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Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes
Theriogenology 57 (2002) 1151±1159
Timing of sequential changes in cumulus cells and ®rst polar body extrusion during in vitro maturation of buffalo oocytes S. Nandi*, B.M. Ravindranatha, P.S.P. Gupta, P.V. Sarma National Institute of Animal Nutrition and Physiology, Indian Council of Agricultural Research, Adugodi, Bangalore 560030, India Received 14 November 2000; accepted 27 July 2001
Abstract Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the ®rst polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 ml droplets of three different culture systems: (1) TCM-199 steer serum (10%); (2) TCM-199 steer serum 10% PMSG (40 IU/ml); and (3) TCM-199 steer serum 10% PMSG 40 IU=ml estradiol 17b (1 mg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 8C) mineral oil and incubated in a CO2 incubator (39 8C, 5% CO2 in air, 90± 95% relative humidity) for 16±18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying ®rst polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9±10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of ®rst polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22±24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded ®rst polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Cumulus cells; Polar body; Time; IVM; Buffalo
* Corresponding author. Tel.: 91-80-571-1304; fax: 91-80-571-1420. E-mail address: [email protected] (S. Nandi).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 1 ) 0 0 7 0 9 - 9
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S. Nandi et al. / Theriogenology 57 (2002) 1151±1159
1. Introduction In vitro production of ruminant embryos provides an excellent opportunity for cheap and abundant embryos for carrying out basic research and for the application of emerging biotechnologies like cloning and transgenesis. In vitro production of ruminant embryos comprises harvesting of good quality immature oocytes, maturing them in vitro in the laboratory, in vitro fertilization of matured oocytes and culture of the embryos up to the transferable stages. The most important step in laboratory embryo production is the maturation of the follicle with enclosed immature oocytes which are arrested at the diplotene stage of prophase of the ®rst meiotic division. Though maturation rate of oocytes are assessed by various methods like staining method (MII stage), degree of cumulus cells expansion [1,2] and identi®cation of extruded ®rst polar body in the perivitteline space, the degree of expansion of cumulus cell mass is routinely employed in buffalo IVM for evaluating oocyte maturation for in vitro fertilization [3]. In conventional in vitro fertilization studies, cumulus±oocyte complexes (COCs) with nearly 70% of homogeneously expanded cumulus cells were considered. COCs with slight or no cumulus expansion were discarded though they may have extruded ®rst polar body, thus underestimating the maturation rate. After removing the cumulus cells mass oocytes with the ®rst polar body can be used for IVF studies, as almost equal fertilization rate was observed with cumulus-intact and cumulus-free buffalo oocytes [4]. Expansion of cumulus cells depends largely on the culture media used for maturation of the oocytes. Supplementation of serum of any source, gonadotrophins and steroids in the culture media enhances the expansion of cumulus cells. Information regarding the degree of cumulus cell expansion and ®rst polar body extrusion of buffalo COCs in relation to time of incubation in different culture media during in vitro maturation is lacking. The aims of our present study were: (1) to investigate the degree of the cumulus cell expansion in relation to time of incubation in different culture media during in vitro maturation of buffalo oocytes; (2) to ascertain the start of expulsion of the ®rst polar body of buffalo oocytes during IVM and to ®nd out the time period for complete expulsion; and (3) to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. 2. Materials and methods All media and chemicals were procured from Sigma Chemical, St. Louis, MO, USA unless otherwise stated. Pregnant mare serum gonadotrophin (PMSG) (FOLLIGON1) was purchased from Intervet International B.V., Boxmeer, The Netherlands. Steer serum was collected from the National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, India herd by clotting the blood from long-term castrated bullock which was then heat inactivated at 50 8C for 30 min, ®lter (0.22 mm) sterilized and stored in 2 ml aliquots at 20 8C until use. The same pool of sera was used throughout the study. 2.1. Collection of oocytes Buffalo ovaries were obtained at a local slaughterhouse (2±3 h after slaughter) and transported to laboratory at 27±30 8C in normal saline supplemented with 50 mg
S. Nandi et al. / Theriogenology 57 (2002) 1151±1159
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gentamycin/ml. Ovaries were washed three±four times using fresh phosphate buffered saline (PBS). Follicles (3±6 mm diameter) were aspirated in to 10 ml syringe ®tted with 18gauge needle. The aspiration media consisted of TCM-199 supplemented with 10% steer serum and PBS mixed with 0.3% fraction V bovine serum albumin (BSA) at 1:1 ratio. COCs were isolated under a zoom stereo microscope (SZ-PT, Olympus, Japan). The COCs were graded by morphological appearance of the cumulus cells investments and homogeneity of ooplasm under the zoom stereo microscope as per the following criteria: (a) Grade A (good)ÐCOCs with more than ®ve layers of compact cumulus cells and with evenly granular homogenous ooplasm; (b) Grade B (fair)ÐCOCs with two to four layers of compact cumulus cells and with evenly granular homogenous ooplasm; (c) Grade C (poor)ÐCOCs with one layer of cumulus cells or denuded oocytes; and (d) Grade D (poor)ÐCOCs with highly scattered cumulus cells and with irregular dark ooplasm. Only Grades A and B oocytes were chosen for in vitro maturation studies. 2.2. Maturation of oocytes in vitro The COCs were washed once with the aspiration medium and twice in the medium in which they would be cultured. COCs (8±10/50 ml droplet) were cultured in three different maturation media: (1) TCM199 steer serum (10%); (2) TCM199 steer serum 10% PMSG (40 IU/ml); and (3) TCM-199 steer serum 10% PMSG (40 IU=ml estradiol 17b (1 mg /ml) under sterile mineral oil at 39 8C and 5% CO2 in air for 16, 17, 18, 20, 22, and 24 h. Commercially available PMSG FOLLIGON1 was used as the gonadotrophin source at the level of 40 IU/ml as this was the optimum for in vitro maturation of buffalo oocytes in an earlier study in this laboratory (Ravindranatha et al. personal communications). 2.3. Experiment 1: evaluating degree of the cumulus cell expansion in relation to the time of incubation in different culture media during IVM of buffalo oocytes The criteria used for assessing the degree of cumulus cells expansion were as described by Kobayashi et al. [2]. Brie¯y, Degree 2, full cumulus cells expansion, all cumulus cells homogeneously spread. Degree 1, moderate cumulus cells expansion, approximately 70% cumulus cells homogeneously spread. Degree 0, slight or no expansion, cumulus cells were adherent to the zona pellucida. COCs were evaluated for the expansion of cumulus cells after culturing for the desired time period. 2.4. Experiment 2: ascertain the time of start and complete expulsion of the first polar body of buffalo oocytes during IVM Oocytes were examined for ®rst polar body by identifying them in the perivitteline space after denuding the oocytes by repeated pipetting the COCs after culturing for the desired time period in different media. The maturation rate was assessed in two ways: Criteria 1Ðoocytes with Degrees 1 and 2 cumulus cells expansion; and Criteria 2Ðoocytes with Degrees 1 and 2 cumulus cells expansion and extruded ®rst polar body in Degree 0 oocytes.
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2.5. Experiment 3: comparison of embryo yield from matured buffalo oocytes considering both criteria of maturation rates Oocytes with more than ®ve layers of cumulus cells and homogenous ooplasm following aspiration were cultured in TCM-199 steer serum 10% PMSG (40 IU/ml) and TCM-199 steer serum 10% PMSG (40 IU=ml estradiol 17b (1 mg/ml) for 24 h for maturation. 2.6. Oviductal cell culture, in vitro fertilization and embryo culture The oviductal cells (20±40 cells per droplet) were cultured in 100 ml droplets of TCM-199 containing 10% steer serum in CO2 incubator at 39 8C for use during in vitro culture of embryos. A monolayer of the oviductal cells was formed on the basement of the culture dish after 3 days of culture. The methods for processing spermatozoa for use in IVF have been described by Nandi et al. [4]. Brie¯y, 0.5 ml of frozen thawed buffalo semen were washed with Brackett and Oliphant (BO) medium (without BSA) containing 10 mg/ml heparin. The spermatozoa were suspended for swim-up in BO medium containing 10 mg/ml heparin and 2.5 mM caffeine. The maturation media containing the matured oocytes were removed 24 h later and replaced by progressively motile spermatozoa in 100 ml droplets of BO medium containing 0.5% BSA, 10 mg/ml heparin and 2.5 mM caffeine. The dishes were then placed in 5% CO2 incubator at 39 8C for 16±18 h. After 16±18 h of incubation, the BO medium and unattached spermatozoa were removed and replaced by TCM-199 supplemented with 10% steer serum along with 10±15 motile buffalo oviductal cells. The dishes were then again placed in CO2 incubator at 39 8C for 24 h. The cleavage rate (oocytes cleaved to two cells or more) was recorded 48 h after insemination commenced. The cleaved embryos were washed three times with TCM-199 10% steer serum and were cultured in 100 ml droplets of TCM-199 10% steer serum containing a monolayer of buffalo oviductal epithelial cells formed on the basement of culture dishes for 8 days in a CO 2 incubator at 39 8C. 2.7. Statistical analysis Degrees of cumulus expansion of buffalo COCs cultured in three different media in relation to time of incubation was analyzed using analysis of variance (ANOVA). Proportions (percentage of oocytes matured, oocytes cleaved, morulae and blastocysts produced of cleaved embryos) were transformed by arcsine square root and data were analyzed by ANOVA. Differences were considered to be signi®cant at P < 0:05.
3. Results A total of 1517 oocytes were analyzed for cumulus expansion and polar body extrusion, and results are presented in Table 1.
Table 1 Effect of incubation time on in vitro cultured buffalo oocytes in three different media on degree of cumulus expansion and first polar body extrusion Cultured media used
Oocytes cultured
Cumulus cells expansion in degrees, n (% S:D:)
Extrusion of First polar body in
D-0
D-2
Degree 0
16
A B C
75 78 81
74 (98.6 9.6) a 70 (89.7 11.9) ab 75 (92.5 7.4) ab
1 (1 0.0) a 7 (9 2.6) b 5 (6 3.9) b
±(0) a 1 (1 0.0) a 1 (1 0.0) a
± ± ±
± ± ±
± ± ±
17
A B C
82 81 77
80 (97.5 5.6) a 71 (87.6 8.9) b 71 (92.2 7.2) ab
2 (2 2.2) a 8 (10 3.9) b 5 (7 4.7) b
±(0) a 2 (2 5.5) a 1 (1 0.0) a
± ± ±
± ± ±
± 2 1
18
A B C
80 92 90
78 ( 97.5 5.6) a 65 (70.6 6.8) c 75 (83.3 6.8) b
2 (3 2.1) a 19 (20 5.6) c 10 (11 4.9) b
±(0) a 8 (9 2.1) b 5 (6 2.6) b
± ± ±
± 3 1
± 8 3
20
A B C
80 93 86
70 (87.5 9.5) ab 47 (50.5 5.1) d 58 (67.4 14.3) c
6 (8 2.2) b 29 (12 3.1) b 20 (23 6.3) c
4 (5 8.6) b 17 (18 4.3) c 8 (9 6.5) b
4 1 1
± 7 6
3 16 11
22
A B C
80 95 83
60 (75.0 5.0) c 30 (31.5 3.8) f 43 (51.8 7.7) d
14 (17 5.3) cb 39 (41 7.4) d 26 (31 9.3) e
6 (8 13.4) b 26 (27 6.9) d 14 (17 11.5) c
7 2 2
6 11 9
9 26 21
24
A B C
85 92 87
52 (61.1 6.3) d 15 (16.3 9.2) g 31 (35.6 12.6) f
9 (11 7.8) b 25 (27 4.7) ce 29 (33 11.7) ce
19 (22 14.6) c 52 (57 8.7) e 27 (31 12.3) d
9 7 8
8 14 15
16 37 29
D-1
Degree 1
Degree 2 S. Nandi et al. / Theriogenology 57 (2002) 1151±1159
Duration of culture (h)
A: TCM-199 steer serum (10%); B: TCM-199 steer serum 10% PMSG (40 IU/ml); C: TCM-199 steer serum 10% PMSG 40 IU=ml estradiol17b (1 mg/ml). D-0: Degree 0 cumulus expansion; D-1: Degree 1 cumulus expansion; D-2: Degree 2 cumulus expansion. Values with different letters in the same column differs significantly (P < 0:05). 1155
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Media used
Oocytes cultured (n)
Types of matured oocytes inseminateda
A
119
B
111
D-1 D-1 D-0 D-1 D-1 D-0
D-2 cumulus expanded D-2 cumulus expanded oocytes with polar body D-2 cumulus expanded D-2 cumulus expanded oocytes with polar body
Maturation rate (% S:D:)
Cleavage rate (% S:D:)
Compact Morulae (% S:D:)
Blastocyst (% S:D:)
oocytes oocytes
83.0 9.5 a 91.4 12.3 b
51.8 10.6 a 57.2 8.7 b
32.4 8.4 a 37.8 7.6 b
28.5 7.9 a 35.2 8.2 b
oocytes oocytes
66.2 11.5 c 74.7 10.6 d
33.3 9.6 c 40.1 9.6 d
20.4 9.6 c 25.1 11.6 d
17.1 7.8 c 22.5 9.8 d
A: TCM-199 steer serum 10% PMSG (40 IU/ml); B: TCM-199 steer serum 10% PMSG (40 IU=ml estradiol 17b (1 mg/ml). Values with different letters in the same column differ significantly (P < 0:05), (b > a > c > d). a D-1: Degree 1 cumulus expansion; D-2: Degree 2 cumulus expansion.
S. Nandi et al. / Theriogenology 57 (2002) 1151±1159
Table 2 Comparison of embryo yield from matured buffalo oocytes considering both criteria of maturation rates
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3.1. Experiment 1 Addition of gonadotrophin alone or with steroid in basic media supplemented with steer serum causes signi®cant increment in cumulus cells expansion. Commencement of cumulus cells expansion was recorded as early as 16 h after culture; the maximum expansion was exhibited during 22±24 h after culture. Degree 2 cumulus expansion was signi®cantly higher in oocytes cultured in media supplemented with steer serum and PMSG than in media containing combination of PMSG and estradiol. The combination of Degrees 1 and 2 cumulus expanded oocytes were signi®cantly higher in media supplemented with PMSG than with PMSG estradiol (68% versus 48% and 84% versus 64%) in both 22 and 24 h of culture. 3.2. Experiment 2 The extrusion of ®rst polar body in the perivitteline space commenced at 17 h of oocytes culture in media supplemented with exogenous hormones. In absence of any exogenous hormones, the ®rst polar body was extruded at 20 h of culture. Complete extrusion of the ®rst polar body was noticed between 22 and 24 h of oocytes culture in all media studied. A high maturation rate was observed at 24 h of culture in all media studied. Maximum maturation rate was found in oocytes cultured for 24 h in media supplemented with steer serum and PMSG. 3.3. Experiment 3 Cleavage, morulae and blastocyst yield were signi®cantly higher in oocytes when Degrees 1 and 2 cumulus expanded oocytes and Degree 0 oocytes with polar body were considered as matured after 24 h of culture (Table 2). Cleavage, morulae and blastocyst yield were signi®cantly higher in oocytes matured in media supplemented with PMSG than in media supplemented with PMSG estradiol. 4. Discussion The criteria used for selecting an ideal IVM oocyte is: (1) the surrounding cumulus cells are fully expanded without evidence of degeneration of ooplasm; (2) the dimension of the perivitelline space is increased; and (3) the ®rst polar body extruded into that space. Degenerated oocytes were excluded from the results. The oocytes were designated as degenerated when it was not possible to determine the exact developmental stage and having abnormal shape after culture. Large variations in the timing of occurrence of oocyte maturation process in vitro have been reported in cattle, ranging from 20 [5], 20±24 [6], 24±30 [7] and 31 h [8]. In buffaloes, oocytes were shown to mature after 24 h of culture [9,10]. Cumulus expansion of oocytes was recorded as early as 16 h of culture in our study, culminating maximum expansion at 22±24 h of culture. Such difference may be due to different culture conditions, oocyte quality, different procedures of evaluation of maturation rate and recording procedures.
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Hormonal in¯uences on oocyte maturation are mediated by the cells of the corona radiata and cumulus cells surrounding the oocytes. In our study, higher maturation of oocytes were observed when PMSG was supplemented rather than the combination of PMSG and estradiol in the media. This may be due to PMSG is having maximum FSH and some LH bioactivity. Cumulus cells stimulated by gonadotrophins (FSH and LH) produce and secrete hyaluronic acid [11], phosphorylated glycosylated polypeptide, Mw 3000 [12] causing dispersion of cells, a process called expansion or muci®cation. Increased maturation rates of the oocytes when matured with PMSG in our study may be due to the differential mitogenic effect of FSH on bovine cumulus cells and granulosa cells [13] and the combined action of FSH and LH on cumulus cells to synthesize pyruvate, thus stimulating the tetraacetic acid cycle leading to an increased availability of ATP for the energy requirement of the oocytes [14]. Since a good maturation rate of oocytes by using PMSG alone was observed in the present study, it is inferred that there is no need to use steroids or commercially available, costly, pure gonadotrophin. Our results also suggest that the addition of estradiol in the medium inhibited the action of PMSG on the fair quality grade of oocytes, con®rming earlier observations of the inhibitory action of estradiol on maturation rates of cattle oocytes [15]. Some discrepancies do exist with regard to the time of extrusion of ®rst polar body in cattle [16]. It was observed to have occurred at 18±24 h after the start of maturation [17±19]. Information in buffalo in this regard is totally lacking. This is the ®rst report wherein expulsion of ®rst polar body was shown to occur at 17 and 20 h of oocyte culture in media supplemented with and without any exogenous hormones, respectively. In contrast to cattle, where expulsion of ®rst polar body was related to the thickness and compaction of cumulus investments, with a delay in extrusion in heavily compacted COCs, our study in buffaloes revealed that the time taken for polar body to be extruded is equal in both COCs with three or four layers of cumulus cells and COCs with more than ®ve layers of cumulus cells. Degree 0 oocytes were also found to contain ®rst polar body extruded in the perivitteline space after culture, thus they were considered matured though with less or no cumulus cell expansion. For Degree 0 oocytes having an extruded ®rst polar body when considered as matured, and then inseminated along with Degrees 1 and 2 cumulus expanded oocytes, there was marked increment in overall maturation rate, cleavage rate and subsequent embryo development. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded ®rst polar body in Degree 0 can be considered matured, thereby overcoming the underestimated maturation rate that is important in commercial embryo production. In conclusion, cumulus expansion and extrusion of ®rst polar body commence at 16± 17 h of buffalo oocyte culture, respectively. Maximum cumulus expansion and extrusion of ®rst polar body occur during 22±24 h of culture. Oocytes with Degrees 1 and 2 cumulus cell expansion and Degree 0 oocytes with extruded ®rst polar body may be considered matured and can be used in IVF studies. Acknowledgements The authors thank Dr. Khub Singh, Director, National Institute of Animal Nutrition and Physiology for providing the necessary facilities for carrying out this work.
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References [1] Chauhan MS, Palta P, Das K, Katiyar PK, Madan ML. Replacement of serum and hormone additives with follicular ¯uid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro. Theriogenology 1997;48:461±9. [2] Kobayashi K, Yanagamachi S, Hoshi H. In¯uence of epidermic growth factor and transforming growth factor-alpha on in vitro maturation of cumulus enclosed bovine oocytes in de®ned medium. J Reprod Fertil 1994;100:439±46. [3] Palta P, Chauhan MS. Laboratory production of buffalo (Bubalus bubalis) embryos. Reprod Fertil Dev 1998;10:379±91. [4] Nandi S, Chauhan MS, Palta P. In¯uence of cumulus cells and sperm concentrations on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro. Theriogenology 1998;50:1251±62. [5] Fulka J, Motlik J. In vitro maturation. In: Proceedings of the Ninth International Congress of Animal Reproduction and Arti®cial Insemination, Madrid, vol. 2. 1980. p. 55±62. [6] Suss U, Wuthrich K. Stages of the ®rst meiotic division in bovine oocytes matured in vitro. Theriogenology 1985;23:281 [abstract]. [7] Sato E, Iritani A, Nishikawa Y. Factors involving in vitro maturation of pig and cattle follicular oocytes cultured in vitro. Jpn J Anim Reprod 1977;23:12±8. [8] Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 1965;196:349±51. [9] Singh R, Majumdar AC. Chronological changes of buffalo follicular oocyte maturation in vitro. Indian J Anim Sci 1992;82:205±9. [10] Yadav BR, Katiyar PK, Chauhan MS, Madan ML. Chromosome con®guration during in vitro maturation of goat, sheep and buffalo oocytes. Theriogenology 1997;47:943±51. [11] Chen L, Wert SE, Hendrix EM, Russel PT, Cannon M, Larsen WJ. Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass. Mol Reprod Dev 1990;26:236±47. [12] Moor RM, Crosby IM. Cellular origin hormonal regulation and biochemical characterization of polypeptide secreted by graa®an follicles of sheep. J Reprod Fertil 1987;79:469±83. [13] Armstrong DT, Xia P. Differential mitogenic action of insulin-like growth factor 1 and FSH on bovine cumulus cells and granulosa cells. Theriogenology 1993;39: 181 [abstract]. [14] Brackett BG, Zuelka KA. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 1993;39:43±64. [15] Robertson JE, Baker RD. Role of sex steroids and Possible regulators of oocyte maturation. Proc Soc Stud Reprod 1969;57 [abstract]. [16] Hyttel P, Xu KP, Smith S, Greve T. Ultrastructure of in vitro maturation in cattle. J Reprod Fertil 1986;78:615±25. [17] Greve T, Goff A, King WA, Bousquet D. LH surge oocyte maturation and in vitro fertilization in superovulated heifers. J Fertil Embryo Transfer 1986;4:136 [abstract]. [18] King WA, Bousquet D, Greve T, Goff AK. Meiosis in bovine oocytes matured in vitro and in vivo. Acta Vet Scand 1986;27:267±79. [19] Xu KP, Greve T, Smith S, Hyttel P. Chronological changes of bovine follicular oocyte maturation in vitro. Acta Vet Scand 1986;27:505±19.
Timing of sequential changes in cumulus cells and ®rst polar body extrusion during in vitro maturation of buffalo oocytes S. Nandi*, B.M. Ravindranatha, P.S.P. Gupta, P.V. Sarma National Institute of Animal Nutrition and Physiology, Indian Council of Agricultural Research, Adugodi, Bangalore 560030, India Received 14 November 2000; accepted 27 July 2001
Abstract Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the ®rst polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 ml droplets of three different culture systems: (1) TCM-199 steer serum (10%); (2) TCM-199 steer serum 10% PMSG (40 IU/ml); and (3) TCM-199 steer serum 10% PMSG 40 IU=ml estradiol 17b (1 mg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 8C) mineral oil and incubated in a CO2 incubator (39 8C, 5% CO2 in air, 90± 95% relative humidity) for 16±18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying ®rst polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9±10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of ®rst polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22±24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded ®rst polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Cumulus cells; Polar body; Time; IVM; Buffalo
* Corresponding author. Tel.: 91-80-571-1304; fax: 91-80-571-1420. E-mail address: [email protected] (S. Nandi).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 1 ) 0 0 7 0 9 - 9
1152
S. Nandi et al. / Theriogenology 57 (2002) 1151±1159
1. Introduction In vitro production of ruminant embryos provides an excellent opportunity for cheap and abundant embryos for carrying out basic research and for the application of emerging biotechnologies like cloning and transgenesis. In vitro production of ruminant embryos comprises harvesting of good quality immature oocytes, maturing them in vitro in the laboratory, in vitro fertilization of matured oocytes and culture of the embryos up to the transferable stages. The most important step in laboratory embryo production is the maturation of the follicle with enclosed immature oocytes which are arrested at the diplotene stage of prophase of the ®rst meiotic division. Though maturation rate of oocytes are assessed by various methods like staining method (MII stage), degree of cumulus cells expansion [1,2] and identi®cation of extruded ®rst polar body in the perivitteline space, the degree of expansion of cumulus cell mass is routinely employed in buffalo IVM for evaluating oocyte maturation for in vitro fertilization [3]. In conventional in vitro fertilization studies, cumulus±oocyte complexes (COCs) with nearly 70% of homogeneously expanded cumulus cells were considered. COCs with slight or no cumulus expansion were discarded though they may have extruded ®rst polar body, thus underestimating the maturation rate. After removing the cumulus cells mass oocytes with the ®rst polar body can be used for IVF studies, as almost equal fertilization rate was observed with cumulus-intact and cumulus-free buffalo oocytes [4]. Expansion of cumulus cells depends largely on the culture media used for maturation of the oocytes. Supplementation of serum of any source, gonadotrophins and steroids in the culture media enhances the expansion of cumulus cells. Information regarding the degree of cumulus cell expansion and ®rst polar body extrusion of buffalo COCs in relation to time of incubation in different culture media during in vitro maturation is lacking. The aims of our present study were: (1) to investigate the degree of the cumulus cell expansion in relation to time of incubation in different culture media during in vitro maturation of buffalo oocytes; (2) to ascertain the start of expulsion of the ®rst polar body of buffalo oocytes during IVM and to ®nd out the time period for complete expulsion; and (3) to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. 2. Materials and methods All media and chemicals were procured from Sigma Chemical, St. Louis, MO, USA unless otherwise stated. Pregnant mare serum gonadotrophin (PMSG) (FOLLIGON1) was purchased from Intervet International B.V., Boxmeer, The Netherlands. Steer serum was collected from the National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, India herd by clotting the blood from long-term castrated bullock which was then heat inactivated at 50 8C for 30 min, ®lter (0.22 mm) sterilized and stored in 2 ml aliquots at 20 8C until use. The same pool of sera was used throughout the study. 2.1. Collection of oocytes Buffalo ovaries were obtained at a local slaughterhouse (2±3 h after slaughter) and transported to laboratory at 27±30 8C in normal saline supplemented with 50 mg
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gentamycin/ml. Ovaries were washed three±four times using fresh phosphate buffered saline (PBS). Follicles (3±6 mm diameter) were aspirated in to 10 ml syringe ®tted with 18gauge needle. The aspiration media consisted of TCM-199 supplemented with 10% steer serum and PBS mixed with 0.3% fraction V bovine serum albumin (BSA) at 1:1 ratio. COCs were isolated under a zoom stereo microscope (SZ-PT, Olympus, Japan). The COCs were graded by morphological appearance of the cumulus cells investments and homogeneity of ooplasm under the zoom stereo microscope as per the following criteria: (a) Grade A (good)ÐCOCs with more than ®ve layers of compact cumulus cells and with evenly granular homogenous ooplasm; (b) Grade B (fair)ÐCOCs with two to four layers of compact cumulus cells and with evenly granular homogenous ooplasm; (c) Grade C (poor)ÐCOCs with one layer of cumulus cells or denuded oocytes; and (d) Grade D (poor)ÐCOCs with highly scattered cumulus cells and with irregular dark ooplasm. Only Grades A and B oocytes were chosen for in vitro maturation studies. 2.2. Maturation of oocytes in vitro The COCs were washed once with the aspiration medium and twice in the medium in which they would be cultured. COCs (8±10/50 ml droplet) were cultured in three different maturation media: (1) TCM199 steer serum (10%); (2) TCM199 steer serum 10% PMSG (40 IU/ml); and (3) TCM-199 steer serum 10% PMSG (40 IU=ml estradiol 17b (1 mg /ml) under sterile mineral oil at 39 8C and 5% CO2 in air for 16, 17, 18, 20, 22, and 24 h. Commercially available PMSG FOLLIGON1 was used as the gonadotrophin source at the level of 40 IU/ml as this was the optimum for in vitro maturation of buffalo oocytes in an earlier study in this laboratory (Ravindranatha et al. personal communications). 2.3. Experiment 1: evaluating degree of the cumulus cell expansion in relation to the time of incubation in different culture media during IVM of buffalo oocytes The criteria used for assessing the degree of cumulus cells expansion were as described by Kobayashi et al. [2]. Brie¯y, Degree 2, full cumulus cells expansion, all cumulus cells homogeneously spread. Degree 1, moderate cumulus cells expansion, approximately 70% cumulus cells homogeneously spread. Degree 0, slight or no expansion, cumulus cells were adherent to the zona pellucida. COCs were evaluated for the expansion of cumulus cells after culturing for the desired time period. 2.4. Experiment 2: ascertain the time of start and complete expulsion of the first polar body of buffalo oocytes during IVM Oocytes were examined for ®rst polar body by identifying them in the perivitteline space after denuding the oocytes by repeated pipetting the COCs after culturing for the desired time period in different media. The maturation rate was assessed in two ways: Criteria 1Ðoocytes with Degrees 1 and 2 cumulus cells expansion; and Criteria 2Ðoocytes with Degrees 1 and 2 cumulus cells expansion and extruded ®rst polar body in Degree 0 oocytes.
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2.5. Experiment 3: comparison of embryo yield from matured buffalo oocytes considering both criteria of maturation rates Oocytes with more than ®ve layers of cumulus cells and homogenous ooplasm following aspiration were cultured in TCM-199 steer serum 10% PMSG (40 IU/ml) and TCM-199 steer serum 10% PMSG (40 IU=ml estradiol 17b (1 mg/ml) for 24 h for maturation. 2.6. Oviductal cell culture, in vitro fertilization and embryo culture The oviductal cells (20±40 cells per droplet) were cultured in 100 ml droplets of TCM-199 containing 10% steer serum in CO2 incubator at 39 8C for use during in vitro culture of embryos. A monolayer of the oviductal cells was formed on the basement of the culture dish after 3 days of culture. The methods for processing spermatozoa for use in IVF have been described by Nandi et al. [4]. Brie¯y, 0.5 ml of frozen thawed buffalo semen were washed with Brackett and Oliphant (BO) medium (without BSA) containing 10 mg/ml heparin. The spermatozoa were suspended for swim-up in BO medium containing 10 mg/ml heparin and 2.5 mM caffeine. The maturation media containing the matured oocytes were removed 24 h later and replaced by progressively motile spermatozoa in 100 ml droplets of BO medium containing 0.5% BSA, 10 mg/ml heparin and 2.5 mM caffeine. The dishes were then placed in 5% CO2 incubator at 39 8C for 16±18 h. After 16±18 h of incubation, the BO medium and unattached spermatozoa were removed and replaced by TCM-199 supplemented with 10% steer serum along with 10±15 motile buffalo oviductal cells. The dishes were then again placed in CO2 incubator at 39 8C for 24 h. The cleavage rate (oocytes cleaved to two cells or more) was recorded 48 h after insemination commenced. The cleaved embryos were washed three times with TCM-199 10% steer serum and were cultured in 100 ml droplets of TCM-199 10% steer serum containing a monolayer of buffalo oviductal epithelial cells formed on the basement of culture dishes for 8 days in a CO 2 incubator at 39 8C. 2.7. Statistical analysis Degrees of cumulus expansion of buffalo COCs cultured in three different media in relation to time of incubation was analyzed using analysis of variance (ANOVA). Proportions (percentage of oocytes matured, oocytes cleaved, morulae and blastocysts produced of cleaved embryos) were transformed by arcsine square root and data were analyzed by ANOVA. Differences were considered to be signi®cant at P < 0:05.
3. Results A total of 1517 oocytes were analyzed for cumulus expansion and polar body extrusion, and results are presented in Table 1.
Table 1 Effect of incubation time on in vitro cultured buffalo oocytes in three different media on degree of cumulus expansion and first polar body extrusion Cultured media used
Oocytes cultured
Cumulus cells expansion in degrees, n (% S:D:)
Extrusion of First polar body in
D-0
D-2
Degree 0
16
A B C
75 78 81
74 (98.6 9.6) a 70 (89.7 11.9) ab 75 (92.5 7.4) ab
1 (1 0.0) a 7 (9 2.6) b 5 (6 3.9) b
±(0) a 1 (1 0.0) a 1 (1 0.0) a
± ± ±
± ± ±
± ± ±
17
A B C
82 81 77
80 (97.5 5.6) a 71 (87.6 8.9) b 71 (92.2 7.2) ab
2 (2 2.2) a 8 (10 3.9) b 5 (7 4.7) b
±(0) a 2 (2 5.5) a 1 (1 0.0) a
± ± ±
± ± ±
± 2 1
18
A B C
80 92 90
78 ( 97.5 5.6) a 65 (70.6 6.8) c 75 (83.3 6.8) b
2 (3 2.1) a 19 (20 5.6) c 10 (11 4.9) b
±(0) a 8 (9 2.1) b 5 (6 2.6) b
± ± ±
± 3 1
± 8 3
20
A B C
80 93 86
70 (87.5 9.5) ab 47 (50.5 5.1) d 58 (67.4 14.3) c
6 (8 2.2) b 29 (12 3.1) b 20 (23 6.3) c
4 (5 8.6) b 17 (18 4.3) c 8 (9 6.5) b
4 1 1
± 7 6
3 16 11
22
A B C
80 95 83
60 (75.0 5.0) c 30 (31.5 3.8) f 43 (51.8 7.7) d
14 (17 5.3) cb 39 (41 7.4) d 26 (31 9.3) e
6 (8 13.4) b 26 (27 6.9) d 14 (17 11.5) c
7 2 2
6 11 9
9 26 21
24
A B C
85 92 87
52 (61.1 6.3) d 15 (16.3 9.2) g 31 (35.6 12.6) f
9 (11 7.8) b 25 (27 4.7) ce 29 (33 11.7) ce
19 (22 14.6) c 52 (57 8.7) e 27 (31 12.3) d
9 7 8
8 14 15
16 37 29
D-1
Degree 1
Degree 2 S. Nandi et al. / Theriogenology 57 (2002) 1151±1159
Duration of culture (h)
A: TCM-199 steer serum (10%); B: TCM-199 steer serum 10% PMSG (40 IU/ml); C: TCM-199 steer serum 10% PMSG 40 IU=ml estradiol17b (1 mg/ml). D-0: Degree 0 cumulus expansion; D-1: Degree 1 cumulus expansion; D-2: Degree 2 cumulus expansion. Values with different letters in the same column differs significantly (P < 0:05). 1155
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Media used
Oocytes cultured (n)
Types of matured oocytes inseminateda
A
119
B
111
D-1 D-1 D-0 D-1 D-1 D-0
D-2 cumulus expanded D-2 cumulus expanded oocytes with polar body D-2 cumulus expanded D-2 cumulus expanded oocytes with polar body
Maturation rate (% S:D:)
Cleavage rate (% S:D:)
Compact Morulae (% S:D:)
Blastocyst (% S:D:)
oocytes oocytes
83.0 9.5 a 91.4 12.3 b
51.8 10.6 a 57.2 8.7 b
32.4 8.4 a 37.8 7.6 b
28.5 7.9 a 35.2 8.2 b
oocytes oocytes
66.2 11.5 c 74.7 10.6 d
33.3 9.6 c 40.1 9.6 d
20.4 9.6 c 25.1 11.6 d
17.1 7.8 c 22.5 9.8 d
A: TCM-199 steer serum 10% PMSG (40 IU/ml); B: TCM-199 steer serum 10% PMSG (40 IU=ml estradiol 17b (1 mg/ml). Values with different letters in the same column differ significantly (P < 0:05), (b > a > c > d). a D-1: Degree 1 cumulus expansion; D-2: Degree 2 cumulus expansion.
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Table 2 Comparison of embryo yield from matured buffalo oocytes considering both criteria of maturation rates
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3.1. Experiment 1 Addition of gonadotrophin alone or with steroid in basic media supplemented with steer serum causes signi®cant increment in cumulus cells expansion. Commencement of cumulus cells expansion was recorded as early as 16 h after culture; the maximum expansion was exhibited during 22±24 h after culture. Degree 2 cumulus expansion was signi®cantly higher in oocytes cultured in media supplemented with steer serum and PMSG than in media containing combination of PMSG and estradiol. The combination of Degrees 1 and 2 cumulus expanded oocytes were signi®cantly higher in media supplemented with PMSG than with PMSG estradiol (68% versus 48% and 84% versus 64%) in both 22 and 24 h of culture. 3.2. Experiment 2 The extrusion of ®rst polar body in the perivitteline space commenced at 17 h of oocytes culture in media supplemented with exogenous hormones. In absence of any exogenous hormones, the ®rst polar body was extruded at 20 h of culture. Complete extrusion of the ®rst polar body was noticed between 22 and 24 h of oocytes culture in all media studied. A high maturation rate was observed at 24 h of culture in all media studied. Maximum maturation rate was found in oocytes cultured for 24 h in media supplemented with steer serum and PMSG. 3.3. Experiment 3 Cleavage, morulae and blastocyst yield were signi®cantly higher in oocytes when Degrees 1 and 2 cumulus expanded oocytes and Degree 0 oocytes with polar body were considered as matured after 24 h of culture (Table 2). Cleavage, morulae and blastocyst yield were signi®cantly higher in oocytes matured in media supplemented with PMSG than in media supplemented with PMSG estradiol. 4. Discussion The criteria used for selecting an ideal IVM oocyte is: (1) the surrounding cumulus cells are fully expanded without evidence of degeneration of ooplasm; (2) the dimension of the perivitelline space is increased; and (3) the ®rst polar body extruded into that space. Degenerated oocytes were excluded from the results. The oocytes were designated as degenerated when it was not possible to determine the exact developmental stage and having abnormal shape after culture. Large variations in the timing of occurrence of oocyte maturation process in vitro have been reported in cattle, ranging from 20 [5], 20±24 [6], 24±30 [7] and 31 h [8]. In buffaloes, oocytes were shown to mature after 24 h of culture [9,10]. Cumulus expansion of oocytes was recorded as early as 16 h of culture in our study, culminating maximum expansion at 22±24 h of culture. Such difference may be due to different culture conditions, oocyte quality, different procedures of evaluation of maturation rate and recording procedures.
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Hormonal in¯uences on oocyte maturation are mediated by the cells of the corona radiata and cumulus cells surrounding the oocytes. In our study, higher maturation of oocytes were observed when PMSG was supplemented rather than the combination of PMSG and estradiol in the media. This may be due to PMSG is having maximum FSH and some LH bioactivity. Cumulus cells stimulated by gonadotrophins (FSH and LH) produce and secrete hyaluronic acid [11], phosphorylated glycosylated polypeptide, Mw 3000 [12] causing dispersion of cells, a process called expansion or muci®cation. Increased maturation rates of the oocytes when matured with PMSG in our study may be due to the differential mitogenic effect of FSH on bovine cumulus cells and granulosa cells [13] and the combined action of FSH and LH on cumulus cells to synthesize pyruvate, thus stimulating the tetraacetic acid cycle leading to an increased availability of ATP for the energy requirement of the oocytes [14]. Since a good maturation rate of oocytes by using PMSG alone was observed in the present study, it is inferred that there is no need to use steroids or commercially available, costly, pure gonadotrophin. Our results also suggest that the addition of estradiol in the medium inhibited the action of PMSG on the fair quality grade of oocytes, con®rming earlier observations of the inhibitory action of estradiol on maturation rates of cattle oocytes [15]. Some discrepancies do exist with regard to the time of extrusion of ®rst polar body in cattle [16]. It was observed to have occurred at 18±24 h after the start of maturation [17±19]. Information in buffalo in this regard is totally lacking. This is the ®rst report wherein expulsion of ®rst polar body was shown to occur at 17 and 20 h of oocyte culture in media supplemented with and without any exogenous hormones, respectively. In contrast to cattle, where expulsion of ®rst polar body was related to the thickness and compaction of cumulus investments, with a delay in extrusion in heavily compacted COCs, our study in buffaloes revealed that the time taken for polar body to be extruded is equal in both COCs with three or four layers of cumulus cells and COCs with more than ®ve layers of cumulus cells. Degree 0 oocytes were also found to contain ®rst polar body extruded in the perivitteline space after culture, thus they were considered matured though with less or no cumulus cell expansion. For Degree 0 oocytes having an extruded ®rst polar body when considered as matured, and then inseminated along with Degrees 1 and 2 cumulus expanded oocytes, there was marked increment in overall maturation rate, cleavage rate and subsequent embryo development. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded ®rst polar body in Degree 0 can be considered matured, thereby overcoming the underestimated maturation rate that is important in commercial embryo production. In conclusion, cumulus expansion and extrusion of ®rst polar body commence at 16± 17 h of buffalo oocyte culture, respectively. Maximum cumulus expansion and extrusion of ®rst polar body occur during 22±24 h of culture. Oocytes with Degrees 1 and 2 cumulus cell expansion and Degree 0 oocytes with extruded ®rst polar body may be considered matured and can be used in IVF studies. Acknowledgements The authors thank Dr. Khub Singh, Director, National Institute of Animal Nutrition and Physiology for providing the necessary facilities for carrying out this work.
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