4 mm, 5 - 8 mm and 9 - 13 mm groups of follicular sizes (Ectors et al., 1996; Hagemann, 1999). However, in all mammalian species, there are strong links between follicular growth and oocyte maturation. For example, in cattle, only 1.4% of the oocytes originating from follicles smaller than 1 mm in diameter have the ability to reach M-II (Füher et al., 1989). This proportion is at least 10 times lower than the level reached when oocytes are obtained from follicles 1 to 3 mm in diameter. Moreover, the diameter of oocyte increases with the progressive phases of folliculogenesis inside the follicle (Rüsse, 1983; Chappel and Howles, 1991; Hyttel et al., 1999). Under this condition, the rate of M-II oocytes are lower when their diameter <120 um (30.0%) than >120 um (76.0%) as recorded by Majerus et al. (1999) and Yong et al. (1997). Salamone et al. (1999) observed a higher proportion of oocytes collected on day 5 and 6 (regression phase) showed evidence of nuclear maturation (50.0%) than on day 3 and 4 (static phase) (8.3%; P<0.05). Also, the findings of McGaughey et al. (1979) confirmed these results. They found that, oocytes with good quality recovered from large follicles completed maturation in vitro (i.e. underwent the first meiotic division) at significantly higher incidence (55.0%) than did oocytes from small (11.0 to 20.0%) or medium (16.0%) follicles. Our results are consistent with the hypophesis that a high proportion of COCs from small antral follicles are atretic, and that a maturational and developmental program controls the molecular and cytogenetic changes occuring in oocytes during follicular growth.

In each follicular size, there are differences in the rate of cumulus expansion and M-II between all oocytes categories. Similarly, More (P<0.05) oocytes of grade I COCs matured (59.3%) than from all other grades, but maturation success did not differ (P>0.05) for grade II (32.4 and 11.6%) and grade III (21.9 and 5.1%) oocytes (Wood and Wildt, 1997). These values were superior (P<0.05) to those of grade IV (5.1 and 1.4%). So highly heterogeneous population of COCs can be separated on the basis of grossly apparent morphological characteristics that reflect functional in the ability to mature in vitro.

To determine the role of GCs in oocyte maturation, we carried out an investigation on the effects of addition of GCs either from own follicles or >10 mm follicles to cat.IV COCs in maturation medium. It was found that, addition of GCs from >10 mm follicles to cat.IV COCs recovered from 2-3 mm follicles significantly increase the rate of M-II than with own GCs (20.0 and 10.0%), dut no differences were observed in the other groups of follicular sizes (4-8 mm and 8-10 mm groups). This phenomenon was observed by Hashimoto et al. (1998), who added granulosa and cumulus cells to oocytes denuded from their somatic cells and found no effect of these cells on their maturation. It is known that the GCs from dominant follicles at least from adult animals in comparison to all other follicle sizes exhibited the highest border with gonadotropin receptors (Khatir et al., 1997). The results obtained in this work and by Konishi et al. (1996) proved that the maturation of bovine oocytes was improved by supplementation of the maturation medium with GCs from large sized follicles comparable with own GCs. However, the negative effect of GCs collected from own follicles on the progressive development of oocytes and embryos in vitro was observed by Telfer (1998), who found the most oocytes co-matured with own GCs degenerated and did not sustain progressive development during in-vitro culture. These observations were recently confirmed by Götze et al. (1990); Galli and Lazzari (1995) and Hinrichs (1996), who proved that the supplementation with GCs in the maturation medium improves the in-vitro development by increasing of hormonal concentration, in consequence enhances the resumption of meiosis that requires synthesis of new proteins mittled by GCs.

CONCLUSION

These results support the hypophesis that the oocyte morphology and maturation up to M-II change during the recruitment, selection and dominance phases of follicular development. Thus, COCs from different follicle subclasses may be differ in their nutrient requirements, so current IVM technology needs further improvement to assist those oocytes that are developmentally challenged. From these technologies are the determination of the size of follicle from which the oocytes will be recovered and creation an optimum environment during IVM by addition of well developed GCs to the maturation medium.

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