Abstract: The present study was undertaken to investigate the effects of Luteinizing Hormone (LH, 100 ng mL-1) on bovine oocyte maturation in vitro (Experiment 1) and the relationship between the gap junction and LH (Experiment-2). In experiment 1, cumulus-enclosed oocyte (CO) and Denuded Oocytes (DO) were cultured in the presence or absence of LH. After 42-43 h in vitro culture, nuclear maturation rates which reached the metaphase stage of the second meiotic division (M-ll stage) were observed. In Experiment 2, we evaluated whether the gap junction, formed between cumulus cells and oocytes, was associated with the presence of LH in the maturation medium. The addition of LH to the maturation medium significantly enhanced nuclear maturation of both CO and DO (p<0.05) compared with those oocytes, which were cultured without LH. The addition of 1-heptanol (5 mM), a blocker of coupling in the gap junction, to the maturation medium supplemented with LH significantly inhibited (p<0.05) the number of CO and DO reaching the M-ll stage. These results suggest that the addition of LH to the maturation medium could improve the nuclear maturation of bovine oocytes.
INTRODUCTION
In Bangladesh, many heifer and cows are slaughter in every day to fulfill the meat requirements. A lot of immature bovine oocytes are abused in each day. There is a great opportunity to exploit these abused oocytes for further utilization in vitro in the laboratory. Bovine embryos can be derived by in vitro maturation and fertilization in the laboratory. It is well known that the addition of luteinizing hormone can enhance nuclear and cytoplasmic maturation of oocytes in vitro in cow (Zuelke and Brackett, 1990; Yunis and Brackett, 1992) and pig oocytes (Mattioli et al., 1991). It has been suggested that LH may lead to germinal vesicle breakdown (GVBD) of oocytes in vitro if granulose or cumulus cells are present (Downs, 1993). Some researchers suggested that gap injections formed between oocytes and cumulus cells or cumulus and granulose cells are related to gonadotropin-stimulated meiotic resumption of mouse oocytes (Downs, 1993; Fagbohun and Down, 1991), but no sufficient research report on the relationship between gonadotropin stimulation and gap injections in the bovine was not found. For this reason, further investigation seemed to be needed to determine how LH acts on oocyte maturation.
The present study was undertaken to investigate the influence of LH on the maturation of bovine cumulus -enclosed oocytes and denuded oocytes in vitro.
MATERIALS AND METHODS
Oocyte Maturation
Bovine oocytes were obtained from the local slaughterhouse and brought to
the AI and ET project laboratory at Central Cattle Breeding and Dairy Farm in
Savar, Dhaka, immersed in physiological saline (0.9% NaCl, 27-33°C)
supplemented with 400 μ mL-1 penicillin, G potassium salt and
500 μg mL-1 streptomycin sulfate within 4 h. The oocytes were
aspirated from superficial follicles (2-6 mm in diameter) with a 20-G needle
(Terumo, Tokyo, Japan) attached to a 6 mL plastic syringe (Top, Tokyo, Japan).
After aspiration, oocytes surrounded by more than one layer of cumulus cells
were selected and washed with TCM-199 (Gibco, BRL products, USA) more than 4
times. The oocytes were transferred to 100 μL drops of maturation medium
and cultured for 42-43 h in 5% CO2, 95% air 100% humidity at 39°C.
The maturation medium contained 25 mM Hepes buffered TCM-199 supplemented with
10% (v/v) fetal calf serum (Gibco), 0.35 mM D-glucose, 2.29 mM calcium lactate,
0.91 mM sodium pyruvate, 1 μg mL-1 estradiol-17 β (Sigma,
E-8875, USA), 5 IU mL-1 pregnant mare’s serum gonadotropin and antibiotics
(Novartis, Bangladesh).
In experiment 1,oocytes were assigned to four groups: oocytes enclosed in cumulus cells (CO) were loaded into the maturation medium mentioned above, with (+CO) or without (-CO) 100 ng mL-1 LH. Another group of CO were loaded into Dulbecco’s phosphate buffer saline (Gibco product) supplemented with 0.2% sodium citrate and their cumulus cells were removed mechanically with a vortex mixture and by pipetting (denuded oocytes, DO). The oocytes were transferred to the maturation medium, supplemented with (+DO) and without (-DO) LH. The effect of LH on CO and DO was evaluated by observing nuclear maturation. After the maturation culture, the oocytes in each group were fixed with ethanol and acetic acid for more than 6 h and stained with 1% orcein (Merk, Germany) in 40% acetic acid.
To observed the nuclear stage of the oocytes before maturation, some cumulus-enclosed oocytes aspirated from ovaries were immediately denuded, fixed and stained as mentioned before.
In experiment 2, We observed the intact structure of junctional communication
of projections of cumulus cells and oolemma in both CO and DO before maturation
culture by transmission electron microscopy (data not shown). To evaluate whether
gap junctions could influence oocyte maturation, CO and DO were introduced into
maturation medium contained 100 ng mL-1 LH, supplemented with or
without 5 mM 1-heptanol, which blocks the coupling of gap junctions (Fagbohun
and Downs, 1991). In this preliminary experiment, more oocytes were prevented
from reaching the metaphase stage of the second meiotic division (M-
Statistical Analysis
All data were analyzed by chi-square test (Snedecor and Cochran, 1980).
RESULTS
In experiment 1, we observed the nuclear morphology of bovine oocytes before the maturation culture. A large proportion of oocytes were the same as in the germinal vesicle stage (GV stage).
The nuclear maturation of the oocytes cultured with and without LH is shown
in Table 1. Non -CO was arrested at M-l stage although 13.3%
of -DO remained at this stage. On the other hand, the number of -CO which reached
the M-ll stage was significantly (p<0.05) higher than that in -DO. Maturation
of the M-ll stage was higher (p<0.05) in CO and DO cultured with LH than
in those cultured without LH. There were no significant differences in the percentage
of CO and DO oocytes reaching M-ll when they were cultured with LH.
| Table 1: | The effect of Luteinizing Hormone (LH, 100 ng mL-1) on the nuclear maturation of Bovine oocytes in vitro for 42-43 h |
| GV, Germinal Vesicle; PM-l, Prometaphase of the first meiotic division; M-l, Metaphase of the first meiotic division; A-l, Anaphase of the first meiotic division; T-l, Telophase of the first meiotic division; M-ll, Metaphase of the second meiotic division; CO, Cumulus-enclosed oocytes; DO, Denuded oocytes; +, Presence of LH; - , Absence of LH. a,b,c; percentage with different superscripts are significantly (p<0.05) different within a column | |
| Table 2: | The effect of 1-heptanol (5 mM) on the nuclear maturation of Bovine oocytes in vitro for 42-43 h with LH (100 ng mL-1) |
| GV, Germinal Vesicle; PM-l, Prometaphase of the first meiotic division; M-l, Metaphase of the first meiotic division; A-l, Anaphase of the first meiotic division; T-l, Telophase of the first meiotic division; M-ll, Metaphase of the second meiotic division; CO, Cumulus-enclosed oocytes; DO, Denuded oocytes; +, Presence of 1-heptanol; -, Absence of 1-heptanol. a,b,c; percentage with different superscripts are significantly (p<0.05) different within a column | |
In experiment 2, as shown in Table 2, the addition of 1 heptanol to the maturation medium supplemented with LH significantly inhibited (p<0.05) the nuclear maturation in both CO and DO. No significant differences were found (p<0.05) between CO and DO cultured with or without 1-heptanol.
DISCUSSION
In the present study, most of the oocytes were at the GV stage before the maturation culture. The addition of LH to the maturation medium resulted in a considerable improvement in the nuclear maturation to the M-ll stage regardless of the presence or absence of cumulus cells (CO and DO). Downs (1993) postulated that LH can inhibit the coupling of gap junctions forming between granulosa and cumulus cells or cumulus cells and oocytes to induce GVBD in the mouse. Most CO and DO in each group underwent GVBD in the present study. A high percentage of CO and DO cultured without LH reached to the prometaphase stage. These results indicate that bovine oocytes can progress from the GV stage without LH or cumulus cells and that LH could stimulate the nuclear maturation of bovine oocytes after GVBD without cumulus cells.
In experiment 1, the absence of LH, even though some -DO were arrested at M-l stage, more -CO reached the M-ll stage. This suggested that cumulus cells have a stimulating affect in maturing bovine oocytes without adding gonadotropins to the medium. In the light of this, Isobe et al. (1996) indicated that cumulus cells suppress the meiotic progression of pig oocytes matured in vitro, but we could not find any inhibitory effect of cumulus cells during oocytes maturation in vitro. This contradiction might be due to the difference between medium components. Compared to the maturation medium, we added three more reagents, sodium pyruvate, calcium lactate and D-glucose, to the maturation medium. Leese and Barton (1984) indicated that mouse oocytes required sodium pyruvate in the medium. One possibility suggested is that the addition of pyruvate might be needed to stimulate the cumulus-enclosed oocytes in pig.
1-heptanol inhibited nuclear maturation even in the presence of LH in the medium. This indicates that gap junctions can be related to the stimulating effect of LH on the nuclear maturation of oocytes. Gap junctions can pass through components up to 1000 Da in size (Beyer, 1993). It is considered that LH can’t pass through gap junctions formed between cumulus cells and oocytes because of the great molecular weight (approximately 30000). Therefore, it is postulated that uncoupling of the gap junction caused by the addition of 1-heptanol might suppress the passing of important factor(s) less than 1000 Da, not LH, regarded as necessary for bovine oocyte maturation.
In the present study the percentage of nuclear maturation in DO treated with LH and 1-heptanol was shown to be similar to that CO matured with both reagents. Loewastein (1981) suggested that the pores of the gap junctions in cumulus-oocytes complexes are likely to close suddenly after rupture of cumulus cell projections reaching to the oocytes. It is suggested that although DO were mechanically removed from enclosed cumulus cells in the present study, the pores of the gap junctions may not be closed for a longer period like those of CO.
In conclusion, LH has a positive effect on the nuclear maturation of bovine oocytes in vitro. Gap junctions can be associated with stimulation of nuclear maturation of oocytes, but we could not clarify which factor(s) can pass through the gap junctions in the culture medium supplemented with LH. Further investigation seems to be needed to elucidate the factor(s) including molecules.
ACKNOWLEDGMENT
We sincerely thanks to those butchers who supplied the bovine ovaries.